Fig. 4: Effect of NRBF2 knockdown and overexpression in cultured kidney cells.

a Brightfield images of podocytes (Pod), glomerular endothelial cells (GEC), mesangial cells (MC) and proximal tubular cells (PTC) showing changes in cell morphology 4 days after shRNA NRBF2 knockdown compared with scrambled shRNA controls. NRBF2 knockdown induces cell vacuolisation (enlarged images) along with podocyte hypertrophy and loss of GEC, MC and PTC. Scale bar = 100 μm. b Bar chart showing reduced cell number 4 days after shRNA NRBF2 knockdown compared with scrambled controls. Unpaired two-tailed t-test, cells were counted in three fields of view (n = 3 biological repeats), data are presented as mean values ± SEM. c Bar chart showing increased cell area in shRNA NRBF2 knockdown podocytes compared with controls. Area was measured in 10 cells in each of 3 fields of view. Unpaired two-tailed t-test, (n = 3 biological repeats), data are presented as mean values ± SEM. d Images of phalloidin-stained podocytes overexpressing NRBF2 (pod NRBF2 OE) and wild-type controls cultured for 10 days in basal or diabetic media (‘DM’). Diabetic media-induced changes in cell morphology and F-actin distribution (top right) that are attenuated by NRBF2 overexpression (bottom right). e Quantification of F-actin stress fibres, indicating significant F-actin rearrangement in wild-type podocytes exposed to Diabetic media ‘DM’ and no difference in F-actin distribution in NRBF2-overexpressing podocytes, one-way ANOVA with Tukey’s multiple comparisons test (n = 8 technical repeats), data are presented as mean values ± SEM.