Fig. 4: Depletion of Kindlin-2 activates Hippo pathway and inhibits YAP nuclear localization.

A, B Volcano plots showing differential expression in TM4 (A) or 15P-1 (B) cells transfected with Kindlin-2 siRNA or control siRNA. Genes which have greater than or equal twice-fold difference expression among two groups were shown in gray (downregulated) or orange (upregulated), P_adj < 0.05. C Overlap of genes regulated by depletion of Kindlin-2 in TM4 (A) and 15P-1 (B) cells were shown in Venn diagram. Comparing transcript abundances in control cells and Kindlin-2 inhibited cells revealed 3146 and 2802 significantly altering genes in TM4 and 15P-1 cell respectively. A total of 2203 altering genes overlapped in both two cells, in which 1583 genes were down-regulated and 620 genes were up-regulated. P_adj < 0.05. D KEGG enrichment of 1583 down-regulated genes after Kindlin-2 depletion. KEGG analysis was carried out by using DAVID online tool. Enrichment scores were shown as −log10 (P-value). E A heat map analysis of Hippo signaling pathway related genes. Blue and red square frames represent low or high expression levels respectively. F Control and Kindlin-2 siRNAs were transiently transfected into TM4 and 15P-1 cells separately, after 48 h, qPCR was performed to detect the expression of cell junction related genes (Jam2 and Dlg2) and Hippo signaling genes (Wnt4, Wnt10b, TGFβ2 and Cypr61) using specific primers. GAPDH was used as internal reference. Three independent experiments were involved. Data are mean ± s.e.m. The variance was similar between the groups. *p < 0.05, **p < 0.01 and ***p < 0.001 by Student’s t-test. G Representative Western blot analyses of Hippo signaling pathway factors pMST1, MST1, pMOB1, MOB1, pLATS1-909, LATS1, pYAP-127 and YAP using specific antibodies through loss of function of Kindlin-2 in two cell lines TM4 and 15P-1. β-actin acts as an internal reference. H Left: Representative immunohistochemistry detection for YAP in 4 W WT and KO mice testis. The red arrows show SCs nucleus and the black arrows showed the nucleuses of spermatogenic cells. Scale bar, 50 μm. Magnifications were shown on the right panel; red arrows indicate the staining of YAP in Sertoli cells. Right: quantitative analysis to compare the amount of Yap in spermatogenic nuclei and Sertoli nuclei of KO testes to WT testes. The variance was similar between the groups. ***p < 0.001 by Student’s t-test. I Nuclear and cytoplasmic proteins isolated from TM4 and 15P-1 cells transfected with control or Kindlin-2 siRNAs were subjected to immunoblot analysis using antibodies against Kindlin-2, YAP and pYAP (S127). LamB1 and GAPDH were used as loading controls for nucleus and cytoplasm respectively. J Quantifications of nuclear YAP of two Sertoli cell lines upon Kindlin-2 silencing or not. K Confocal images of Kindlin-2 (purple), YAP (green), CTGF (red) and DAPI (blue) immunofluorescence staining of 15P-1 Ctrl si cells and 15P-1 Kindlin-2 si cells (transient transfection). Scale bar, 50 µm.