Fig. 5

The drug combination enhanced cell cycle arrest at the G1/S phase. a A table summarizing the changes in the expression of genes (induced by the drug combination) associated with the estrogen-mediated S-phase entry pathway. The mRNAs were extracted from different BC cell lines 72 h after treatment with the drug combination prior to RT-qPCR analysis. b A correlation plot of the differential gene expression levels in MDA-MB-468 cell lines determined using RNA-seq and RT-qPCR methods. c Plots of cell counts versus the propidium iodide fluorescence (FL2). MDA-MB-468 cells were treated with PBS (control), Sal (0.5 μM), Das (15 μM), or the drug combination for 72 h, fixed in ethanol, and incubated with propidium iodide and RNase staining solution prior to FACS analysis. All the experiments were performed in triplicate. d Graph bars showing the percentage of cells at G1, S, and G2/M phases after the drug treatments. e The drug combination also modulated the estrogen-mediated S-phase entry pathway at the translational level. Western Blot analysis of cyclin D1, cyclin E2, E2F2, and ESR2 protein expression in MDA-MB-468 cells after exposure to the drugs alone or in combination for 72 h. GAPDH and β-actin were used as loading controls based on the molecular weight of the proteins of interest. The blots were processed and cropped using Image Studio Lite 5.2 software.

Full-length blots are available Fig.S13. f Graph bar showing the expression level of each selected protein. The relative protein expression levels were quantified using Image Studio software and normalized to the control cells treated with PBS. All the experiments were performed in triplicate. Data were presented as mean ± standard deviation (SD) and statistical differences were analyzed using Student’s t-test (*P < 0.05, **P < 0.01)