Fig. 3

H2A1H overexpression leads to increase in cell proliferation. a AUT-PAGE analysis (silver stained) showing the enrichment of the H2A isoforms in chromatin upon their overexpression in CL38 cells. b Quantitative analysis of the isoforms enrichment in the chromatin. Quantification of bands of H2A.1 and H2A.2 was performed by using the software GelAnalyzer. Normalization was done with respect to H2B as it appears as a single discrete band on AUT-PAGE. The data were plotted after taking the densitometric readings of three independent experiments. Error bars represent SEM of three independent experiments. c Cell proliferation curves by MTT assay of H2A1H and H2A2A3 overexpressing CL38 cells in comparison with control CL38 cells. Error bars represent SEM of six independent experiments. d Colony formation assay of CL38 cells upon H2A1H and H2A2A3 overexpression. e Quantitative analysis of the colony sizes of 20 colonies each performed using ImageJ. Error bar represents SEM. f qRT-PCR for the cell proliferation markers Ki67 and PCNA on H2A1H and H2A2A3 overexpression normalized to 18S rRNA. Error bars represent SEM of three independent experiments. g Cell cycle analysis of the CL38 cells exogenously overexpressing H2A isoform post-serum starvation and release. h The expression level analysis of the CL38 cells expressing H2A1H single or double mutants with anti-FLAG antibody. i Bar graph depicting the proliferation of the CL38 cells expressing H2A1H single mutants by the MTT assay. Error bars represent SEM of 6 independent experiments. j Bar graph depicting the proliferation of the CL38 cells expressing H2A1H double mutants by MTT assay. Error bars represent SEM of six independent experiments. VC—vector control. H2A1H, H2A2A3 and their mutants in the figure are the genes cloned and expressed as FLAG tagged proteins in pcDNA3.1(+) vector