Fig. 4

Functional assays to establish inhibitory effects of c28MS and standard treatments in RAFLS in glucose and glucose-free/low-glucose medium (CB839: 300 nm, 3BrPy: 25uM, c28MS: 2uM). A–D, H–K RAFLS (n = 3) were plated for matrigel invasion and migration scratch assay in glucose and glucose-free medium. Quantification of the results (A, C, H, J) and representative images of RAFLS invasion and migration (B, D, I, K) are shown. E, L RAFLS (n = 3) were plated and starved overnight with 0.1% fetal bovine serum (FBS) and inhibitors added the following day in high-glucose (25 mM) and low-glucose (2 mM) medium. MTT was added on day 5 after the addition of conditions. RAFLS (n = 3) were starved overnight 0.1% FBS after plating. Compound and standard treatments were added in 1% and 10% FBS media under high-glucose (25 mM) and low-glucose (2 mM) and left to proliferate for 4 days in the presence of EdU and consequently counterstained with Hoechst 33,342 (blue) dye to assess the total proportion of cells. F, M Results were quantified as the percentage of EdU-positive cells among the total number of Hoechst 33,342-positive cells and representative images shown in G and N. Horizontal lines and error bars show the mean ± SD. * = p < 0.05; **** = p < 0.0001