Fig. 2. LIN28B-AS1 silencing inhibits HCC cell progression in vitro.

HepG2 cells were transfected with LIN28B-AS1 siRNA (“si-LIN28B-AS1-S1/S2”, 0.5 μM) or the non-sense control siRNA (“siR-C”) for 48 h, expression of LIN28B-AS1 a and listed genes b, c was shown; Cells were further cultured for applied time periods, and cell viability was tested by MTT d, with cell proliferation examined by EdU staining assay e; Cell migration and invasion were tested by “Transwell” and “Matrigel Transwell” assays, respectively f. Cell apoptosis and mitochondrial depolarization were examined by TUNEL staining/Annexin V FACS g and JC-1 staining h assays, respectively. Huh7 cells and primary human HCC cells (“HCC1/2”) as well as L02 or primary human hepatocytes (“Hepatocytes”, same for all figures) were transfected with si-LIN28B-AS1-S1 (“kd”) or non-sense control siRNA (“siR-C”) for 48 h. Cells were further cultured for applied time periods, cell survival and proliferation were, respectively, tested by MTT i and BrdU incorporation j assays, with cell apoptosis examined by TUNEL staining k. For EdU-staining assays, five randomly selected views (of each condition) with total 1000 cells were included to calculate EdU/DAPI ratios (same for all figures). For “Transwell” and “Matrigel Transwell” assays, five randomly selected views in each condition were included to calculate the average number of migrated/invaded cells (same for all figures). For all in vitro functional assays, the exact same number of viable cells of different genetic treatment/s were initially seeded onto each well/dish (“Day-0”/“0 h”, same for all figures). Listed proteins were quantified and normalized c. “Ctrl” stands for the parental control cells (same for all figures). Data were presented as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “siR-C” cells. The experiments were repeated three times, and similar results were obtained. Bar = 100 μm.