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Figures
Figure 1. Morphology of cultured podocytes and expression of podocyte-specific markers. Undifferentiated (left panels) and differentiated (right panels) cultured podocytes. (A) Light microscopy images. (B) Scanning electron microscopy showing undifferentiated cell without processes and differentiated cell demonstrating fine processes (arrows) with interdigitations between cells and presumed slit-diaphragm–like structures. (C) WT-1 showing nuclear expression in both undifferentiated and differentiated cells. (D) Synaptopodin, absent in undifferentiated cells, and appearing along actin filaments in differentiated cells. Magnifications: ×40 in A and C; ×60 in D.
Figure 2. Immunofluorescence microscopy of slit diaphragm-associated proteins. Undifferentiated (left panels) and differentiated (right panels) podocytes. Immunofluorescence staining for podocyte-specific proteins. (A) Weak nuclear expression of P-cadherin in undifferentiated cells (left panel), but clear expression at cell-cell junction (arrow) and in the nucleus of differentiated cells (right panel). (B) Weak diffuse cytoplasmic expression of nephrin in undifferentiated cells (left panel), changing to punctuated cell surface and cytoplasmic distribution in differentiated cells. (C) Podocin, no expression in undifferentiated cells; filamentous and cell surface (arrows) expression in differentiated cells. (D and E) Controls without primary antibodies. (D) Rabbit serum for podocin antibody, showing unspecific nuclear staining. (E) Mouse IgG1 control for nephrin and P-cadherin antibodies, showing specificity of nuclear nephrin and P-cadherin expression. Magnification, ×40
Figure 3. Reverse transcription-PCR (RT-PCR) and Western blot analysis of slit diaphragm–associated proteins. (A) RT-PCR for CD2AP and nephrin using cDNA from undifferentiated (33°C) and differentiated (37°C) immortalized podocytes. Control lanes are from reactions without reverse transcription (no RT). As a positive control, CD2AP was also amplified from a mouse glomerular cDNA library). CD2AP mRNA is expressed in both undifferentiated and differentiated cells (left panel); nephrin mRNA is expressed in differentiated and in undifferentiated podocytes (right panel). (B) Western blot analysis of nephrin (left panel) and podocin (right panel) using total protein extracts from undifferentiated and differentiated immortalized podocytes. Nephrin is detected in undifferentiated and differentiated cells at a size of approximately 180 kD; podocin is expressed only in differentiated cells at a size of approximately 45 kD.
Figure 4. Immunofluorescence microscopy of cell cycle–associated proteins. Undifferentiated (left panels) and differentiated (right panels) cultured podocytes. Nuclear staining of proliferating cell nuclear antigen (PCNA) (A) and cyclin A (B) in undifferentiated cells, switching off in differentiated cells. (C and D) Absence of p27 (C) and cyclin D1 (D) in undifferentiated cells (left panels), and strong expression in differentiated cells (right panels). Magnifications: ×40 in A and D; ×60 in B and C.
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