Fig. 4

Knockdown of ocrl1 increased podocyte migration and decreased endocytosis activity. A, Mouse MCP5 podocytes were transfected with shRNAs 848, 1816, and 2208 targeting ocrl1. Western blot analysis was performed to evaluate knockdown efficiency (n = 3). B, MCP5 cells transfected with GFP-tagged OCRL1 protein showed a primarily intracellular distribution of green fluorescence. Scale bar: 10 μm. C, Confocal microscopy analysis indicated that knockout of ocrl1 did not impact the structure of microtubules and microfilaments in podocytes, as evidenced by staining for α-tubulin. Scale bar: 10 μm. D, Transwell analysis was conducted to assess podocyte migration. Scale bar: 100 μm. E, An internalization assay was conducted using Alexa-568-transferrin to evaluate endocytosis in MCP5 cells. The images were captured 4 h after the cells were incubated with transferrin. F, Flow cytometry was used to measure the percentage of cells in the S phase, G1 phase, and G2 phase. Data were expressed as the mean ± SEM. *P < 0.05; †P < 0.01; ‡P < 0 .001 vs. control; n = 6