phenformin  (MedChemExpress)

Journal: Advanced Science

Article Title: Inhibition of Endoplasmic Reticulum Stress Cooperates with SLC7A11 to Promote Disulfidptosis and Suppress Tumor Growth upon Glucose Limitation

doi: 10.1002/advs.202408789

Figure Lengend Snippet: SLC7A11 promotes p38 phosphorylation during disulfidptosis induced by glucose deprivation. A–E) Cell morphological changes (A,C) and cell viability (B,D,E) measured by CCK8 assay in SLC7A11 high UMRC6, H460, and 7402 cells cultured in glucose‐containing (Ctrl) or glucose‐free (‐Glc) medium with or without Z‐VAD (5 µ m ), Nec‐1s (2 µ m ), Liprox‐1 (5 µ m ), CQ (20 µ m ) and NAC (2 m m ) for 4–6 h. Scale bars, 100 µm. F) Western blotting analysis of apoptotic markers in UMRC6 and H460 cells cultured in medium without glucose (Glc) or glutamine (Gln) or with energy stress inducers 2‐DG (10 m m ), metformin (2 m m ) and phenformin (2 m m ) for 8 h. G) Reducing and non‐reducing Western blotting analysis of Drebrin in UMRC6 and 7402 cells cultured in glucose‐containing/‐free medium for 4–6 h. H) Western blotting analysis of SLC7A11 expression in control and SLC7A11 knockout UMRC6 cell lines. I) Antibody microarray analysis of involved signaling molecules in sgCon and sgSLC7A11 UMRC6 cells cultured in glucose‐containing/‐free medium. J) Western blotting analysis of p38 phosphorylation of indicated cells cultured in glucose‐containing/‐free medium. K) Western blotting analysis of SLC7A11 expression and p38 phosphoryation in 786‐O empty vector (EV), wild type (WT), and C86S mutant cell lines cultured in glucose‐containing/‐free medium. L,M) Western blotting analysis of p38 phosphoryation in UMRC6 (L) and 7402 (M) cells cultured in glucose‐containing/‐free medium with or without Cystine, Erastin (10 µ m ) and Sulfasalazine (SAS, 10 µ m ) for 4–6 h. N,O) Cell viability measured by CCK8 assay in UMRC6 (N) and 7402 (O) cells cultured in glucose‐containing/‐free medium with or without indicated concentrations of Cystine, Erastin and SAS for 4–6 h. P) Western blotting analysis of p38 phosphoryation in UMRC6 cells cultured in glucose‐containing/‐free medium with or without p38 inhibitor SB203580 (50 µ m ) for 6 h. Q) Cell viability measured by CCK8 assay in UMRC6 cells cultured in glucose‐containing/‐free medium with or without SB203580 treatment. R) Reducing and non‐reducing Western blotting analysis of Drebrin in UMRC6 and 7402 cells cultured in glucose‐containing/‐free medium with or without SB203580 treatment. S–X) Western blotting analysis of MAPK12 (S) and MAPK14 (W) expression, and RT‐PCR analysis of MAPK13 (U) levels in control and corresponding knockout UMRC6 cell lines. Cell viability was measured by CCK8 assay in sgCon and sgMAPK12/13/14 (T/V/X) UMRC6 cells cultured in glucose‐containing/‐free medium. All p values were calculated using a two‐tailed unpaired Student's t ‐test. Data are mean ± SD, n ≥ 3 independent repeats unless specified. ns: not significant ( p > 0.05). All Western blotting was repeated at least twice, independently, with similar results.

Article Snippet: The reagents were purchased as followings: Z‐VAD‐FMK (T7020), Necrostain 2 racemate (S8641), Rotenone (T2970) and MitoQ10 (T28049) were bought from TargetMol (China); GSH‐MEE (G1404), tert‐Butyl hydroperoxide (458 139), D‐Penicillamine (P4875) and L‐Penicillamine (196 312) were bought from Sigma‐Aldrich (USA); Antimycin A1 (C4452) was from APExBIO (USA); NADP + /NADPH detection kit (S0179) was from Beyotime (China); D‐Glucose (A501991), L‐cystine (A610088), propidium iodide (A601112), 2‐Deoxy‐D‐glucose (A602241), N‐Acetyl‐L‐cysteine (A601127) were obtained from Sangon Biotech (China); RSL3 (S8155), Chloroquine (S6999), Erastin (S7242) and Liproxstain‐1 (S7699) were from Selleck (USA); Staurosporine (HY‐15141), Sulfasalazine (HY‐14655), SB203580 (HY‐10256), BSO (HY‐106376), Metformin (HY‐B0627), Phenformin (HY‐16397A), Brefeldin A (HY16592), Thapsigargin (HY‐13433), Tunicamycin (HY‐A0098), GSK2656157 (HY‐13820), GSK2606414 (HY‐18072), Salubrinal (HY‐15486), Trolox (HY‐101445), BAY‐876 (HY‐100017), KL‐11743 (HY‐145597) were purchased from MedChemExpress (USA).

Techniques: CCK-8 Assay, Cell Culture, Western Blot, Expressing, Control, Knock-Out, Microarray, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test