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. 2017 Jan 19;8(8):13986–14002. doi: 10.18632/oncotarget.14753

Figure 1. PROS1 is expressed in OSCC cells and stimulates cell proliferation.

Figure 1

A. Analysis of PROS1 mRNA levels by realtime qPCR in different OSCC cell lines. Results presented are relative to PROS1 mRNA levels in HaCat immortalized human keratinocytes. Graphs represent mean ± SEM from 3 independent experiments. ***P<0.001. B. Analysis of PROS1 protein levels in whole cell extracts from the indicated cell lines. High PROS1 levels are detected in SCC-1 and SCC-25 OSCC cell lines, but not in the immortalized human Keratinocyte cell line HaCaT. Actin serves a as loading control. One representative blot of three independent experiments is shown. C, D. Dose dependent effects of PROS1 on proliferation of SCC-1 (C) and SCC-25 (D) cells. hPROS1 was added to the cells at the indicated concentrations (nmol/L) 48 hours before performing proliferation assays. Proliferation is plotted as a percentage of growth relative to vehicle-treated cells. The means ± SEM of a representative experiment out of four are shown. *P<0.05, ***P<0.001. E, F. Effective knockdown of PROS1 in SCC1 (E) and SCC-25 (F) OSCC lines by two different sh targeting sequences. RT-qPCR detection of PROS1 mRNA in control (EV)-treated and PROS1-knockdown populations using two different PROS1- targeting sequences (shPS1, shPS2). qPCR data are normalized to β-actin. Graphs represent mean ± SEM from 3 experiments. **P<0.01. G. Analysis of PROS1 protein levels in whole cell extracts by western blot analysis in SCC-1 (left) and SCC-25 (right) parental, control-treated (shEV) and stable PROS1- knockdown cell lines (shPS1, shPS2). Actin serves as a loading control. Results are representative of five independent experiments. H. Analysis of secreted PROS1 levels from conditioned medium (CM) by immunoprecipitation (IP) followed by western blot with anti-PROS1 antibody from control and sh-PS stable cell lines. Purified PROS1 served as a positive control. Purified and secreted PROS1 appear as a doublet, following proteolytic cleavage by serum enzymes. Results are representative of five independent experiments.