Research Paper Volume 12, Issue 6 pp 5399—5410

Figure 3. Forced inhibition increases SphK1 expression, promoting OS cell progression in vitro. Expression of listed genes in parental control OS-1 cells (“Ctr”), OS-1 cells with pre-miR-3677 anti-sense lentivirus (“lv-antagomiR-3677”, L1/ L2, two lines) or non-sense anti-sense construct (“lv-an-C”), was tested by qPCR (A and C) and Western blotting (D) assays, with the relative SphK1 3’-UTR activity examined (B); Cell proliferation and migration were tested by EdU incorporation (E) and “Transwell” (F) assays, respectively. The listed OS cells were infected with lv-antagomiR-3677 or lv-an-C for 48h, expression of mature miR-3677 (-3p, G) and SphK1 mRNA (H) was tested, with cell proliferation examined by EdU incorporation assays (I). Expression of listed proteins was quantified, normalized to the loading control Tubulin (G). Data were presented as mean ± SD (n=5), and results were normalized. ***P< 0.001 vs. “lv-an-C” cells. Experiments in this figure were repeated five times with similar results obtained.