Female mice aged around 6-7 weeks were used for this study, which were purchased through Laboratory Animal Center of Chongqing Medical University from Vital River Co. Ltd (Beijing, China).After one week, each mouse was injected subcutaneously with 100 uL of Huh-7 cell suspension (5 x 10⁶ units) to establish the tumor model. The mice were grouped randomly, and then subjected to different treatments after subcutaneous tumors became visually detectable.

Female mice aged around 6-7 weeks were used for this study, which were purchased through Laboratory Animal Center of Chongqing Medical University from Vital River Co. Ltd (Beijing, China).After one week, each mouse was injected subcutaneously with 100 uL of Huh-7 cell suspension (5 x 10⁶ units) to establish the tumor model. The mice were grouped randomly, and then subjected to different treatments after subcutaneous tumors became visually detectable.

Six-week-old male BALB/c athymic nude mice were purchased from the Experimental Animal Center of Peking (Beijing, China). Stable cells (5 x 10⁶) were seeded into the right flanks of the mice. After the xenografts had grown to 200 mm³, saline as a vehicle or sorafenib (30 mg/kg) was administered by gavage every day, and the mice were euthanized by the cervical dislocation method five weeks later. Before sacrifice, the tumor sizes and body weights were measured twice per week. The tumor volume (V) was calculated as follows: (L x W2)/2 (length, L, and width, W). The xenografts were excised and further assessed.

BALB/c nude mice (age 6 weeks) were brought from the Laboratory Animal Center of Chinese Academy of Sciences (China). HepG2 cell suspension (100 uL, 5 x 10⁵ per site) was hypodermically inoculated into the fat pad of mice. Tumor volume was calculated as follows: tumor volume (mm³) = 0.5 x width (mm)2 x length (mm). When tumor size reached 100 mm³, mice were treated with ketamine (20 mg/kg) or saline intraperitoneally. The mice were succumbed to death when tumor size reached 1000 mm³. Tumors were isolated and weighted. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

BALB/c nude mice (age 6 weeks) were brought from the Laboratory Animal Center of Chinese Academy of Sciences (China). HepG2 cell suspension (100 uL, 5 x 10⁵ per site) was hypodermically inoculated into the fat pad of mice. Tumor volume was calculated as follows: tumor volume (mm³) = 0.5 x width (mm)2 x length (mm). When tumor size reached 100 mm³, mice were treated with ketamine (20 mg/kg) or saline intraperitoneally. The mice were succumbed to death when tumor size reached 1000 mm³. Tumors were isolated and weighted. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

Female Nod-SCID mice of 6-8 weeks old were purchased from HFK BIOSCIENCE (Beijing). Hep3B-vehicle/Hep3B-PSTK-KO cells were harvested and injected subcutaneously (1 x 10⁷ cells in 200 uL PBS) into Nod-SCID mice (upper flank). Treatments were started when tumor volumes reached around 50 mm³. Included mice were randomly divided into four groups and injected intraperitoneally with Abemaciclib (50 mg/kg, every other day) or vehicle. Mice were sacrificed when the tumor volume exceeded 2000 mm³. PSTK-KO or vehicle Hep3B cells were implanted and treated with Sorafenib (50 mg/kg, every other day) or Erastin (50 mg/kg, every other day) for 42 days. Tumor volumes were monitored and quantified by the modified ellipsoidal formula, tumor volume = (length x width2)/2. To check the efficacities and appraisal the side effects of PSTK inhibitors, Hep3B cells were harvested and in injected subcutaneously (5 x 10⁶ cells in 200 uL PBS) into Nod-SCID mice (upper flank). Treatments were started when tumor volumes reached around 50 mm³. Included mice were randomly divided into six groups and intragastrically treated with Punicalin (100 mg/kg, every day), Geraniin (100 mg/kg, every day), Sorafenib (50 mg/kg, every day) with or without PSTK inhibitors (Punicalin/Geraniin) for 30 days. Tumor volumes and mice weights were measured every three days.

To generate murine subcutaneous tumours, 1 x 10⁷ control shRNA or S1R-knockdown Huh7 cells in 200 uL of PBS were injected subcutaneously to the right of the dorsal midline. At day seven, the mice were randomly divided into groups and treated with sorafenib (10 mg/kg/intraperitoneal injection (i.p.), once every other day) for 2 weeks. On day 28, tumours were removed.

BALB/c nude mice (4-6 weeks old) from Beijing Vital River Laboratory Animal Technology (Beijing, China) were reared in a standard laboratory with free access to food and water. Lentivirus LV-sh-NC and LV-sh-HCG18 were from GenePharma (Shanghai, China). In order to establish subcutaneous xenograft tumor models, Huh7-SR cells were infected with lentivirus LV-sh-NC or LV-sh-HCG18 and then resuspended in PBS at 5 x 10⁵/mL. Totally 100 uL cells were subcutaneously injected into the right dorsal area of each nude mouse. When the tumor volume reached 150 mm³, sorafenib (10 mg/kg) was orally administered to nude mice once a day to the end. Tumor volume (V) was calculated: V = 0.5 x L x W2, where L and W were defined as tumor length (L) and width (W). After 28 days of cell injection, the nude mice were euthanized by intraperitoneal injection of excessive pentobarbital sodium (100 mg/kg).

BALB/c nude mice aged 4-6 weeks, weighing 15~20 g, were purchased from Shanghai SLAC Laboratory Animal Co.,Ltd (Shanghai, China). Following acclimation, the right flank of each experimental mouse was subcutaneously injected with HepG2 cells (2 x 10⁶) suspended in PBS (200 uL) and then randomly assigned to: (i) the control group and received no further treatment or (ii) the intervention group and received solasonine (50 mg/kg body weight) in an equal volume of PBS. Tumor volumes were measured every 5 days. After 30 days, the mice were sacrificed and the tumors were resected, weighed, and processed for histological analysis.

C57BL/6J SPF mice were purchased from Huazhong Agricultural University Experimental Animal Center. Mice were given tertian intraperitoneal injections of either PBS (control) or dextriferron (500 mg/kg body weight) for 2 weeks and then sacrificed. Mice were given a daily intraperitoneal injection of either vehicle or ferrostatin-1 (Fer1, 1 mg/kg body weight) for 3 weeks before sacrificed.

Mice were divided into five groups as follows: WT + DMSO (control), WT + Sora, Gstz1-/-+DMSO, Gstz1-/-+Sora, and Gstz1-/-+Sora + RSL3. Each group included three male and three female mice. At 2 weeks of age, all mice were administered an intraperitoneal injection of diethylnitrosamine (DEN; Sigma, St. Louis, MO, USA) at a dose of 75 mg/kg. At the third week, the mice were intraperitoneally administered carbon tetrachloride (CCl4; Macklin, Shanghai, China) at 2 ml/kg twice a week for 12 weeks. In the WT + Sora and Gstz1-/-+Sora group, the mice at 22 weeks were administered intraperitoneally sorafenib (30 mg/kg) every 2 days for 4 weeks until euthanasia. In the Gstz1-/-+Sora + RSL3 group, in addition to sorafenib administration as described above, the mice were injected intraperitoneally with RSL3 (10 mg/kg) every 2 days for 4 weeks at the same weeks.

Animal experiments were conducted under the guidance of Animal Management Regulations in Chongqing University. The tumor volume was calculated as follows: (length x width2)/2. After 24 days, the mice were killed and their tumors were collected, fixed and sectioned, stained by hematoxylin and eosin, and examined by a light microscopy for histological changes.

Animal experiments were conducted under the guidance of Animal Management Regulations in Chongqing University. The tumor volume was calculated as follows: (length x width2)/2. After 24 days, the mice were killed and their tumors were collected, fixed and sectioned, stained by hematoxylin and eosin, and examined by a light microscopy for histological changes.

C57BL/6 mice (4- to 6-week-old male) were fed in a pathogen-free vivarium under standard conditions at the animal care facility at Sun Yat-sen University. Hepa 1-6 cells transduced with RBMS1 or GPX4 or circIDE overexpression lentiviral vectors were subcutaneously injected into the right flank of mice in 100 ul of sterile PBS. IVIS images were taken.

For the proliferation assays, HuCCT1 cells with linc00976 knockdown, linc00976 overexpression, and negative control were subcutaneously injected into BALB/c nude mice. The mice were weighed every week and euthanized 5 weeks after injection. Finally, tumors were dissected and weighed. For the proliferation assays, HuCCT1 cells with linc00976 knockdown, linc00976 overexpression, and negative control were subcutaneously injected into BALB/c nude mice. The mice were weighed every week and euthanized 5 weeks after injection. Finally, tumors were dissected and weighed.

C57BL/6 mice (4- to 6-week-old male) were fed in a pathogen-free vivarium under standard conditions at the animal care facility at Sun Yat-sen University. Hepa 1-6 cells transduced with RBMS1 or GPX4 or circIDE overexpression lentiviral vectors were subcutaneously injected into the right flank of mice in 100 ul of sterile PBS. IVIS images were taken.

The 5-week-old BALB/c male nude mice (n = 10) were purchased from the Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). A number of 2 x 10⁶ HuH-7 cells were transfected with lentiviral vectors containing sh-circIL4R or sh-NC to establish the stably expressed cell lines. Ten mice were subcutaneously injected with HuH-7 cells with sh-circIL4R or sh-NC (five mice per group), constructing the xenograft model of HCC in vivo. Every 5 days after injection, tumor size was measured by a vernier caliper and tumor volume (length x width2 x 0.5) was calculated.

For the proliferation assays, HuCCT1 cells with linc00976 knockdown, linc00976 overexpression, and negative control were subcutaneously injected into BALB/c nude mice. The mice were weighed every week and euthanized 5 weeks after injection. Finally, tumors were dissected and weighed. For the proliferation assays, HuCCT1 cells with linc00976 knockdown, linc00976 overexpression, and negative control were subcutaneously injected into BALB/c nude mice. The mice were weighed every week and euthanized 5 weeks after injection. Finally, tumors were dissected and weighed.

For the proliferation assays, HuCCT1 cells with linc00976 knockdown, linc00976 overexpression, and negative control were subcutaneously injected into BALB/c nude mice. The mice were weighed every week and euthanized 5 weeks after injection. Finally, tumors were dissected and weighed. For the proliferation assays, HuCCT1 cells with linc00976 knockdown, linc00976 overexpression, and negative control were subcutaneously injected into BALB/c nude mice. The mice were weighed every week and euthanized 5 weeks after injection. Finally, tumors were dissected and weighed.

The 5-week-old BALB/c male nude mice (n = 10) were purchased from the Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). A number of 2 x 10⁶ HuH-7 cells were transfected with lentiviral vectors containing sh-circIL4R or sh-NC to establish the stably expressed cell lines. Ten mice were subcutaneously injected with HuH-7 cells with sh-circIL4R or sh-NC (five mice per group), constructing the xenograft model of HCC in vivo. Every 5 days after injection, tumor size was measured by a vernier caliper and tumor volume (length x width2 x 0.5) was calculated.

C57BL/6 mice (4- to 6-week-old male) were fed in a pathogen-free vivarium under standard conditions at the animal care facility at Sun Yat-sen University. Hepa 1-6 cells transduced with RBMS1 or GPX4 or circIDE overexpression lentiviral vectors were subcutaneously injected into the right flank of mice in 100 ul of sterile PBS. IVIS images were taken.

All animal studies were approved by the Committee on Ethics of Animal Experiments of Binzhou Medical University (approval no: BZMU-IACUC-2021-331, date: 09/10/2021). To generate the ectopic HCC mouse models, HepG2-luciferase cells (HepG2 cells transfected with luciferase gene) were suspended in serum-free media and matrigel (BD Biosciences) at a ratio of 1:1 v/v. A total of 2.5 x 10⁶ HepG2-luciferase cells/100 ul were injected into the left axilla of mice. After reaching a tumor size of 100-150 mm³, all mice were randomly divided into four groups: control (vehicle, intraperitoneal [i.p.]), tiliroside (20 mg/kg,i.p.), sorafenib (30 mg/kg,i.p.), or combination treatment (tiliroside and sorafenib,i.p.). All treatments were administered every 3 d, and the length and width of tumor were measured every 4 d. The formula tumor volume = (length x width2)/2 was used to calculate the tumor volume. Body weight was recorded every 7 d, and the morphology of the tumor was photographed using animal in vivo imaging technology (IVIS Spectrum; PerkinElmer) before the day of sacrifice. The mice were sacrificed 40 d after administration, and the tumors were dissected and weighed. The major organs and xenograft tumors were fixed with 4% paraformaldehyde.

All animal studies were approved by the Committee on Ethics of Animal Experiments of Binzhou Medical University (approval no: BZMU-IACUC-2021-331, date: 09/10/2021). To generate the ectopic HCC mouse models, HepG2-luciferase cells (HepG2 cells transfected with luciferase gene) were suspended in serum-free media and matrigel (BD Biosciences) at a ratio of 1:1 v/v. A total of 2.5 x 10⁶ HepG2-luciferase cells/100 ul were injected into the left axilla of mice. After reaching a tumor size of 100-150 mm³, all mice were randomly divided into four groups: control (vehicle, intraperitoneal [i.p.]), tiliroside (20 mg/kg,i.p.), sorafenib (30 mg/kg,i.p.), or combination treatment (tiliroside and sorafenib,i.p.). All treatments were administered every 3 d, and the length and width of tumor were measured every 4 d. The formula tumor volume = (length x width2)/2 was used to calculate the tumor volume. Body weight was recorded every 7 d, and the morphology of the tumor was photographed using animal in vivo imaging technology (IVIS Spectrum; PerkinElmer) before the day of sacrifice. The mice were sacrificed 40 d after administration, and the tumors were dissected and weighed. The major organs and xenograft tumors were fixed with 4% paraformaldehyde.

To generate murine subcutaneous tumors, 1 x 10⁶ Hepa16 cells in control shRNA or NRF2 knockdown cells in 200 ul phosphate buffered saline were injected subcutaneously to the right of the dorsal midline in C57BL/6 mice. Once the tumors reached 80-100 mm³ at day seven, mice were randomly allocated into groups and treated with erastin (30 mg/kg intraperitoneal injection [i.p.], twice every other day) and sorafenib (10 mg/kg i.p., once every other day) for two weeks.

Male 6-week-old BALB/c nude mice were purchased from Charles River Company (Shanghai, China). For subcutaneous mouse model, 5 x10⁶ MHCC97H vector control cells or MHCC97H/QSOX1 cells were implanted into right flanks subcutaneously. At the 7th day following implantation, the mice bear with MHCC97H vector control cells or MHCC97H/QSOX1 cells respectively were randomly separated into two groups: one group were treated with vehicle (0.9% NaCl i.p., once every other day) and another group sorafenib (10 mg/kg i.p., once every other day) for two weeks.

Mice were divided into five groups as follows: WT + DMSO (control), WT + Sora, Gstz1-/-+DMSO, Gstz1-/-+Sora, and Gstz1-/-+Sora + RSL3. Each group included three male and three female mice. At 2 weeks of age, all mice were administered an intraperitoneal injection of diethylnitrosamine (DEN; Sigma, St. Louis, MO, USA) at a dose of 75 mg/kg. At the third week, the mice were intraperitoneally administered carbon tetrachloride (CCl4; Macklin, Shanghai, China) at 2 ml/kg twice a week for 12 weeks. In the WT + Sora and Gstz1-/-+Sora group, the mice at 22 weeks were administered intraperitoneally sorafenib (30 mg/kg) every 2 days for 4 weeks until euthanasia. In the Gstz1-/-+Sora + RSL3 group, in addition to sorafenib administration as described above, the mice were injected intraperitoneally with RSL3 (10 mg/kg) every 2 days for 4 weeks at the same weeks.

Mice weighing between 20 and 23 g were selected and 5 x 10⁶ Hep-G2 cells were subcutaneously injected into their backs. The mice were subsequently divided into the following four groups: control (n = 5), FNDC5 overexpressing (n = 5), FNDC5 overexpressing followed by treatment with the PI3K inhibitor LY294002 (MCE, China), and FNDC5 knockdown (n = 5). Seven days after cell injection, sorafenib (30 mg/kg) was administered to all mice via intraperitoneal injection every alternate day for 4 weeks. The mice in the third group were intraperitoneally injected with LY294002 (25 mg/kg) diluted with DMSO twice a week for 4 weeks.

Mice weighing between 20 and 23 g were selected and 5 x 10⁶ Hep-G2 cells were subcutaneously injected into their backs. The mice were subsequently divided into the following four groups: control (n = 5), FNDC5 overexpressing (n = 5), FNDC5 overexpressing followed by treatment with the PI3K inhibitor LY294002 (MCE, China), and FNDC5 knockdown (n = 5). Seven days after cell injection, sorafenib (30 mg/kg) was administered to all mice via intraperitoneal injection every alternate day for 4 weeks. The mice in the third group were intraperitoneally injected with LY294002 (25 mg/kg) diluted with DMSO twice a week for 4 weeks.

Zebrafish were maintained at 28 and in light cycle conditions (12 h). The Casper mutant fish line was purchased from the Zebrafish International Resource Center (ZIRC). For zebrafish xenotransplantation, 48 hours post-fertilization (hpf) zebrafish embryos were dechorionated and anesthetized in egg water solution containing 0.04 mg/mL tricaine (Sigma-Aldrich, St. Louis, MO, USA) before human cell injection. Approximately 200 to 500 fluorescent cells were injected (Eppendorf Femtojet microinjector) into the ducts of Cuvier of each embryo, and the zebrafish were maintained in 0.3X Danieaus solution for 1 h at 28 . After confirmation of a visible cell mass at the injection site, the zebrafish were transferred to a 24-well plate in 500 uL of a 0.3X Danieaus solution incubator and maintained at 34 . The zebrafish with already formed metastasis at 1 hour post-injection (hpi) were discarded.

Age-matched male BALB/c nude mice (4-6 weeks old) were used for the orthotopic mouse model. Cohorts of mice were randomized into different treatment groups. 4 x 10⁶ tumor cells were suspended in a 50 ul PBS/Matrigel (356234, BD Biosciences) mixture (1:1 (v/v) ratio) for each group of mice and injected into the left liver lobes by surgical implantation.

Mice weighing between 20 and 23 g were selected and 5 x 10⁶ Hep-G2 cells were subcutaneously injected into their backs. The mice were subsequently divided into the following four groups: control (n = 5), FNDC5 overexpressing (n = 5), FNDC5 overexpressing followed by treatment with the PI3K inhibitor LY294002 (MCE, China), and FNDC5 knockdown (n = 5). Seven days after cell injection, sorafenib (30 mg/kg) was administered to all mice via intraperitoneal injection every alternate day for 4 weeks. The mice in the third group were intraperitoneally injected with LY294002 (25 mg/kg) diluted with DMSO twice a week for 4 weeks.

Female mice aged around 6-7 weeks were used for this study, which were purchased through Laboratory Animal Center of Chongqing Medical University from Vital River Co. Ltd (Beijing, China).After one week, each mouse was injected subcutaneously with 100 uL of Huh-7 cell suspension (5 x 10⁶ units) to establish the tumor model. The mice were grouped randomly, and then subjected to different treatments after subcutaneous tumors became visually detectable.

Parental MHCC97L cells (2 x 10⁶ cells/mouse) were subcutaneously injected into the 4-to-5-week-old NOD-SCID mice. When the tumours reached a volume of around 50-100 mm³ (calculated by the formula 4/3(D/2)(d/2)2, where D and d represent the minor and major axis of the tumour, respectively), the maximum tolerated dose of sorafenib (50 mg/kg) was given to the mice by oral gavage daily until the drug resistance occurred, denoted as the drug resistant group. For the control, the wild type group was treated with the vehicle (0.5% CMC-Na). The tumour size and body weight were measured every 3 days.

Parental MHCC97L cells (2 x 10⁶ cells/mouse) were subcutaneously injected into the 4-to-5-week-old NOD-SCID mice. When the tumours reached a volume of around 50-100 mm³ (calculated by the formula 4/3(D/2)(d/2)2, where D and d represent the minor and major axis of the tumour, respectively), the maximum tolerated dose of sorafenib (50 mg/kg) was given to the mice by oral gavage daily until the drug resistance occurred, denoted as the drug resistant group. For the control, the wild type group was treated with the vehicle (0.5% CMC-Na). The tumour size and body weight were measured every 3 days.

A total of 20 male Balb/c nude mice aged 6-8 weeks were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). Five million Huh7 cells were inoculated into the right flanks of the mice. When the tumor size reached 80-100 mm³, the mice were randomly divided into four groups and administered artesunate (30 mg/kg mouse weight) alone, sorafenib (20 mg/kg mouse weight) alone, a combination of artesunate and sorafenib, or the same volume of PBS by gavage every other day.

Age-matched male BALB/c nude mice (4-6 weeks old) were used for the orthotopic mouse model. Cohorts of mice were randomized into different treatment groups. 4 x 10⁶ tumor cells were suspended in a 50 ul PBS/Matrigel (356234, BD Biosciences) mixture (1:1 (v/v) ratio) for each group of mice and injected into the left liver lobes by surgical implantation.

Six-week-old male BALB/c athymic nude mice were purchased from the Experimental Animal Center of Peking (Beijing, China). Stable cells (5 x 10⁶) were seeded into the right flanks of the mice. After the xenografts had grown to 200 mm³, saline as a vehicle or sorafenib (30 mg/kg) was administered by gavage every day, and the mice were euthanized by the cervical dislocation method five weeks later. Before sacrifice, the tumor sizes and body weights were measured twice per week. The tumor volume (V) was calculated as follows: (L x W2)/2 (length, L, and width, W). The xenografts were excised and further assessed.

Xenografts with MiaPaCa-2 and HepG2 cells in nude mice and treated them for 4 or 3 weeks, respectively, with vehicle alone and 2.5 or 5.0 mg/kg/day of ZZW-115. Then, we measured the GPX4 activity and analyzed the mRNA levels of the key genes involved in ferroptosis by qRT-PCR analysis.

Xenografts with MiaPaCa-2 and HepG2 cells in nude mice and treated them for 4 or 3 weeks, respectively, with vehicle alone and 2.5 or 5.0 mg/kg/day of ZZW-115. Then, we measured the GPX4 activity and analyzed the mRNA levels of the key genes involved in ferroptosis by qRT-PCR analysis.

Six-week-old BALB/c female athymic nude mice (SJA Laboratory Animal Co., Ltd, China) were used to construct the xenograft. The mice were randomly grouped: (1) shNC + vehicle; (2) shNC + Erastin; (3) shWTAP + Erastin; (4) shcircCMTM3 + Erastin. Briefly, HCC cells with stable silenced WTAP and circCMTM3 (2 x 10⁶ in 0.1 mL PBS) were injected into the left flank of the mice subcutaneously. Once the tumor size reached approximately > 60 mm³, mice in the groups (2), (3), and (4) were injected with 15 mg/kg erastin. Erastin was injected twice a day for a period of time.

PDX tumors in cold Dulbeccos Modified Eagles Medium (DMEM) were minced into 1-2 mm³ fragments, and each fragment was subcutaneously transplanted into the dorsal flank of 6-week-old male NSG (non-obese diabetic; severe combined immunodeficiency; interleukin-2 receptor gamma chain null) mice. Tumor growth was monitored by bidimensional tumor measurements with a caliper twice a week until the endpoint.

To generate murine subcutaneous tumors, 2 x 10⁶ Huh7 cells in control shRNA or MT-1G knockdown cells in 200 ul phosphate buffered saline were injected subcutaneously to the right of the dorsal midline in nude mice. Once the tumors reached 80-100 mm³ at day seven, mice were randomly allocated into groups and treated with sorafenib (10 mg/kg/intraperitoneal injection (i.p.), once every other day) for two weeks. In another experiment, nude mice were injected subcutaneously with indicated Huh7 cells (2 x 10⁶ cells/mouse) and treated with sorafenib (10 mg/kg/i.p., once every other day) with or without ATRA (0.5 mg/kg/i.p., once every other day) or PPG (10 mg/kg/i.p., once every other day) at day seven for two weeks.

NU/NU nude mice were purchased from Charles River (Beijing). For xenograft models, HepG2 or HuH-7 cells (5 x 10⁶ cells per mouse) that were transfected with the NEAT1 vector or an empty vector were injected into the left posterior flanks of 7-week-old immunodeficient female nude mice. The tumors were measured every 4 days, and tumor volume was calculated using the following formula: volume = (L x W2)/2, among which L and W are the longest and shortest diameters, respectively. When tumors reached a volume of ~50 mm³, mice were randomly allocated into groups and treated with erastin or RSL3 via intraperitoneal injection for 20 days. Mice were then sacrificed, the xenograft tumors were excised and weighted for immunohistochemistry assays. The erastin was dissolved in 5% DMSO + corn oil (C8267, Sigma). To better dissolve erastin, we warmed the tube at 37 water base and shake it gently.

Eight-week-old athymic nude mice were obtained from Bikai Laboratory Animal Crop (Bikai, Shanghai, China) and Bel-7402 cells (initial 5 x 10⁶) were subcutaneously injected into each mouse. Dimethy sulfoxide (DMSO) or piperazine erastin (5 mg/kg, MedChemExpress, Monmouth Junction, NJ, USA) was subcutaneously injected once every day after xenografts were formed.

H22 mouse liver tumor cells were intraperitoneal injected into female ICR mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China). 8 days later, mice with distended abdomen were killed, and the ascites was collected, washed and resuspended with PBS. 0.1ml cell suspension with a density of 1 x 10⁷/ml was subcutaneously injected into the right dorsum of ICR female mice (~20g). The mice bearing H22 tumor cells were then randomized into groups (n = 8) on the following day after the implantation. Then, the mice were injected via a tail vein (i.v.) with PBS, CH004 (10 mg/kg) or cyclophosphamide (CTX, a known anticancer drug; 20 mg/kg) once per day and for 21 days.

Six-week-old BALB/c female athymic nude mice (SJA Laboratory Animal Co., Ltd, China) were used to construct the xenograft. The mice were randomly grouped: (1) shNC + vehicle; (2) shNC + Erastin; (3) shWTAP + Erastin; (4) shcircCMTM3 + Erastin. Briefly, HCC cells with stable silenced WTAP and circCMTM3 (2 x 10⁶ in 0.1 mL PBS) were injected into the left flank of the mice subcutaneously. Once the tumor size reached approximately > 60 mm³, mice in the groups (2), (3), and (4) were injected with 15 mg/kg erastin. Erastin was injected twice a day for a period of time.

PDX tumors in cold Dulbeccos Modified Eagles Medium (DMEM) were minced into 1-2 mm³ fragments, and each fragment was subcutaneously transplanted into the dorsal flank of 6-week-old male NSG (non-obese diabetic; severe combined immunodeficiency; interleukin-2 receptor gamma chain null) mice. Tumor growth was monitored by bidimensional tumor measurements with a caliper twice a week until the endpoint.

PDX tumors in cold Dulbeccos Modified Eagles Medium (DMEM) were minced into 1-2 mm³ fragments, and each fragment was subcutaneously transplanted into the dorsal flank of 6-week-old male NSG (non-obese diabetic; severe combined immunodeficiency; interleukin-2 receptor gamma chain null) mice. Tumor growth was monitored by bidimensional tumor measurements with a caliper twice a week until the endpoint.

NU/NU nude mice were purchased from Charles River (Beijing). For xenograft models, HepG2 or HuH-7 cells (5 x 10⁶ cells per mouse) that were transfected with the NEAT1 vector or an empty vector were injected into the left posterior flanks of 7-week-old immunodeficient female nude mice. The tumors were measured every 4 days, and tumor volume was calculated using the following formula: volume = (L x W2)/2, among which L and W are the longest and shortest diameters, respectively. When tumors reached a volume of ~50 mm³, mice were randomly allocated into groups and treated with erastin or RSL3 via intraperitoneal injection for 20 days. Mice were then sacrificed, the xenograft tumors were excised and weighted for immunohistochemistry assays. The erastin was dissolved in 5% DMSO + corn oil (C8267, Sigma). To better dissolve erastin, we warmed the tube at 37 water base and shake it gently.

NU/NU nude mice were purchased from Charles River (Beijing). For xenograft models, HepG2 or HuH-7 cells (5 x 10⁶ cells per mouse) that were transfected with the NEAT1 vector or an empty vector were injected into the left posterior flanks of 7-week-old immunodeficient female nude mice. The tumors were measured every 4 days, and tumor volume was calculated using the following formula: volume = (L x W2)/2, among which L and W are the longest and shortest diameters, respectively. When tumors reached a volume of ~50 mm³, mice were randomly allocated into groups and treated with erastin or RSL3 via intraperitoneal injection for 20 days. Mice were then sacrificed, the xenograft tumors were excised and weighted for immunohistochemistry assays. The erastin was dissolved in 5% DMSO + corn oil (C8267, Sigma). To better dissolve erastin, we warmed the tube at 37 water base and shake it gently.

To evaluate the effects of drugs to strengthen the effects of IKE, cell-derived xenograft (CDX) mouse models were generated by subcutaneously injecting 2 x 10⁶ cells per athymic nude mouse (BALB/c-nu, Spaefer, Beijing, China). When tumours were 220-250 mm³, mice were administered with IKE (50 mg/kg) daily with or without PTX (20 mg/kg), Clolar (10 mg/kg), OXA (10 mg/kg) or TMZ (40 mg/kg). Tumour growth was monitored, and sizes were calculated by 0.5 x L x W2 (L indicating length and W indicating width).

Xenograft mouse model experiments were used male BALB/c nude mice (4 weeks old) purchased from SPF Biotechnology (Beijing, China). Each mouse was injected 5 x 10⁶ tumor cells at the volume of 100 uL into the subcutaneous tissue. The tumor volume and weight of the mice was observed every 2 days. Mice were monitored daily and the tumor volume calculated according to the equation volume = length x width2 x 1/2.

The 5-week-old nude mice (BALB/c) were purchased from Beijing HFK Bioscience Co., Ltd. (China). Hep3B cells (7 x 10⁶) stably transfected with NC1 or pre-miR-214 plasmid were subcutaneously injected into the nude mice. When the tumor sizes reached approximately >50 mm³, mice in Groups B and C were treated with 15 mg/kg erastin (MCE, China) twice every day for 20 days. Mice in Group A were treated with an equal volume vehicle.

The 5-week-old nude mice (BALB/c) were purchased from Beijing HFK Bioscience Co., Ltd. (China). Hep3B cells (7 x 10⁶) stably transfected with NC1 or pre-miR-214 plasmid were subcutaneously injected into the nude mice. When the tumor sizes reached approximately >50 mm³, mice in Groups B and C were treated with 15 mg/kg erastin (MCE, China) twice every day for 20 days. Mice in Group A were treated with an equal volume vehicle.

Six week-old female Balb/cnude mice (Janvier, Le Genest Saint Isle, France) were injected with HCC cells (10 x 10⁶ cells) stably transfected with the vector pGL4.51 [luc2/CMV/neo] (Promega) encoding the luciferase reporter gene luc2 (Photinus pyralis). When the first tumours appeared, mice were randomly assigned into four groups: control or shRb, receiving sorafenib administered by oral gavage (30 mg/kg/d, resuspended in Cremophore EL) or vehicle alone.

C57BL/6J SPF mice were purchased from Huazhong Agricultural University Experimental Animal Center. Mice were given tertian intraperitoneal injections of either PBS (control) or dextriferron (500 mg/kg body weight) for 2 weeks and then sacrificed. Mice were given a daily intraperitoneal injection of either vehicle or ferrostatin-1 (Fer1, 1 mg/kg body weight) for 3 weeks before sacrificed.

A total of eight male BALB/c nude mice (4-5 weeks old; weight, 13-18 g) were obtained from Beijing Vital River Laboratories. The mice were randomly divided into the following two groups: The NC + SF and sh-PTBP1 + SF groups. Hep3B cells (2 x 10⁶) were subcutaneously injected into the right flank of each mouse. The mice were injected intraperitoneally with SF (10 mg/kg) every 2 days for 2 weeks.

Female mice aged around 6-7 weeks were used for this study, which were purchased through Laboratory Animal Center of Chongqing Medical University from Vital River Co. Ltd (Beijing, China).After one week, each mouse was injected subcutaneously with 100 uL of Huh-7 cell suspension (5 x 10⁶ units) to establish the tumor model. The mice were grouped randomly, and then subjected to different treatments after subcutaneous tumors became visually detectable.

Four-week-old nude mice (BALB/c) were used, and they were housed for 1 week in a specific pathogen-free (SPF) environment before experimentation. Each mouse received subcutaneous injections of 1 x 10⁶ SMMC-7721 or HCCLM3 cells in 200 uL phosphate-buffered saline (PBS) into both flanks. One group was intraperitoneally injected every two days with EB at 25 or 50 mg/mouse body weight, for a total of 3 weeks. As a control, DMSO was used in another group. Tumor size was measured to calculate tumor volume, and mouse body weights were monitored every two days.

A total of 32 male BALB/c nude mice (age, 6-8 weeks; weight, 18-20 g) were obtained from HFK Bioscience Co. Ltd. and housed in a specific pathogen-free facility. The mice were randomly divided into four groups (n = 8/group) and PLC cells were subcutaneously injected into the nude mice. When the xenografted tumors had grown to 80-100 mm³, half of the mice in each group were administered with 100 mg/kg DHA for 5 days/week by gavage.

Six-week-old female BALB/c nude mice were purchased from Byrness Weil Biotechnology Ltd. (Chengdu, China) and housed in a specific pathogen-free environment with a 12-h light/dark cycle and controlled temperature and humidity, and food and water were provided ad libitum. Three million designated treated PLC5 cells were collected and injected subcutaneously into mice. At least 4 mice were used in each group in each experiment. Once the tumours reached a mean volume of 200 mm³, the mice were treated intraperitoneally with sorafenib every 3 days. The mice were then euthanized at the indicated time after injection. Each tumour was dissected, fixed with 4% formaldehyde, and embedded in paraffin. The tumour growth was monitored weekly by calliper measurements.

Six-week-old female BALB/c nude mice were purchased from Byrness Weil Biotechnology Ltd. (Chengdu, China) and housed in a specific pathogen-free environment with a 12-h light/dark cycle and controlled temperature and humidity, and food and water were provided ad libitum. Three million designated treated PLC5 cells were collected and injected subcutaneously into mice. At least 4 mice were used in each group in each experiment. Once the tumours reached a mean volume of 200 mm³, the mice were treated intraperitoneally with sorafenib every 3 days. The mice were then euthanized at the indicated time after injection. Each tumour was dissected, fixed with 4% formaldehyde, and embedded in paraffin. The tumour growth was monitored weekly by calliper measurements.

A total of 20 male Balb/c nude mice aged 6-8 weeks were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). Five million Huh7 cells were inoculated into the right flanks of the mice. When the tumor size reached 80-100 mm³, the mice were randomly divided into four groups and administered artesunate (30 mg/kg mouse weight) alone, sorafenib (20 mg/kg mouse weight) alone, a combination of artesunate and sorafenib, or the same volume of PBS by gavage every other day.

Xenograft mouse model experiments were used male BALB/c nude mice (4 weeks old) purchased from SPF Biotechnology (Beijing, China). Each mouse was injected 5 x 10⁶ tumor cells at the volume of 100 uL into the subcutaneous tissue. The tumor volume and weight of the mice was observed every 2 days. Mice were monitored daily and the tumor volume calculated according to the equation volume = length x width2 x 1/2.