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. 2017 Aug 3;8(51):88332–88344. doi: 10.18632/oncotarget.19917

Figure 4. The podocyte protective activity of valproate is blunted in podocytes expressing a constitutively active mutant of GSK3β.

Figure 4

Differentiated murine podocytes were subjected to liposome-mediated transient transfection with vectors encoding the HA-conjugated wild-type GSK3β (WT) or mutant GSK3β (S9A), in which the regulatory serine 9 residue is replaced by alanine so that GSK3β becomes uninhibitable. Cells were treated with LPS (100 µg/ml) for 24 h after transfection in the presence or absence of sodium valproate (VPA; 1.5 mM). A. Cell lysates were collected and prepared for immunoblotting for phosphorylated NFkB RelA/p65 at serine 467 residue or indicated molecules. B. Immunoblots were subjected to densitometric analysis and arbitrary units were expressed respectively as immunoblot densitometric ratios of p-NFkB RelA/p65 to NFkB RelA/p65 as folds of the control group. *P < 0.05 versus the level of the same molecule in the non-VPA treated WT group; #Not significant versus the level of the same molecule in the non-VPA treated S9A group (n = 3). C. Cell lysates or conditioned media were collected and prepared for immunoblotting for indicated immune molecules. D. Immunoblots were subjected to densitometric analysis and arbitrary units were expressed respectively as immunoblot densitometric ratios of diverse molecules to actin as folds of the control group. *P < 0.05 versus the level of the same molecule in the non-VPA treated WT group; #Not significant versus the level of the same molecule in the non-VPA-treated S9A group (n = 3). E. Cells were fixed and processed for fluorescent labeling of F-actin and counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Represetative fluorescent microscopy shows changes in F-actin cytoskeleton. Bar = 10µm. F. The percentage of stress fibers under fluorescent microscopy were analyzed and estimated. *P < 0.05 versus non-VPA-treated WT cells; #P < 0.05 versus non-VPA-treated WT cells; #Not significant versus VPA-treated S9A cells (n = 4). CTSL, cathepsin L; DAPI, 4’,6-diamidino-2-phenylindole; LPS, lipopolysaccharide; HA, hemagglutinin; VPA, sodium valproate.