Figure 5

Inhibition of TGFβ signaling induces lipogenesis in primary SSG3 cells. ( a, b) SSG3 cells stably expressing a shRNA against TGFβ RII, show accumulation of lipid droplets on brightfield images (scale bars, 20 μm), by Nile red (scale bars, 20 μm) and Oil red O stainings (scale bars, 10 μm). White arrows show the presence of multiple lipid droplets in the shRNA expressing cells compared to the control (Ctr). 24 h of TGFβ1 (5 ng/ml) treatment decreases the basal level of lipid production in control cells but does not affect cells expressing the TGFβ RII shRNA, mainly seen by Oil red O. (c) Electron microscopy showing the increase of lipid droplets in SSG3 cells (denoted by white arrows) expressing the shRNA compared to the control. Scale bars, 2 μm. LD, Lipid Droplets. N, Nucleus. (d) Flow cytometry of SSG3 cells expressing the shRNA labeled with Nile red. FL-1 measures the neutral lipids and SSC reflects the granularity of the cells. 10,000 cells have been acquired for each condition. As a positive control, SSG3 treated by 0.1 mM linoleic acid (LA) for 24 h show increase of fluorescence and granularity representing the lipid droplets. Note the increase of fluorescence and the increase of granularity in shRNA expressing TGFβ RII compared to the cells expressing a shRNA control. We obtained similar result with two different shRNA expressing TGFβ RII (Additional file 4: Figure S4).