Fig. 5
From: PTBP3 contributes to colorectal cancer growth and metastasis via translational activation of HIF-1α
PTBP3 directly binds to HIF-1α mRNA to active its translation in colon cancer cells. a Relative HIF-1α mRNA expression measured by qRT-PCR in HCT116 ± PTBP3 KD under normoxia and hypoxia (1% O2) 4 h. Values were normalized against GAPDH from three independent experiments and are presented as means ± SD. b Relative HIF-1α mRNA expression measured by qRT-PCR in SW620 cells ± PTBP3 KD under normoxia and hypoxia (1% O2) 4 h. Values were normalized against GAPDH from three independent experiments and are presented as means ± SD. c Western blot showing effects of the MG132 proteasome inhibitor on HIF-1α protein accumulation at the indicated time points for the HCT116 cell line ± PTBP3 KD under 1% O2.GAPDH was used as a loading control. d Western blot showing HIF-1α protein decay in HCT116 cells ± PTBP3 KD at the indicated time points after cyclohexamide (CHX) addition under 1% O2. GAPDH was used a loading control. e Graphical representation of HIF1α protein levels from (d) based on densitometry. Half-lives are shown under the curves, representing results of two independent experiments ± SD. f PTBP3 intracellular localization was visualized in colon cancer cells: HCT116, SW480, SW620 and DLD1 by immunofluorescence assays. g The interaction of HIF-1α 5′UTR with PTBP3 was verified by an RNA immunoprecipitation (RIP) assay. Data are presented as the means ± SD for experiments in triplicate. h Western blot of PTBP3 and YBX1 in RNA pull-down samples by in vitro transcribed biotin labeled HIF-1α 5′UTR RNA in HCT116 cells. i Western blot of PTBP3 in RNA pull-down samples by in vitro transcribed biotin labeled HIF-1α 5′UTR RNA and PTBP3 GST fusion protein. j Relative HIF1a-5′UTR -Luc activity in HCT116 cells ± PTBP3 OE was detected by the Dual Luciferase Reporter Assay System. The Rluc activity was used for normalization. Data are presented as the means ± SD for experiments in triplicate. ***p < 0.001