Figure 7

Figure 7

Generation of CRB2 knockout human immortalized podocytes. (a) The endogenous expression of CRB2 in differentiated human culture podocytes was weakly observed in the nucleus and cytoplasm. Scale bar 20 μm. (b) In differentiated human podocytes electroporated with carboxy-terminal FLAG-tagged mouse full-length Crb2, the staining with rabbit anti-CRB2 antibody and mouse anti-FLAG antibody was co-localized in the cytoplasm and was weak in the plasma membrane. Scale bars 20 μm. (c) While there was a 627-bp product in wild-type podocytes, there were 614- and 388-bp products in CRB2 knockout t17 clone and 628- and 464-bp products in CRB2 knockout t32 clone. The left lane was the 100-bp marker. (d) The CRB2 knockout t17 clone possessed mutations in an allele with c.336_348del TGCATCCCGGCCG, which caused a frameshift mutation and premature stop codon due to a 13-bp deletion, and in the other allele with c.114–352del, which caused a frameshift mutation and premature stop codon due to a 239-bp deletion. The CRB2 knockout t32 clone possessed mutations in an allele with c.195insC, which caused a frameshift mutation and premature stop codon due to a 1-bp insertion, and in the other allele with c.188–350del, which caused a frameshift mutation and premature stop codon due to a 163-bp deletion. (e) There were weak bands above and below 140 kDa marker with CRB2 antibody in the lysates of wild-type podocytes, while these bands were invisible in those of CRB2 knockout t17 and t32 clones. The positive control was a lysate from human embryonic kidney 293 cells transfected with CRB2-DYK. β-actin served as a loading control. (f) The protein expressions of NPHS2, PODXL, and NPHS1 were comparable among wild-type, CRB2 knockout t17, and t32 clones. (g) The F-actin positive area in CRB2 knockout t17 or t32 clones significantly decreased compared to wild-type human podocytes. Scale bars 50 μm. Data are presented as mean ± SD. Statistical analysis by one-way analysis of variance, followed by Scheffe's multiple comparison test; n = 6 in each group. *P < 0.05, **P < 0.01. CRB2KO CRB2 knockout, WT wild-type.