A total of 30 male BALB/c nude mice (4-6 weeks old; 18-23 g) were randomized into four groups (7-8 mice per group): i) control group; ii) treated with 15 mg/kg ICS II; iii) treated with 25 mg/kg ICS II; and, iv) treated with 35 mg/kg ICS II. ACHN and Caki-1 cells (1 x 10⁷) were suspended in 50 ul MEM media mixed with 50 ul Matrigel (BD Biosciences) and injected subcutaneously into the right flank of mice with 1.5%pentobarbital sodium (60 mg/kg body weight; intraperitoneal injection) under anesthesia. Weight lossof more than 20% was considered a humane endpoint.

BALB/c mice were purchased from the Animal Core Facility of Nanjing Medical University. Injection into the tail vein of 6-week-old male mice with Renca-luci (luciferase) cells (1 x 10⁵ cells) was adopted to build the model oflung metastasis. Before lungs were harvested after 1 month to assess pulmonary metastasis, lung metastatic nodules were tracked with IVIS spectrum imaging system in vivo or not (n = 5/group). For survival analysis, the time of death was recorded in each group (n = 10/group) after cells were injected. For assessing the effect of liproxstatin-1 (Lipro, Sigma), one week after injection of cells in the tail vein, mice were tail intravenous administrated with 2.5 mg/kg Lipro three times on a weekly basis for two weeks, then lungs were harvested ( n= 5/group).

5 x 10⁶ HT-1080 or 1 x 10⁷ NCI-H226 cells were injected into mice subcutaneously. When the tumor reached 50-100 mm³, the mice were assigned randomly into different treatment groups. Brequinar or sulfasalazine was dissolved in dimethyl sulfoxide (DMSO) and diluted in PBS. Brequinar was intraperitoneally injected into mice at a dose of 30 mg/kg every three days. Sulfasalazine was intraperitoneally injected daily at a dose of 100 mg/kg. Liproxstatin-1 diluted in PBS was intraperitoneally injected daily at a dose of 10 mg/kg. The daily injection of brequinar, sulfasalazine, or liproxstatin-1 was continued until the endpoint as indicated in the corresponding figures.

PDX tumor derived from lung cancer patient rinsed in cold DMEM media were minced into fragments 1-2 mm³ in volume. Then tumor fragment was subcutaneously inoculated into the dorsal flank of NSG mice. The tumor growth in mice was monitored by bi-dimensional tumor measurements. When tumors grew to a volume of 200 mm³, the mice were divided randomly into four groups (n = 5/group) and treated with vehicle, 10 mg/kg AZD8055, 30 mg/kg IKE, or both (10% dimethyl sulfoxide/90% corn oil) by daily intraperitoneal administration. Body weights of mice in each group during treatment were also recorded accordingly.

Ten million NCCFH1 cells suspended in a 1:1 ratio of PBS:Matrigel were injected into the right flank of each animal. Tumors were measured twice a week using a digital caliper, and tumor volumes were calculated according to the volume of an ellipsoid. When mean tumor volumes reached 100 mm³, an equal number of male and female mice were randomly assigned into four different groups. Thirty three of the 40 mice used in the experiment developed tumors. Thus, the final group assignment was as follow: vehicle (control group, n = 6, 3 female & 3 male), rapamycin (rapamycin only group, n = 7, 4 female & 3 male), Cyst(e)inase (Cyst(e)inase only group, n = 10, 5 female & 5 male), rapamycin+Cyst(e)inase (combination group, n = 10, 5 female & 5 male). Treatments were administered once every three days via intraperitoneal injection of either vehicle (Phosphate buffered saline containing 30% PEG300), rapamycin (0.6 mg/ml suspended in vehicle, injection dose = 0.6 mg per mouse), Cyst(e)inase (7.1 mg/ml suspended in vehicle, injection dose = 7.1 mg per mouse), and rapamycin+Cyst(e)inase combination (0.6 mg/ml rapamycin, 7.1 mg/ml Cyst(e)inase suspended in vehicle.

A total of thirty-two healthy specificpathogenfree C57BL/6J male mice at seven weeks of age and weighted 20-24 g were purchased from the Experimental Animal Center of Chongqing Medical University. After administration of arsenite via drinking water, the animals were euthanized by pentobarbital sodium, three of the animals were subjected to the perfusion fixation, and subsequently, the hippocampus tissues were rapidly dissected on ice and immersed into 4% paraformaldehyde for pre-fixation.

Female SCID mice (4-6 weeks old) were purchased and used for the xenograft models. Approximately 5 x 10⁶ ccRCC cells were injected subcutaneously into the flank. The mice were treated with vehicle (control) or chaetocin (0.5 mg/kg/day) by daily intraperitoneal injection.

For the xenograft of RCC4 cells, four-week-old female NOD. At 6th week, mice were injected with 100 uL of stable RCC4-EV or RCC4-KDM5C cells suspended in Matrigel Basement Membrane Matrix (Corning, 356234) at a population of 1 x 10⁷ cells into the left or right dorsal flank subcutaneously after alcohol sterilization of injection site skin surface. On day 7 after injection, the mice were randomly divided into 2 groups and treated with Liproxstatin-1 (10 mg/kg, MCE HY-12726) or vehicle control (1% DMSO in PBS) each other day by i.p. injection.

Female SCID mice (4-6 weeks old) were purchased and used for the xenograft models. Approximately 5 x 10⁶ ccRCC cells were injected subcutaneously into the flank. The mice were treated with vehicle (control) or chaetocin (0.5 mg/kg/day) by daily intraperitoneal injection.

Six-week-old female athymic nude mice (Charles River Laboratories) were used for xenograft studies. For subcutaneous tumor growth model, cells were pelleted and resuspended in a PBS/Matrigel Matrix (Corning, Cat# 356234) mix at 1:1 ratio. 2 x 10⁶ cells in a 100 uL solution were injected subcutaneously into each flank.

3-4 week-old, male athymic nude mice were used for hosting the 786-O tumor xenografts. In the experiment that leads to isolation of ferroptosis-resistant 1 (FR1) cells , 5 x 10⁶ wildtype or GPX4-/-single-cell clone (originally named as #3A7 clone3, then renamed as ferroptosis-sensitive (FS) GPX4-/-cells in the present study) of 786-O-Cas9 cells were resuspended in 50 ul sterile PBS containing 50 uM Fer-1, mixed with 50 ul Matrigel (BD Biosciences), and subcutaneously injected into both flanks of the mouse.

Female Balb/c nude mice aged 6 weeks were obtained from Vital River Laboratory (Beijing, China). To generate xenografts of RCC, mice were randomized into two groups of 8 that were subcutaneously inoculated with 1 x 10^7 of OS-RC-2 cells stably transfected with USP35 Tet-on shRNA constructs or empty vector. After 7 days, mice were administered with doxycycline every day (20 mg/kg) to induce shRNA expression through oral gavage without blinding.

One million 786O cells with or without shTAZ were implanted subcutaneously into the healthy 8-week-old JAX NOD.CB17-PrkdcSCID-J mice; both male and female mice were used. Once tumor volume reached 120 mm³, mice were randomized into control or erastin treatment group. The vehicle (ORA-plus) or erastin (0.1 ml of 4 mg/ml erastin) was administrated by oral gavage twice daily for 20 days.

One million 786O cells with or without shTAZ were implanted subcutaneously into the healthy 8-week-old JAX NOD.CB17-PrkdcSCID-J mice; both male and female mice were used. Once tumor volume reached 120 mm³, mice were randomized into control or erastin treatment group. The vehicle (ORA-plus) or erastin (0.1 ml of 4 mg/ml erastin) was administrated by oral gavage twice daily for 20 days.