Fig. 6
Sal-induced the targeting of TNBC. a Sal induced ESR2 in MDA-MB-468 cell line. A flow cytometry graph showing an upregulation of ESR2 expression in response to Sal or the drug combination of Sal and Das treatment. The cells were treated with drugs alone (at the corresponding IC50 concentration (Fig. 1c)) or 2-drugs combination prior to incubation with phycoerythrin-labeled anti-ESR2 for FACS analysis. b Representative fluorescence microscopic images showing Sal induced ESR2 in MDA-MB-468 cells. Prior to imaging, the cells were incubated with phycoerythrin-labeled anti-ESR2 and DAPI for staining the ESR2 receptors (green) and the nucleus (blue), respectively. Scale bar is 60 μm. c Flow cytometry graph showing that the ESR2 expression induced by the salinomycin treatment was knocked down with siRNA. MDA-MB-468 cells were simultaneously treated with Sal (0.5 μM) and transfected with Silencer Select ESR2 siRNA oligonucleotides for 72 h. Ctl1 and ctl2 siRNA were used as negative controls. d Plots of the relative cell viability of MDA-MB-468 cells 72 h after treatment with Sal, Tamo, or the drug combination (S + T) with no siRNA transfection or in the presence of ctl1 siRNA (negative control) or with siRNA of ESR2. Data were presented as mean ± standard deviation (SD) and statistical differences were analyzed using Student’s t-test (*P < 0.05). e Synergistic cytotoxicity of Sal and Tamo. Stimulated plots of the CI values of the drug combination at a drug ratio of 1:2 versus the cellular fraction affected (Fa) values. The plots were generated using the CompuSyn software. All the experiments were independently performed in triplicate. f Comparing the cytotoxic effect of Tamo on MDA-MB-468 cells pre-treated with Sal and PBS. The cells were pre-treated with PBS (control) or Sal (0.5 μM) for 72 h and then be trypsinized. Same number of cells were re-seeded and immediately treated with Tamo (1 μM) for 72 h. The results were fit into sigmoidal dose response curves for calculating the IC50 values. The experiments were independently performed in triplicate