Fig 2: SCD5 is downregulated in VHL-deficient ccRCC tumors and cell lines. (a) Expression of SCD5 mRNA in TCGA ccRCC (n = 533) and normal kidney samples (n = 72). **** p < 0.0001. The analysis was performed using the UALCAN/TCGA platform. (b) SCD5 protein expression is reduced in ccRCC (n = 110) compared to normal kidney samples (n = 84) (CPTAC dataset). **** p < 0.0001. (c) Representative result of SCD5 expression using renal cancer (https://www.proteinatlas.org/ENSG00000145284-SCD5/pathology/renal+cancer#img, accessed on 9 June 2021) and normal kidney samples (https://www.proteinatlas.org/ENSG00000145284-SCD5/tissue/kidney#img, accessed on 9 June 2021) from the Human Protein Atlas. (d) SCD5 protein levels are reduced in RCC4 and 786-O ccRCC cells compared to RPTEC. Cell lysates of RCC4, 786-O, and RPTEC cells were analyzed by Western blot using anti-SCD5 antibody. Protein levels of SCD5 were quantified using LabImage 1D software and normalized to ß-actin protein levels. The right panel shows quantification of relative SCD5 protein levels from three independent experiments. Data are presented as mean ± SEM. * p < 0.05 (t-test). kD, kilodalton. Full-length blots are presented in Supplementary Figure S2. (e) low SCD5 expression (n = 134) correlates with shorter overall survival (high SCD5 expression, n = 397). The analysis was performed using the UALCAN platform.

Fig 3: SCD5 affects cell proliferation. (a) RT-qPCR analysis of SCD5 and SCD in RCC4 cells infected with inducible control or SCD5-specific shRNA. Two different primer sets were used for SCD5 mRNA analysis. SCD5 shRNA does specifically affect SCD5, as SCD was not down regulated. HSPCB was used as the reference gene, n = three. Data are presented as mean ± SEM. * < 0.05 (t-test). (b) Western blot of shRNA-mediated knockdown of SCD5 in RCC4 cells. (c) mRNA levels of SCD5 in 786-O cells infected with control-shRNA or SCD5-specific shRNA, assayed by qPCR; β-actin was used as the reference gene. n = three. Data are presented as mean ± SEM. *** p < 0.001 (t-test). (d) Western blot of shRNA-mediated knockdown of SCD5 in 786-O. (e) Knockdown of SCD5 induces cell proliferation. Cell viability of RCC4 cells upon shRNA SCD5 knockdown was assayed compared to control. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 (t-test). (f) Cell viability of 786-O cells upon shRNA SCD5 knockdown was assayed compared to control. Data are presented as mean ± SEM of three independent experiments. ** p < 0.01 (t-test). (g) Overexpression of SCD5 in 293T and RCC4 cells reduces cell proliferation. Cell viability was assayed in 293T and RCC4 cells transfected with Flag-tagged SCD5, or transduced with Venus-tagged SCD5, as indicated. Data are presented as mean ± SEM of three independent experiments. ** p < 0.01 (t-test).

Fig 4: SCD5 expression in ccRCC cells is VHL dependent. (a) mRNA levels of VHL in HeLa cells infected with inducible control-shRNA or VHL-specific shRNA assayed by RT-qPCR; HSPCB was used as the reference gene. n = four. Data are presented as mean ± SEM. *** p < 0.001 (t-test). (b) Representative blot showing VHL expression in HeLa cells infected with lenti-control shRNA or lenti-VHL shRNA. Full-length blots are presented in Supplementary Figure S2. (c) SCD5 mRNA levels are affected by VHL. qPCR analysis of HeLa cells expressing shRNA against control or VHL. Two different primer sets were used for SCD5 mRNA analysis; HSPCB was used as the reference gene. n = four for each experiment. Data are mean ± SEM. *** p < 0.001 (t-test). (d) Analysis of SCD5 mRNA levels in RCC4 cells and RCC4 cells expressing VHL. Cell lysates were prepared from VHL-deficient RCC4 cells and cell lines transduced with VHL (RCC4 + VHL); GAPDH was used as the reference. n = four for each experiment. Data are mean ± SEM. *** p < 0.001 (t-test). (e) Western blot demonstrating SCD5 expression in RCC4 cells (VHL deficient) and RCC4 cells transduced with VHL. Full-length blots are presented in Supplementary Figure S2. Right panel shows quantification of relative SCD5 levels from three independent experiments. * p < 0.05 (t-test).

Fig 5: SCD5 expression depends on HIF2α. (a) 786-O cells were incubated with the HIF2α inhibitor PT2385 (1 µmolar) for 72 h. RNA expression of SCD5, VEGF, and PAI-1 were assessed by RT-qPCR relative to HSPCB. n = three for each experiment. Data are represented as mean ± SEM. * p < 0.05 compared to untreated cells (t-test). (b) SCD5 protein levels are restored after treatment with the HIF2α inhibitor PT2385. The 786-O cells were incubated with the HIF2α inhibitor PT2385 (1 µmolar) or DMSO (control) for 72 h, and cell lysates were assessed by immunoblotting with anti-SCD5 and anti-HIF2α antibody. Quantification of three independent experiments. Data represent mean ± SEM. * p < 0.05 (t-test). kD, kilodalton. Full-length blots are presented in Supplementary Figure S2. (c) Immunoblot of RCC4 cells that were treated with PT2385 (1 µM) or DMSO control. Quantification of relative SCD5 levels from three independent experiments. Data are presented as mean ± SEM. * p < 0.05 (t-test). Full-length blots are presented in Supplementary Figure S2. (d) SCD5 protein levels after hypoxia treatment of RPTEC cells. Quantification from three independent experiments. Data represent mean ± SEM. * p < 0.05 (t-test). kD, kilodalton. Full-length blots are presented in Supplementary Figure S2.