Figure 7. miR-29 repression and KLF4 upregulation contribute to maximal promotion of CK5⁺ and CD44⁺ cells.

A. Transient miR-29 and siKLF4 transfection decreases P4 induced activation of CK5 promoter. T47D cells stably expressing a luciferase reporter driven by the KRT5-promoter were plated at 10000 cells/well in 96-well plates. After 24h cells were transfected with 50 nM miR-29a (29a) or miR-29b mimics or negative control (NC), 5 nM on-target pool siKLF4 or siNC in combination with a renilla-luciferase plasmid (pRL-SV40) using Dual-transfection reagent. Cells were treated for additional 24h with ethanol (OH) or 100 nM P4 and luciferase activity measured 24h after transfection and normalized to renilla-luciferase control. *P<0.05 B. T47D cells were transfected with 5 or 10 nM siKLF4-smart pool or si-NC control in combination with pGFP plasmid to control for transfection efficiency. After 24h, cells were treated with either ethanol (OH) or 100 nM P4 for 24h and western blotting was performed to detect KLF4 and CK5. C. T47D were transfected with 10nM siNC or si-KLF4. After 24h, cells were treated with vehicle (OH) or 100nM P4 for 24h and CD44⁺ expression was measured by flow cytometry. Left: Representative flow-charts. Right. Percentage of CD44⁺ cells from biological triplicates *P<0.05.