Ab37185

We utilized cell lysis buffer (Beyotime), containing 1% PMSF (Amresco), to cleave proteins for 30 minutes on ice. Then, we centrifuged the lysed cells at 12 000 g for 10 minutes at 4°C to extract the supernatant for protein quantification. We utilized the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) to quantify protein concentration. The obtained supernatant was then boiled for 10 minutes by adding 5X SDS. Protein (50 μg) was added to the prepared 12% SDS‐PAGE gels for electrophoretic separation and transferred to 0.45 µm PVDF membranes (Amersham Hybond, GE Healthcare). We then utilized 1% albumin from bovine serum (Amresco) to block the PVDF membranes for 2 hours. Then, the membranes were incubated overnight with diluted xCT (1:1000, ab37185; Abcam) and β‐actin (1:1000, ab179467; Abcam) antibodies on a shaker at 4°C. We washed the membranes with TBS‐T (0.1% Tween‐20) at room temperature three times for 10 minutes. The goat anti‐rabbit IgG H&L (HRP) (1:2000, ab7090; Abcam) for 1 hour was utilized to incubate the membranes. After washed, the membranes were exposed to enhanced chemiluminescence substrate detection solution (Lulong Biotech) subsequently.

Western blot was performed as per standard protocol. Briefly, the protein was extracted from the tumour tissue-cell lysate. 30 μg proteins were separated by SDS-PAGE, and the proteins were transferred onto polyvinylidene fluoride membranes. The following primary antibodies were used: Nrf2 at 1:1,000 dilution (ab137550, Abcam, United Kingdom), SLC7A11 at 1:1,000 dilution (ab37185, Abcam, United Kingdom), GPX4 at 1:1,000 dilution (ab125066, Abcam, United Kingdom), HO-1 and β-Actin at 1:1,000 dilution (70,081 and 4970S, respectively from Cell Signalling Tech, United States). The protein bands were detected using chemiluminescence (Millipore, MA, United States) and exposed to X-ray film (RX-U; Fujifilm, China).

Proteins with equal concentration were subjected into the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred in PVDF membrane (Bio-Rad, USA, 162-0177). The sample was blocked by non-fat milk (5%) and cultured by the antibodies of SLC7A11 (Abcam, USA, ab37185) and β-actin (Abcam, USA, ab8227) overnight at 4°C, followed by the secondary antibody incubation for 2 hours at 25°C. The expression was detected by ECL reagent (Sigma, USA, WBULS0100).

Total protein was isolated by lysing cells with RIPA lysis buffer supplemented with protease inhibitor cocktail. Protein quantification was done with BCA protein assay kit (PC0020, Solarbio, China). Equal quantities of protein samples were then mixed with loading buffer, heated for 10 min at 100 °C, separated on 10% SDS-PAGE gels, and subsequently transferred to PVDF membranes, which were then blocked with 5% nonfat milk for 1 h, exposed overnight (O/N) to specified antibodies at 4 °C, followed by three 15-min TBST rinses, and further exposure to anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (2° Ab, 1:5000) at room temperature (RT) for 1 h, before visualization of the protein bands with ECL reagent and Amersham Imager 600 (General Electric Company, USA). Finally, the protein bands were quantified with Image J gel analysis software. All experiments were repeated three times. Among the primary antibodies (1° Abs) used were: Rabbit monoclonal anti-TERT (ab191523, Abcam, 1:1000); anti-Nrf2 (ab137550, Abcam, 1:1000); anti-SLC7A11 (ab37185, Abcam, 1:1000); anti-Ferritin (ab75973, Abcam, 1:1000); anti-Ferroportin (ab239583, Abcam, 1:1000); anti-GPX4 (ab125066, Abcam, 1:1000); and anti-β-Actin (4970S, Cell Signaling Tech, 1:1000).

All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise indicated. For western blots, primary rabbit antibodies against xCT, EAAC1, GLAST or GLT1 (ab37185, ab124802, ab416 or ab41621 respectively) were obtained from Abcam, Cambridge, MA, USA. Anti-LAT1 (sc-34554) from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Rabbit anti-NR2A, anti-NR2B or mouse anti-GAPDH (AB1555P or AB1557P, MAB374 respectively) from Millipore, Bedford, MA, USA. Rabbit anti mouse-β-tubulin (T4026) from Sigma-Aldrich. Secondary goat anti-rabbit antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). For immunofluorescence staining chicken anti-MAP2 (ab5392) from Abcam. Anti-rabbit Alexa Fluor 594 (A11039) and anti-chicken Alexa 488 (A21207) secondary antibodies were obtained from Life Technologies, Carlsbad, CA, USA.

Treated cells were fixed with 4% paraformaldehydees, permeabilized in Triton X-100, and then blocked with bovine serum albumin. Immunocytochemical analysis was performed using antibodies specific for xCT (#ab37185, Abcam) and S100 (#ab868, Abcam). Nuclear counterstaining was conducted using DAPI (#C1002, Beyotime). Images were acquired using a confocal microscope (Leica, Germany).