Figure 7.

Cmas^−/− podocytes generated by use of CRISPR/Cas9 technology are able to differentiate but show impaired adhesion to collagen IV in vitro. (A) Western blot analysis of CMAS expression in wild-type and Cmas^−/− podocyte cell lines. Actin staining served as loading control. (B) Indirect immunofluorescence staining of podocyte lines grown under permissive (WT1) and nonpermissive (synaptopodin) conditions. Exemplarily, one CMAS-knockout cell line is shown. (C) Cell proliferation of wild-type and Cmas^−/− podocytes was assessed in a WST-1 assay over a time period of 3 days by colorimetric quantification of formazan dye produced by metabolically active cells. One experiment representative of three independent experiments is shown. (D) Cell adhesion assay of wild-type and Cmas^−/− podocytes to GBM component collagen IV and BSA as control. Three individual experiments were conducted with each six technical replicates per cell line. Significance was determined by unpaired t test (*** P<0.001). (E) Western blot analysis of β1-integrin expression in wild-type and Cmas^−/− podocyte cell lines. The upper band represents the mature, and the lower band the immature glycoform of the protein. Actin staining served as loading control.