Fig. 4: AR-Y534D mutant but not wild-type AR rescues Kindlin-2 deficiency-induced inhibition on AR signaling and breast cancer progression.

Control (Ctrl siRNA) or Kindlin-2 knockdown (K2 siRNA) BT549 cells was infected with lentiviral vectors encoding wild-type AR (AR-WT), phosphor-mimic mutant of AR (AR-Y534D) or empty vector 3×Flag-tagged pLVX-IRES-Hyg. A Representative immunofluorescence staining of AR in cells (as specified in the figure). Arrows mark the nuclear regions. Scale bar: 50 µm. Cell nuclei were visualized with DAPI (blue). Quantification analysis of the percentage of cells with AR nuclear staining was shown in the lower panel. ***p < 0.001 vs. Ctrl siRNA, n = 4 independent experiments. B Luciferase analysis of AR transcriptional activity in cells (as specified in the figure). Relative luminance was calculated by luminance signal relative to total protein concentration. **p < 0.01, ***p < 0.001 vs. Ctrl siRNA + EGF, n = 4. C qPCR analysis of cyclin D1 mRNA expression. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 4. D Immunoblotting analysis of cyclin D1 protein expression in cells (as specified in the figure) (left panel). Quantification of cyclin D1 protein expression was shown at the right panel. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 4. E Quantification analysis of cell cycle transition in cells (as specified in the figure). Cells with 2n signal were in G0/G1 phases; with 4n signal were in G2/M phases; between 2n to 4n signal were in S phase. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Ctrl siRNA + EGF, n = 4. F Quantification analysis of cell proliferation assay of indicated cells. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 4. G Cell migration was measured by transwell cell migration assay, as described in “Materials and Methods”. Representative images (left panel) and quantification analysis (right panel) were shown. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 7. Original magnification, ×100. K2 Kindlin-2, AR androgen receptor.