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. 2011 Sep 12;121(10):3965–3980. doi: 10.1172/JCI58552

Figure 5. Prolonged CD2AP loss leads to dendrin translocation into the nucleus.

Figure 5

(A) Ddn mRNA levels, determined by RT-PCR, in high-Tgfb1 Cd2ap–/– podocytes infected with different shRNA constructs (D2–D4) downregulating endogenous dendrin. Con, uninfected high-Tgfb1 Cd2ap–/– podocytes; Scr, high-Tgfb1 Cd2ap–/– podocytes infected with lentiviruses expressing a scrambled oligo. (B) Dendrin levels in high-Tgfb1 Cd2ap–/– podocytes infected with lentiviruses expressing the 2 most efficient shRNA constructs, D3 and D4. (C) Organization of the actin cytoskeleton and FAs in high-Tgfb1 Cd2ap–/– podocytes with dendrin downregulation. FAs and F-actin were visualized with anti-paxillin and rhodamine-phalloidin, respectively. (D) Number of FAs within WT and high-Tgfb1 Cd2ap–/– podocytes with dendrin downregulation. Data (mean ± standard deviation) represent measurements of >50 cells in C. ***P < 0.001. (E) Ctsl mRNA levels, determined by RT-PCR, in high-Tgfb1 Cd2ap–/– podocytes with dendrin downregulation. (F) Protein levels of CatL, dynamin, synaptopodin, RhoA, and GAPDH in high-Tgfb1 Cd2ap–/– podocytes and cells infected with lentiviruses. (G) Time course of CatL activity in high-Tgfb1 Cd2ap–/– podocytes, high-Tgfb1 Cd2ap–/– podocytes infected with lentiviruses, and low-Tgfb1 Cd2ap–/– podocytes at neutral pH. (H) Loss of CD2AP rendered podocytes hypersensitive to proapoptotic signals, as shown by specific enrichment of mono- and oligonucleosomes released into the cytoplasm of WT and high-Tgfb1 Cd2ap–/– podocytes treated with different apoptosis inducers. Stauro, staurosporine; Actino D, actinomycin D; Angio II, angiotensin II. (I) TGF-β1–induced apoptosis was reversed by downregulation of CatL or dendrin in high-Tgfb1 Cd2ap–/– podocytes. Scale bars: 20 μm.