Fig. 5

GPD1 and HIF1α form a positive feedback loop to inhibit the expression of GPD2 and mitochondrial function. (A) Western blotting of HIF1α in Caki1-NC, Caki1-GPD1, 769P-NC, and 769P-GPD1 cells. (B) Immunofluorescence stainings of GPD1 (green signal) and HIF1α (red signal) were analyzed by confocal laser scanning microscopy (magnification × 100). The nuclei of cells were stained by DAPI (blue signal). (C) Western blotting of GPD1 and HIF1α in 769P cells transfected with three different GPD1-specific siRNAs. (D) Western blotting of PHD3 in Caki1-NC, Caki1-GPD1, 769P -NC, and 769P-GPD1 cells. (E) Western blotting of HIF1α IP and PHD3 Co-IP in 769P-NC and 769P-GPD1 cells. (F) Half-lives of HIF1α in 769P-NC and 769P-GPD1 cells transfected with two different GPD1-specific siRNAs (siGPD1). Cycloheximide was used to inhibit new protein synthesis. (G) Western blotting of GPD2 and HIF1α in 769P-NC and 769P-GPD1 cells transfected with two different HIF1α-specific siRNAs (siHIF1α). (H) Oxygen consumption rate (OCR) of 769P-NC and 769P-GPD1 cells transfected with HIF1α-specific siRNAs. Baseline respiration, spare respiratory capacity, and ATP production in 769P-NC and 769P-GPD1 cells transfected with HIF1α-specific siRNAs (siHIF1α) are shown. (I) Cell proliferation in 769P-NC and 769P-GPD1 cells transfected with HIF1α-specific siRNAs (siHIF1α) respectively examined by CCK-8 assays. (J-K) Cell migration and invasion in 769P-NC and 769P-GPD1 cells transfected with HIF1α-specific siRNAs (siHIF1α) respectively examined by wound healing and Transwell assays. Data are shown as the mean + SD (n = 3). Statistical analysis was performed using an unpaired Student’s t test with a two-tailed distribution (NS p > 0.05, * p < 0.05, **p < 0.01). Abbreviations: HIF, hypoxia inducible factor; GPD1, glycerol-3-phosphate dehydrogenase 1; GPD2, glycerol-3-phosphate dehydrogenase 2; Co-IP, coimmunoprecipitation; PHD3, prolyl hydroxylase 3