Fig 1: Modulating PKC alters cell morphology and signalling. (A) Western blot demonstrates PKC activity in both total cell lysates and integrin adhesion complexes from podocytes spread onto collagen IV or laminin for 210 min. (B) The colour-coded shape outlines indicate representative protrusive activities at 5-min intervals recorded over 100-min period. For measurements of protrusive activity live cell imaging was performed between 180 and 280 min of cell spreading. Podocytes were treated with either G?6976 or PMA to suppress or induce PKC activity respectively during cell spreading on matrix ligand. (C,D) PKC modulation impacts podocyte morphology and pseudopodial projection formation. (E,F) PKCa localization to adhesions is controlled through ligand specificity. Podocytes were spread on circles and lines coated with collagen IV or laminin for 3.5 h in serum-free media and stained for PKCa and paxillin and ratiometric imaging was performed. Scale bar represents 20 µm. Data are pooled from three independent experiments. G?6976 induced inhibition of PKCa localization to FA was significant for collagen circles, lines and laminin lines. For ratiometric imaging 20–40 cells were analysed per experiment and each experiment was performed four times. All bar graph measurements are shown as mean ± standard deviation. Box plots indicate 25th and 75th percentiles (lower and upper bounds, respectively), 1.5 × interquartile range (whiskers) and median (black line). Scale bar in (e) represents 5 µm; ****, p < 0.0001; NS, not significant; LAM511, laminin-511; COL4, collagen IV. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: VEGF-A165 expression and synaptic integrity in RA and OIR.Vegfa165 antibody (green) staining was observed to be increased in both RA (n = 4) and OIR (n = 4) mice at P19. While some retinal areas in OIR mice appeared to have increased VEGF-A expression, there was no difference between total VEGF-A164 expression in RA and OIR mice at any age. PKCalpha (red) showed mis-aligned rod bipolar cells in OIR mice, while RA mice had uniformly aligned synapses. Vegfa165 vascular endothelial growth factor-a164, RA room air, OIR oxygen-induced retinopathy, P postnatal day age.
Fig 3: Activity of GFP reporters in the mouse postnatal retina. Retinas electroporated with a Cag::Nucß-gal and the GFP reporter shown to the left of panels and imaged by confocal microscopy for the expression of GFP (green, chicken antibody), Nucß-gal (orange, mouse antibody), and PKCalpha (purple). The scleral portion of the retina is located near the top of the image. Scale bar in top left panel represents 20 µm and applies to all panels
Fig 4: Quantitation of reporter activity in the mouse postnatal retina. Fluorescent cells from confocal images as shown in Fig. 3 were quantified using ImageJ. All electroporated cells (either ß-gal-positive or GFP-positive) were identified and categorized as having their cell body located in either the ONL or the INL. For those cells located in the INL, cells were also examined for expression of PKCalpha, a rod bipolar marker. a The percentage of GFP-positive cells out of all electroporated cells is plotted with blue bars. b The percentage of GFP-positive cells out of all electroporated cells in the INL is plotted with blue bars. The percentage of GFP-positive cells that were identified as rod bipolar cells is shown with red bars
Fig 5: Detection of neuronal and microglial cells in adult Cdc42-KD retinas.Retinal cryosections of central and peripheral regions of Cdc42-KD mice were stained for rod photoreceptors (GNAT1), rod bipolar cells (PRKCA), ganglion cells (POU4F1), and microglia (IBA1). Shown are representative images of N = 3. Arrows point to amoeboid shape microglia in the periphery. ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer, Ap: apical, Ba: basal. Scale bars: 50 µm.
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