Fig 2: A model of p53–p21 activity in ferroptosis suppression.Stabilization of wild-type p53 leads to reduction of ribonucleotide reductase expression and function, and conservation of intracellular glutathione and potentially other reducing agents (e.g., NADPH), which can then be re-directed towards the inhibition of ferroptosis. Note: p53 may down-regulate RRM1 and RRM2 in a partly p21-independent manner.

Fig 3: RRM2 increases the chemotherapy resistance of NSCLC cells via EGFR/AKT signaling pathway. Cancer cells were transfected with empty vector, RRM2, RRM2+DMSO and RRM2+AST1306, and then incubated with the EGFR inhibitor AST1306 at 1 μM for 24 h. (A,B) In NSCLC cells incubated with DDP for 36 h, overexpression of RRM2 enhanced chemotherapy resistance, and AST1306 reversed this effect. (C,D) In NSCLC cells incubated with 5-FU for 36 h, overexpression of RRM2 enhanced chemotherapy resistance and AST1306 reversed this effect. (E,F) In A549 and SPCA-1 cells, overexpression of RRM2 elevated the protein expression of EGFR and the level of p-AKT, and AST1306 reversed this effect. All data shown as means ± S.D. #P < 0.05 indicates a significant difference between the two indicated groups.

Fig 4: UBIAD1 loss leads to ROS increase, impaired cell proliferation and decreased survival in melanoma cells.(A) Growth curves of melanoma cell lines SkMel28, A375 and Mel Juso upon UBIAD1KD compared to control conditions (ctrl). 2-way ANOVA with Sidak's multiple comparisons test was used to quantify statistical significance. Error bars represent SEM, n = 3.(B) Western blot analysis of cyclin A and RRM2 proteins upon UBIAD1KD in SkMel28, A375 and Mel Juso melanoma cell lines. ß-actin was used as loading control.(C) FACS assay of DHE (left) and DCFH-DA (right) staining in SkMel28 and A375 upon UBIAD1KD Low or UBIAD1KD. Data are reported as percentage of positive cells over singlets. Incubation with 50 µM of menadione and 1 mM of H2O2 for 90 min were used as positive controls for DHE and DCFH-DA, respectively. One-way ANOVA with Sidak’s multiple comparisons test was used to quantify statistical significance. Error bars represent SEM, n = 3.(D) HPLC-MS analyses of total CoQ10 level upon UBIAD1KD in SkMel28 and A375 lines, in comparison control conditions (ctrl), normalized to total protein (TP) concentration. Unpaired Student two-tailed t-test was used to quantify statistical significance between control and UBIAD1KD samples. Error bars represent SEM, n = 6.(E) HPLC-MS analyses of CoQ10 in plasma membrane fraction (PL) upon UBIAD1KD in SkMel28 and A375 lines normalized to membrane ceramide abundance (Cer16:0). Unpaired Student two-tailed t-test was used to quantify statistical significance between control conditions (ctrl) and UBIAD1KD samples. Error bars represent SEM, n = 6. Below, Western blot analysis of subcellular fractionation in SkMel28 and A375 lines. M, Mitochondria-enriched fraction; W, Whole cell; PL, Plasma membrane-enriched fraction. PMCA was used as marker of plasma membrane, Tom20 as mitochondrial marker, ERK1/2 and ß-actin as cytosolic markers.(F) Growth curves of melanoma cell lines SkMel28, A375 and Mel Juso upon UBIAD1KD Low in presence or absence of idebenone treatment (1 µM idebenone for SkMel28, 10 µM idebenone for A375 and 50 nM idebenone for Mel Juso). 2-way ANOVA with Sidak's multiple comparisons test was used to quantify statistical significance between UBIAD1KD Low and UBIAD1KD Low + idebenone. Error bars represent SEM, n = 3.(G) Western blot analysis of cell proliferation markers (RRM2 and cyclin A) and DNA-damage marker (pH2AX) in melanoma cell lines SkMel28, A375 and Mel Juso upon UBIAD1KD Low in presence or absence of idebenone (100 nM of idebenone for SkMel28, 200 nM of idebenone for A375 and 200 nM idebenone for Mel Juso). Three biological replicates are shown. Unpaired Student two-tailed t-test was used to quantify statistical significance between UBIAD1KD Low and UBIAD1KD Low + idebenone samples. Error bars represent SEM, n = 3.

Fig 5: Effects of BKM120 and rucaparib as single agents or in combination on HR repair.A U251 and U87MG cells were transfected with DR-GFP plasmid and pCBASceI plasmid using Lipofectamine 3000. Cells were treated with BKM120 or/and rucaparib for 72 h and then collected and resuspended in ice-cold PBS. GFP intensity was analyzed by flow cytometry. B The percentage of GFP-positive cells represents HR repair efficiency. C, D U251 and U87MG cells were treated with BKM120 and/or rucaparib for 48 h. C Cells were collected for western blotting detection of DNA damage- and HR repair-related proteins, including BRCA1, BRCA2, RAD51, RRM2, p-ATR, p-CHK1, p-AKT, ATR, CHK1, and PAR. D Quantitative reverse transcription PCR analysis of BRCA1, BRCA2, and RAD51 expression in two cancer cell lines. Gene expression was normalized to 18 s rRNA. E, F Cells were treated with BKM120 or/and Rucaparib for 12/24 h. Representative images of immunofluorescence staining of RAD51 and γ-H2AX in GBM cells Cell nuclei were stained with DAPI. Scale bar: 20 μm. The percentage of cells with RAD51/γ-H2AX foci was shown in (F). All data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.