To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 cells in 100 ul PBS were injected subcutaneously to the right of the dorsal midline in 6- to 8-week-oldathymic nude mice(n = 5 mice/group). After the tumor reached 60-80 mm³ on day 7, the mice were randomly grouped and then given intratumoral treatment with RSL3 (50 mg/kg, once every other day) at day 7 for 2 weeks.

Female Balb/c mice were purchased from Envigo. At the time of implantation, all mice were aged 5-6 weeks. Mice were implanted subcutaneously in the right flank with 1 x 10⁶ CT-26 cells in 100 uL DPBS. Seven days after implantation, mice were randomized into groups (n = 5) with mean tumor volumes ranging from 97 to 117 mm³. The negative control group was dosed daily in the intraperitoneal cavity (IP) with the same vehicle used for QD394. QD394 was dosed at 10 mg/kg IP, and QD394-Me was dosed 3 times weekly intravenously (IV) at 20 mg/kg.

Female Balb/c mice were purchased from Envigo. At the time of implantation, all mice were aged 5-6 weeks. Mice were implanted subcutaneously in the right flank with 1 x 10⁶ CT-26 cells in 100 uL DPBS. Seven days after implantation, mice were randomized into groups (n = 5) with mean tumor volumes ranging from 97 to 117 mm³. The negative control group was dosed daily in the intraperitoneal cavity (IP) with the same vehicle used for QD394. QD394 was dosed at 10 mg/kg IP, and QD394-Me was dosed 3 times weekly intravenously (IV) at 20 mg/kg.

Female BALB/c nude mice (5 weeks old) were procured from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd (Zhejiang, China) and given 5 days to acclimate to their surroundings. PANC-1 cells (1 x 10⁷) in 100 uL PBS at the logarithmic growth phase were administered to mice subcutaneously in the left flank. The mice were treated with indicated treatments after nearly 10 days when the tumour size was approximately 1,000 mm³. In the control group, mice (n = 5) received intraperitoneal injections of the vehicle. In the treatment group, the mice (n = 5) were administered 50 uL of 60 mg/kg body weight of wogonin once a day for 12 days. A slide calliper size was used to measure the tumour size. The equation for calculating tumour volume is as follows: tumour volume = AB2/2, wherein A is the length, and B is the width of the tumour. The mice were sacrificed the next day after the treatment procedure was complete by cervical dislocation. The tumour tissues were harvested and snap-frozen using liquid nitrogen for subsequent analyses.

To generate murine subcutaneous tumors, 2 x 10⁶ PANC1 cells were injected subcutaneously to the right of the dorsal midline in nude mice. Once the tumors reached ~50 mm³ at day seven, mice were randomly allocated into groups and treated with chemotherapy for two weeks (n = 5 mice/group). To generate orthotopic tumors, B6 mice were surgically implanted with 1 x 10⁶ Panc02 into the tail of the pancreas. Two weeks after implantation, mice were randomly allocated into groups and treated with chemotherapy for three weeks (n = 6 mice/group).

To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 or MIAPaCa2 cells in 100 ul PBS were injected subcutaneously into the right of the dorsal midline in 6- to 8-week-old athymic nude or B6 mice (female). Once the tumors reached 50-70 mm³ at day 7, mice were randomly allocated into groups and treated with rapamycin (20 mg/kg; i.p., once every other day) in the absence or presence of liproxstatin-1 (10 mg/kg; i.p., once every other day) or hydroxychloroquine (50 mg/kg; i.p., once every other day) for 2 weeks.

All animal experiments were approved by the Ethics Committee of Jiangsu University. To investigate the role of the combination of erastin and vitamin C in inducing ferroptosis, Panc02 cells (1 x 10⁵ cells/site) were transfected and subcutaneously injected into 4-week-old C57BL/6 mice to generate xenografts. When the tumors reached a volume of 50-100 mm³, the mice were randomly divided into four groups (five mice per group) and treated with DMSO (control), imidazole ketone erastin (IKE, MedChemExpress), vitamin C, or a combination of erastin and vitamin C. Mice were treated with 80 ul (400M) erastin by intratumoral injection and/or 4 g/kg vitamin C by intraperitoneal injection every 2 days.

To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 cells in 100 ul PBS were injected subcutaneously into the right of the dorsal midline in 6- to 8-week-old femaleathymic nude mice(n = 6 mice/group). After the tumor reached 60-80 mm³ on day 7, the mice were randomly grouped and then given intraperitoneal injections with itaconic acid (50 mg/kg, once every other day) at day 7 for 2 weeks.

Female BALB/c nude mice (5 weeks old) were procured from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd (Zhejiang, China) and given 5 days to acclimate to their surroundings. PANC-1 cells (1 x 10⁷) in 100 uL PBS at the logarithmic growth phase were administered to mice subcutaneously in the left flank. The mice were treated with indicated treatments after nearly 10 days when the tumour size was approximately 1,000 mm³. In the control group, mice (n = 5) received intraperitoneal injections of the vehicle. In the treatment group, the mice (n = 5) were administered 50 uL of 60 mg/kg body weight of wogonin once a day for 12 days. A slide calliper size was used to measure the tumour size. The equation for calculating tumour volume is as follows: tumour volume = AB2/2, wherein A is the length, and B is the width of the tumour. The mice were sacrificed the next day after the treatment procedure was complete by cervical dislocation. The tumour tissues were harvested and snap-frozen using liquid nitrogen for subsequent analyses.

To generate murine subcutaneous tumors, 5 x 10⁶ CFPAC1 cells in 100 ul PBS were injected subcutaneously to the right of the dorsal midline in 6- to 8-week-old athymic nude female mice. Once the tumors reached around 70-80 mm³ at day 7, mice were randomly allocated into groups and then treated with imidazole ketone erastin (IKE; 40 mg/kg, i.p., once every other day) in the absence or presence of liproxstatin-1 (10 mg/kg, i.p., once every other day) for 2 weeks.

All animal experiments were approved by the Ethics Committee of Jiangsu University. To investigate the role of the combination of erastin and vitamin C in inducing ferroptosis, Panc02 cells (1 x 10⁵ cells/site) were transfected and subcutaneously injected into 4-week-old C57BL/6 mice to generate xenografts. When the tumors reached a volume of 50-100 mm³, the mice were randomly divided into four groups (five mice per group) and treated with DMSO (control), imidazole ketone erastin (IKE, MedChemExpress), vitamin C, or a combination of erastin and vitamin C. Mice were treated with 80 ul (400M) erastin by intratumoral injection and/or 4 g/kg vitamin C by intraperitoneal injection every 2 days.

Male BALB/c nude mice (5 weeks old, weighing 18-20 g) were provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Nanjing, China). The mice were subcutaneously transplanted with non-targeting shRNA (shcontrol) or CBR1-targeting shRNA (shCBR1)-transfected PANC-1 cells (200 uL, 1 x 10⁷ cells). Tumor volumes and body weights were measured every 4 days (n = 4, each), tumor volume = 0.5 x (a x a x b) (a, smallest diameter; b, largest diameter). The mice were subcutaneously inoculated with PANC-1 cells (200 uL, 1 x 10⁷ cells) in the combination treatment. When the tumor volume reached 80-100 mm³, the mice were treated with chrysin (30 mg/kg/i.P., daily), gemcitabine (20 mg/kg/i.p., once every other day), or in combination for four weeks.

For xenograft assays, we subcutaneously injected 1 x 10⁶ PANC-1 and CFPAC-1 into the right side of each male nude mouse (n = 6). Tumor volumes (length x width2 x 0.5) were measured at specified time points. For one treatment cycle in a week (starting from week 1 to week 5), solasonine (40 or 80 mg/kg, oral administration, 2 times) were given. A total of five treatment cycles were conducted in this experiment.

For xenograft assays, we subcutaneously injected 1 x 10⁶ PANC-1 and CFPAC-1 into the right side of each male nude mouse (n = 6). Tumor volumes (length x width2 x 0.5) were measured at specified time points. For one treatment cycle in a week (starting from week 1 to week 5), solasonine (40 or 80 mg/kg, oral administration, 2 times) were given. A total of five treatment cycles were conducted in this experiment.

AsPC-1 cells (1 x 10⁶) with a control or GRP78 shRNA transfection were injected into right subcutaneous flank of nude mice (five mice per group). The nude mice were randomized into two groups and treated with DMSO or artesunate (30 mg/kg/i.p.), respectively. Artesunate was administered every two days. The tumor growth speed and volume were monitored every two days until the end point at day 35. All the tumor size and weight in the artesunate-treated groups were measured by using a caliper and an electronic balance.

Six to eight-week-old female C57BL/6 mice were purchased from the Experimental Animal Center of Military Medical Sciences (Beijing, China). C57BL/6 mice were anesthetized and the tail of the pancreas was exposed. Panc 02 cells were resuspended in PBS at a concentration of 1 x 10⁶ cells/0.1 ml and 50 ul cells were injected into the tail of the pancreas. Tumor-bearing mice were randomly divided into two groups (3 days after implantation). The control group was intraperitoneally injected 200 ul PBS daily for 10 days, and the DHA group was intraperitoneally injected with 100 mg/kg DHA daily for 10 days. The pancreatic tumors and spleens of the mice were collected for subsequent analysis.

1 x 10⁶ BCAT2 overexpression and control Panc02 cancer cells were implanted subcutaneously into the right dorsal flanks of C57BL/6 mice (five mice per group), respectively. To investigate the role of combination sorafenib with sulfasalazine inducing ferroptosis, 1 x 10⁶ Panc02 were implanted subcutaneously into the right dorsal flanks of C57BL/6 mice. To generate orthotopic tumors, forty C57BL/6 mice were surgically implanted with 1 x 10⁶ H22 cells into left lobe of livers.

NOD SCID mice (394) were purchased from Charles River Laboratories. Indicated wild-type or gene knockdown PANC1 cells (5 x 10⁶ cells) were subcutaneously injected into the dorsal side of NOD SCID mice. At day 7, these mice were administrated with the indicated drug (zalcitabine [50 mg/kg, per day by i.p.], H-151 [750 nM per mouse, once every other day by i.p.], chloroquine [50 mg/kg, once every other day by i.p.], or liproxstatin-1 [10 mg/kg, once every other day by i.p.]) for 2 weeks.

NOD SCID mice (394) were purchased from Charles River Laboratories. Indicated wild-type or gene knockdown PANC1 cells (5 x 10⁶ cells) were subcutaneously injected into the dorsal side of NOD SCID mice. At day 7, these mice were administrated with the indicated drug (zalcitabine [50 mg/kg, per day by i.p.], H-151 [750 nM per mouse, once every other day by i.p.], chloroquine [50 mg/kg, once every other day by i.p.], or liproxstatin-1 [10 mg/kg, once every other day by i.p.]) for 2 weeks.

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.

A total of 15 six-week-old female BALB/c nude mice (25.2-25.9 g) were purchased from SLAC Animal Laboratories. Approximately 1 x 10⁶ BxPC-3 cells were subcutaneously implanted into the right flank of the nude mice. The tumors were allowed to grow to approximately 120 mm³ in size and a total of 15 tumor-bearing mice were divided randomly into 3 groups (n = 5 per group). Group 1 was injected with 0.1 ml PBS as control; group 2 and 3 received 5 and 10 mg/kg ruscogenin, respectively twice per week.

Male BALB/c nude mice (5 weeks old; 16-18 g) were obtained from the Animal Center of Guangxi Medical University and grown under specific pathogen-free conditions. The mice (n = 20) were randomly divided into four groups; Vector group, A2M-AS1 group, Vector + Erastin group, and A2M-AS1 + Erastin group. The Vector and Vector + Erastin groups were injected with 10⁶ cells that stably expressing the lentiviral vector, whereas the other two groups were injected with 10⁶ cells that stably overexpressing A2M-AS1. On the 7th day after inoculation, the Vector + Erastin and A2M-AS1 + Erastin groups were intraperitoneally injected with a 100-uL Erastin solution (40 mg/kg) once every 2 days.

At 6 weeks of age, male C57BL/6J mice received standard diet (SCD) or high-fat diet (HFD; 5.24 kcal/g with 20% energy derived from protein, 60% from fat, and 20% from carbohydrate; Research Diets; D12492) for 12 weeks. Then the mouse PDAC cell lineKPC (male) was implanted subcutaneously into the right abdomen of SCD and HFD mice. Once the tumors reached 60-80 mm³ at day 7, tumor-bearing mice were treated with IKE (40 mg/kg, i.p.,once every other day) or the ferroptosis inhibitor liproxstatin-1 (10 mg/kg, i.p., once every other day) or the PDK inhibitor dichloroacetate (DCA, 50 mg/kg, i.p., once every other day) under the corresponding diet.

A total of 20 SPF BALB/C nude mice were randomly divided into two groups [SIRT6 group (n = 10) and vector group (n = 10)]. PANC-1 cells (1 x 10⁶) in 200 ul phosphate-buffered saline were subcutaneously inoculated in nude mice of each group. The tumor volume was recorded every 7 days.

Male BALB/c nude mice (5 weeks old; 16-18 g) were obtained from the Animal Center of Guangxi Medical University and grown under specific pathogen-free conditions. The mice (n = 20) were randomly divided into four groups; Vector group, A2M-AS1 group, Vector + Erastin group, and A2M-AS1 + Erastin group. The Vector and Vector + Erastin groups were injected with 10⁶ cells that stably expressing the lentiviral vector, whereas the other two groups were injected with 10⁶ cells that stably overexpressing A2M-AS1. On the 7th day after inoculation, the Vector + Erastin and A2M-AS1 + Erastin groups were intraperitoneally injected with a 100-uL Erastin solution (40 mg/kg) once every 2 days.

To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 or HepG2 cells in 100 ul PBS were injected subcutaneously to the right of the dorsal midline in 6- to 8-week-old athymic nude or B6 mice. Once the tumors reached 50-70 mm³ at day 7, mice were randomly allocated into groups and treated with (1S-3R)-RSL3 (30 mg/kg; i.p., once every other day) for 2 weeks.

To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 or HepG2 cells in 100 ul PBS were injected subcutaneously to the right of the dorsal midline in 6- to 8-week-old athymic nude or B6 mice. Once the tumors reached 50-70 mm³ at day 7, mice were randomly allocated into groups and treated with (1S-3R)-RSL3 (30 mg/kg; i.p., once every other day) for 2 weeks.

Six to eight-week-old female C57BL/6 mice were purchased from the Experimental Animal Center of Military Medical Sciences (Beijing, China). C57BL/6 mice were anesthetized and the tail of the pancreas was exposed. Panc 02 cells were resuspended in PBS at a concentration of 1 x 10⁶ cells/0.1 ml and 50 ul cells were injected into the tail of the pancreas. Tumor-bearing mice were randomly divided into two groups (3 days after implantation). The control group was intraperitoneally injected 200 ul PBS daily for 10 days, and the DHA group was intraperitoneally injected with 100 mg/kg DHA daily for 10 days. The pancreatic tumors and spleens of the mice were collected for subsequent analysis.

To generate murine subcutaneous tumors, 5 x 10⁶ CFPAC1 cells in 100 ul PBS were injected subcutaneously to the right of the dorsal midline in 6- to 8-week-old athymic nude female mice. Once the tumors reached around 70-80 mm³ at day 7, mice were randomly allocated into groups and then treated with imidazole ketone erastin (IKE; 40 mg/kg, i.p., once every other day) in the absence or presence of liproxstatin-1 (10 mg/kg, i.p., once every other day) for 2 weeks.

To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 cells in 100 ul PBS were injected subcutaneously into the right of the dorsal midline in 6- to 8-week-old femaleathymic nude mice(n = 6 mice/group). After the tumor reached 60-80 mm³ on day 7, the mice were randomly grouped and then given intraperitoneal injections with itaconic acid (50 mg/kg, once every other day) at day 7 for 2 weeks.

To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 cells in 100 ul PBS were injected subcutaneously to the right of the dorsal midline in 6- to 8-week-oldathymic nude mice(n = 5 mice/group). After the tumor reached 60-80 mm³ on day 7, the mice were randomly grouped and treated with IKE (imidazole ketone erastin; 40 mg/kg; i.p., once every other day) for 3 weeks, and then samples were collected and assayed.

To generate murine subcutaneous tumors, 5 x 10⁶ PANC1 cells in 100 ul PBS were injected subcutaneously to the right of the dorsal midline in 6- to 8-week-oldathymic nude mice(n = 5 mice/group). After the tumor reached 60-80 mm³ on day 7, the mice were randomly grouped and treated with IKE (imidazole ketone erastin; 40 mg/kg; i.p., once every other day) for 3 weeks, and then samples were collected and assayed.