Figure 5. HSD3B7 is essential for ccRCC viability by regulating oxysterols homeostasis.

A, Simplified schematic of oxysterols production through cholesterol catabolism.

B, Metabolomics analysis of 7α-hydroxy-3-oxo-4-cholestenoic acid in 97 normal kidney tissues and 135 ccRCC tumors.

C, Quantification of the HSD3B7 upstream product, 7α-hydroxy-cholesterol in shSCR and shHSD3B7 A498 cells after 72h, using liquid chromatography-high resolution mass spectrometry (LC/HRMS).

D, Quantification of the HSD3B7 downstream product, 7α-hydroxy-3-oxo-4-cholestenoic acid in shSCR and shHSD3B7 A498 cells after 72h, using LC/HRMS.

E, Representative photographs of CTR and HSD3B7 short hairpin resistant cDNA (HSD3B7^R) expressing A498 cells treated with 10μM 7α-hydroxy-cholesterol from supplementary Figure 6J. Magnification (100X).

F, Annexin-V/PI staining and flow cytometry analysis performed on shSCR and shHSD3B7 A498 cells expressing empty vector or HSD3B7 short hairpin resistant cDNA (HSD3B7^R) cultured in media with 10% FBS supplemented with vehicle (DMSO) or 7α-hydroxycholesterol (10μM) every other day for 96h.

G, Annexin-V/PI staining and flow cytometry analysis performed on A498 cells cultured in media supplemented with or without 7α-hydroxycholesterol (10μM) every other day and treated with vehicle (DMSO) or Z-Vad-fmk (50μM) for 96h.

H, Real-time qPCR analysis performed on A498 cells cultured in media supplemented with or without 7α-hydroxycholesterol (10μM) every other day for 96h, evaluating expression of TP53 target genes; NOXA, PUMA and p21. Beta-2 microglobulin (B2M) was used as the housekeeping gene.

I, TP53, PARP, γ-H2A.X, and Cleaved Caspase 3 protein expression assessed by immunoblots in A498 cells cultured in media supplemented with or without 7α-hydroxycholesterol (10μM) every other day for 96h. GAPDH was used as the loading control.

J, HSD3B7 protein expression assessed by immunoblots in shSCR and shHSD3B7 A498 cells expressing HSD3B7 short hairpin resistant cDNA (HSD3B7^R) or catalytic inactive HSD3B7^R (HSD3B7^R-Y159S). HSP90 was used as the loading control.

K, Annexin-V/PI staining and flow cytometry analysis performed on shSCR and shHSD3B7 A498 cells expressing HSD3B7 short hairpin resistant cDNA (HSD3B7^R) or catalytic inactive HSD3B7^R (HSD3B7^R-Y159S) after 96h.

(All experiments were performed in at least triplicates and statistical analysis was applied with *=P<0.05, **=P<0.01, ***=<0.001, n.s=non-significant).