Figure 1.
Kindlin 2 is activated during muscle cell differentiation.
(A) C2C12 cells were cultured in growth medium (GM) or differentiation medium (DM) for 0, 1, 3, 5 days. Proteins were extracted from the cells at different stages, and Western blot (WB) assays were performed using the indicated antibodies. β-actin was used as a loading control. (B) Protein bands in A were scanned, and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments. (C–D) Total RNA was extracted from the cells at different stages. The mRNA levels of Kindlin 2 and MyHC were examined by qPCR. Error bars indicate s.d. values, n = 3; * indicates p<0.05 by Student’s t-test.
Figure 2.
Knockdown of Kindlin 2 inhibits muscle cell differentiation.
(A) The efficacy of Kindlin 2 siRNA was detected by Western blot. (B) Control or Kindlin 2 siRNA was transfected into C2C12 cells. After 24 hr, C2C12 cells were induced myogenic differentiation. At day 3 of differentiation, Western blot analysis was carried out with the indicated antibodies. (C) Protein bands in C were scanned, and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments.
Figure 3.
Kindlin 2 is required for activating Wnt signaling.
(A) 100 ng Super8xTOPFlash/FOPFlash plasmid with 1 ng of pRL were transfected into C2C12 cells. After 24 hr, C2C12 cells were induced myogenic differentiation for two days. The luciferase reporter activity was measured in undifferentiated cells (GM) and differentiated cells (DM day 2). (B) SuperTop/Fopflash reporter assays were performed in GM cells and DM cells with or without Kindlin 2 knockdown. Error bars indicate S.D. values, n = 3; * indicates p<0.05 by Student’s t-test.
Figure 4.
Kindlin 2 mediates the activation of β-catenin idifferentiation.
(A) Proteins were extracted from the cells at different stages, and WB assays were performed using the indicated antibodies. (B) Protein bands in A were scanned and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments. (C) Control or Kindlin 2 siRNA was transfected into C2C12 cells. After 24 hr, C2C12 cells were induced myogenic differentiation for 3 days. WB analysis was carried out with the indicated antibodies. (D) Protein bands in C were scanned, and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments.
Figure 5.
Kindin 2 is enriched in the nucleus in myogenic differentiation.
(A) Immunofluorescence staining was performed in GM cells and DM cells (day 3) with or without Kindlin 2 knockdown using anti-Kindlin 2 (Alexa Flour 488, green) and anti-β-catenin (Alexa Flour 568, red) antibodies. Images were captured with a confocal microscopy. (B) Immunofluorescence staining for Kindlin 2 (Alexa Flour 488, green) and active β-catenin (Alexa Flour 568, red). (C) Both cytoplasmic and nuclear lysates were extracted from GM and DM cells (day 3) for WB analysis using the indicated antibodies. The absence of α-tubulin demonstrates that the fraction was from the nucleus. The absence of YY1 indicates that the fraction was from the cytoplasm. (D) Both cytoplasmic and nuclear lysates were extracted from DM cells with or without Kindlin 2 knockdown, and WB was performed.
Figure 6.
Kindlin 2 interacts with β-catenin.
(A–C) Lysates from C2C12 cells (DM day 3) were extracted and then anti-Kindlin 2 (A), anti-β-catenin (B) or anti-active β-catenin (C) antibodies were used for Co-IP assays. (D–E) Immunofluorescence staining for Kindlin 2 (Alexa Flour 488, green) and β-catenin (D) or active β-catenin (E) (Alexa Flour 568, red) was performed in C2C12 cells (DM day 3). Images were captured with a confocal microscopy.
Figure 7.
Kindlin 2 forms a complex with active β-catenin and TCF4.
(A) Control or Kindlin 2 siRNA was transfected into C2C12 cells. After 24 hr, C2C12 cells were induced myogenic differentiation. At different time points, WB analysis was performed. (B) Protein bands in A were scanned, and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments. (C–E) Nuclear lysates from C2C12 cells (DM day 3) were prepared and then anti-TCF4 (C), anti-Kindlin 2 (D) or anti-active β-catenin (E) antibodies were used for Co-IP assays. (F) Immunofluorescence staining for Kindlin 2 (Alexa Flour 488, green) or TCF4 (Alexa Flour 568, red) was performed in C2C12 cells (DM day 3).
Figure 8.
Kindlin 2 activates myogenin expression.
(A) Control or Kindlin 2 siRNA was transfected into C2C12 cells. After 24 hr, C2C12 cells were induced myogenic differentiation. At different time points, WB analysis was performed. (B) Protein bands in A were scanned, and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments. (C) C2C12 cells were induced myogenic differentiation for five days. ChIP assay was performed for detecting the occupancy of the tripartite complex at myogenin promoter. The indicated antibodies were used to perform ChIP assays. To quantify the ChIP-enriched DNA, qPCR was carried out. Each data point is the average of duplicates from a representative experiment. Experiments were repeated three times. * indicates p<0.05 by Student’s t-test.