Fig 2: PTBP3 directly binds to HIF-1a mRNA to active its translation in colon cancer cells. a Relative HIF-1a 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-1a 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-1a 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-1a 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 HIF1a 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-1a 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-1a 5'UTR RNA in HCT116 cells. i Western blot of PTBP3 in RNA pull-down samples by in vitro transcribed biotin labeled HIF-1a 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

Fig 3: LCN2-HIF1A pathway regulates EDN1 expression in human HSCs. (a) Representative IF images of co-localization LCN2 receptor (SLC22A17) and a-SMA in AH livers. Hoechst 33,258 (blue) was used to label nuclei, TSA-Cy5 visualized SLC22A17 (red) and TSA-Cy3 visualized a-SMA (green). Overlay of SLC22A17 and a-SMA showed co-expression (yellow). (b) LX-2 cells were exposed to 50 µM human recombinant LCN2. ACTA2, COL1A1, AGT and EDN1 gene expression was measured by qPCR (n = 3). (c) Intracellular free calcium concentration of LX-2 cells was detected using Fura-2 fluorescence. (d) Human primary HSCs transduced with adenovirus-mediated overexpression of GFP or LCN2. ACTA2, COL1A1, AGT and EDN1 gene expression were measured by qPCR (n = 3). (e) Human primary HSCs transduced with adenovirus-mediated overexpression of GFP or LCN2. HIF1A expression was detected by Western Blot (n = 3) and the original blots are presented in Supplementary Figure 5. (f) HIF1A siRNA was transfected into human primary HSCs and EDN1 gene expression measured by qPCR (n = 3). (g) ChIP-qPCR was used to detect binding affinity of HIF1A to the EDN1 gene promoter in human primary HSCs (n = 3). *p < 0.05 by two-tailed Student’s t test. Scale bar: 100 µm.