Fig. 3: Kindlin-2-ΔF1 and Kindlin-2-ΔF0 mutants fail to rescue AR Tyr-534 phosphorylation and downstream signaling.

Control (Ctrl siRNA) or Kindlin-2 knockdown (K2 siRNA) BT549 cells was infected with lentiviral vectors encoding wild-type Kindlin-2 (K2-WT), F1-domain-deleted mutant of Kindlin-2 (K2-ΔF1), F0-domain-deleted mutant of Kindlin-2 (K2-ΔF0) or empty vector 3×Flag-tagged pLVX-IRES-Hyg. A Cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting with antibodies as indicated. The presence of Flag-tagged-wild-type Kindlin-2 (Flag-K2-WT), Flag-tagged-F1-domain-deleted mutant of Kindlin-2 (Flag-K2-ΔF1), AR and Src in cell lysates was shown as input. B Immunoblotting analysis of AR Tyr-534 phosphorylation level in cells as specified in the figure (left panel). Quantification analysis of the normalized ratio of AR Tyr-534 phosphorylation level to total AR was shown in the right panel. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 5. C Cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting with antibodies as indicated. The presence of Flag-K2-WT, Flag-tagged-F0-domain-deleted mutant of Kindlin-2 (Flag-K2-ΔF0), AR and Src in cell lysates was shown as input. D Immunoblotting analysis of AR Tyr-534 phosphorylation level in cells as specified in the figure (left panel). Quantification analysis of the normalized ratio of AR Tyr-534 phosphorylation level to total AR was shown in the right panel. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 4. E 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). F Quantification analysis of the percentage of cells (as specified in the figure) with AR nuclear staining was shown. ***p < 0.001 vs. Ctrl siRNA, n = 8 for Ctrl siRNA + EGF, K2 siRNA + EGF and K2 siRNA + K2-WT + EGF; n = 4 for K2 siRNA + K2-ΔF1 + EGF and K2 siRNA + K2-ΔF0 + EGF. G 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 < 0.001 vs. Ctrl siRNA + EGF, n = 10 for Ctrl siRNA + EGF, K2 siRNA + EGF and K2 siRNA + K2-WT + EGF; n = 5 for K2 siRNA + K2-ΔF1 + EGF and K2 siRNA + K2-ΔF0 + EGF. H qPCR analysis of cyclin D1 mRNA expression. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 8 for Ctrl siRNA + EGF, K2 siRNA + EGF and K2 siRNA+ K2-WT + EGF; n = 4 for K2 siRNA + K2-ΔF1 + EGF and K2 siRNA + K2-ΔF0 + EGF. I 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 = 7 for Ctrl siRNA + EGF, K2 siRNA + EGF and K2 siRNA + K2-WT + EGF; n = 3 for K2 siRNA + K2-ΔF1 + EGF; n = 4 for K2 siRNA + K2-ΔF0 + EGF. J 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 < 0.001 vs. Ctrl siRNA + EGF, n = 8 for Ctrl siRNA + EGF, K2 siRNA + EGF and K2 siRNA + K2-WT + EGF; n = 4 for K2 siRNA + K2-ΔF1 + EGF and K2 siRNA + K2-ΔF0 + EGF. K Quantification analysis of cell proliferation assay in the indicated cells, as described in “Materials and Methods”. ***p < 0.001 vs. Ctrl siRNA + EGF, n = 12 for Ctrl siRNA + EGF, K2 siRNA + EGF and K2 siRNA+ K2-WT + EGF; n = 6 for K2 siRNA + K2-ΔF1 + EGF and K2 siRNA + K2-ΔF0 + EGF. L 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 = 14 for Ctrl siRNA + EGF, K2 siRNA + EGF and K2 siRNA+ K2-WT + EGF; n = 7 for K2 siRNA + K2-ΔF1 + EGF and K2 siRNA + K2-ΔF0 + EGF. Original magnification, ×100. K2 Kindlin-2, AR androgen receptor.