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. 2020 Feb 28;14(4):742–762. doi: 10.1002/1878-0261.12649

Figure 4.

Figure 4

Effect of P4HA1 protein downregulation or treatment with a prolyl 4‐hydroxylase inhibitor on CTHRC1 secretion in WM239 and SKMEL‐28 cells. (A, B) Western blot analyses of CTHRC1 protein expression in cell extracts (A) and in the conditioned medium (B) of parental WM239, control (ctrl shRNA), and P4HA1‐KD (P4HA1 shRNA) cells transduced with lentiviral shRNAs from Sigma‐Aldrich/Merck. (C–F) Western blot analyses of CTHRC1 protein expression in cell extracts of WM239 (C) and SKMEL‐28 (E) cells and in the conditioned medium of WM239 (D) and SKMEL‐28 (F) cells incubated without or with a prolyl 4‐hydroxylase inhibitor 3,4‐dihydroxybenzoic acid (DHB) for 18 h. Alpha‐tubulin was used as a loading control for cell extracts (A, C, E) and silver staining to analyze the total secreted proteins in the conditioned medium (B, D, F). The lower‐molecular‐weight bands of secreted CTHRC1 in the high‐expressing control cells may represent some degradation products of CTHRC1.