Fig 1: RNF146 is a hypoxia-responsive gene. (A) Heatmap of ten known hypoxia target genes and RNF146 expression in Hep3B cells under normoxia (20% O2) and hypoxia (1% O2) conditions. (B) The expression of RNF146 mRNA in HCC cells under normoxia and hypoxia conditions was measured by RT-qPCR. (C) HIF-1α, HIF-2α and RNF146 protein levels were determined by WB in HCC cells under normoxia and hypoxia conditions. (D) The lentivirus-mediated shRNAs against HIF-2α (sh2α), HIF-1α (sh1α), HIF-1α/2α (DKD) and non-targeting shRNA (NTC) were transduced into HCC cells. After incubation under hypoxia conditions, RNF146 mRNA level in HCC cells was measured by RT-qPCR. (E) HIF-1α, HIF-2α and RNF146 protein levels were determined by WB in transfected HCC cells under hypoxia conditions. *p < 0.05.
Fig 2: RNF146 expression is increased in HCC. (A) TCGA data analysis using the GEPIA platform showed the expression difference of RNF146 mRNA between HCC and normal liver tissues. (B) IHC analysis indicated that RNF146 was highly expressed in HCC compared to adjacent nontumor tissues. (C) The RNF146 mRNA levels were detected in MIHA, Hep3B, Huh7, HepG2 and HCCLM3 cells by RT-qPCR. (D) The RNF146 protein levels were detected in MIHA, Hep3B, Huh7, HepG2 and HCCLM3 cells by WB. (E) The comparison of 3-years overall survival of patients with high or low RNF146 expression.
Fig 3: RNF146 knockdown reduces HCC growth in vivo. HCCLM3 cells with or without RNF146 knockdown were subcutaneously injected into nude mice. (A) Tumor volume and (B) weight were consistently reduced by RNF146 knockdown. (C) Tumor tissues from the RNF146 knockdown group showed fewer Ki-67 staining cells compared to the control group. (D) Tumor tissues from the RNF146 knockdown group showed lower RNF146 protein levels than the control group. *p < 0.05.
Fig 4: Reducing PTEN expression abolishes the effects of RNF146 knockdown in HCCLM3 cells. (A) HCCLM3 cells that were transfected with NTC, shRNF#1 or shRNF#1 + shPTEN were detected by WB for the RNF146, PTEN, p-AKT (Ser473), AKT, p-mTOR (Ser2448) and mTOR levels. (B) CCK-8, (C) EdU, (D) colony formation, (E) glucose consumption and (F) lactate production assays were performed to detect the proliferation, colony formation and glycolysis of HCCLM3 cells with or without RNF146 overexpression. *p < 0.05.
Fig 5: RNF146 knockdown suppresses the proliferation and glycolysis of HCCLM3 cells. (A) The lentivirus-mediated shRNAs targeting RNF146 (shRNF#1 and shRNF#2) and non-targeting shRNA (NTC) were transduced into HCCLM3 cells. WB was performed to determine the RNF146 protein level. (B) RNF146 knockdown reduced the viability of HCCLM3 cells. (C) RNF146 silencing inhibited the proliferation of HCCLM3 cells. (D) The inhibitory effects of RNF146 knockdown on HCCLM3 cell colony formation were confirmed. (E) RNF146 silencing reduced the glucose consumption of HCCLM3 cells. (F) HCCLM3 cells’ lactate production was decreased by RNF146 knockdown. *p < 0.05.
Supplier Page from Abcam for Anti-RNF146 antibody