Journal: bioRxiv

Article Title: PBRM1-VHL cooperation rewires lipid and iron metabolism to promote ferroptosis resistance in clear cell renal cell carcinoma

doi: 10.64898/2026.05.02.722429

Figure Lengend Snippet: (A) Representative flow cytometry histograms and quantification of BODIPY 581/591 C11 oxidation ratio (oxidized/reduced) in Caki-2 cells expressing vector, PBRM1, VHL, or PBRM1+VHL treated with DMSO or RSL3 (0.33 µM) for 12 hours. (B) As in (A), BODIPY 581/591 C11 oxidation ratio in RCC4 cells reconstituted cells treated DMSO or RSL3 (0.11 µM) for 12 hours. (C) BODIPY 581/591 C11 oxidation ratio in CRISPR-edited HEK293T cells (sgControl, sgPBRM1, sgVHL, sgPBRM1+sgVHL) treated with DMSO or RSL3 (0.33 µM) for 8 hours. (D-E) Representative histograms and mean fluorescence intensity (MFI) quantification of Liperfluo staining in Caki-2 (D) and RCC4 (E) cells following RSL3 treatment as in (A-B). (F) Liperfluo fluorescence as mean fluorescence intensity in CRISPR-edited HEK293T cells following RSL3 treatment as in (C). (G) Representative flow cytometry histograms of BODIPY 581/591 C11 oxidation in Caki-2 reconstituted cells treated with RSL3 (0.33 µM) ± ferrostatin-1 (Fer-1; 1 µM) for 8 h. (H) Schematic illustrating the relationship between the NAD⁺/NADH pool, NADP⁺/NADPH generation via NAD kinase, GSH/GSSG redox cycling, and GPX4-dependent detoxification of lipid peroxides. GSTs: Glutathione S-transferases; Prdxs: Peroxiredoxins. (I) Glutathione redox status (GSH/GSSG ratio) in Caki-2 cells treated with DMSO or RSL3 (0.33 µM) for 8 hours, quantified using the GSH/GSSG-Glo™ Assay. (J) Total NAD (NAD⁺ + NADH) in Caki-2 cells treated with DMSO or RSL3 (0.33 µM) for 8 hours, quantified using the NAD/NADH-Glo™ Assay. Data are presented as mean ± SD with individual replicates shown ( n = 3 biologically independent replicates for panels A, J, I; n = 4 for panels B, C; n = 5 for panels I; n = 6 for panels (D, E, F). For panels comparing ≥3 genotype groups, statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test using vector as the reference group. For panels I and J, comparisons between DMSO and RSL3 within each genotype were assessed by unpaired two-sided Student’s t -test. Exact P -values are shown in the figure for comparisons that did not reach the threshold for asterisk annotation. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Lipid peroxidation was assessed using BODIPYTM 581/591 C11 (Invitrogen, Waltham, MA, USA, D3861) and Liperfluo (Dojindo, Kumamoto, Japan; L248).

Techniques: Flow Cytometry, Expressing, Plasmid Preparation, CRISPR, Fluorescence, Staining, Glo Assay