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. Author manuscript; available in PMC: 2014 Jul 24.

Published in final edited form as: J Mol Med (Berl). 2012 Sep 27;90(12):1407–1420. doi: 10.1007/s00109-012-0960-6

Fig. 5 — NFATc1 mediates the regulation of uPAR expression in podocytes. a Ionomycin activation of NFAT was used as a positive control. Ionomycin (0.5–2 μM) induced the expression of Plaur mRNA (encoding uPAR) in a dose-dependent manner (p<0.05). b, c Flow cytometry of uPAR expression showed an ionomycin-induced uPAR cell surface expression (p<0.01). d–f Same as ionomycin, LPS induced uPAR expression in podocytes. In contrast, 11R-VIVIT (NFAT inhibitor, 10–1,000 nM) inhibited the LPS-induced expression of Plaur mRNA (encoding uPAR (d); p<0.05 and p<0.01, respectively) and uPAR protein (e, f) (p<0.01) in a dose-dependent manner. NFATc1 was silenced using NFATc1-siRNA. Differentiated podocytes were identified by synaptopodin, a marker of this cell type (g). h, i Western blotting and quantitative real-time RT-PCR analysis showed that NFATc1-siRNA inhibited the expression of the NFATc1 protein (h) and mRNA (i) (p<0.01). j–l NFATc1-siRNA-treated podocytes showed a substantial reduction of Plaur mRNA (encoding uPAR (j); p<0.01) and uPAR protein (k, l) (p<0.01). m Schematic of constructs used for the generation of podocyte-specific, Dox-inducible NFATc1-^nuc-transgenic mice. rTetR reverse Tet repressor, rtTA reverse tetracycline-controlled transactivator, VP16 herpes simplex VP16 protein, tetO tet operator sequences, pCMV cytomegalovirus promoter. Western blot analysis of isolated glomeruli from double-transgenic NFATc1^nuc mice treated with Dox for 4 days revealed an increase in uPAR protein levels when compared to Dox-treated single transgenic rtTA mice used as the negative control. The protein levels of synaptopodin were not affected by NFATc1^nuc transgene expression. The intensity of immunoblot signals was quantified by densitometry and the uPAR protein levels are depicted relative to the GAPDH levels in arbitrary units. n ChIP analysis in podocytes using antibody to NFATc1, followed by qPCR using the Plaur gene promoter-specific primer. The Amplified promoter sequence is designed at the region 455–572 bp upstream of TSS (chr7:25246946+25247063). Fold enrichment=[%(ChIP/Input)]/[%(Negative control/Input)]. All values are expressed as the means±SD. ^§p<0.01, ^§§p<0.05 versus control podocytes; *p<0.01, **p<0.05 versus LPS-treated podocytes

Fig. 5 — NFATc1 mediates the regulation of uPAR expression in podocytes. a Ionomycin activation of NFAT was used as a positive control. Ionomycin (0.5–2 μM) induced the expression of Plaur mRNA (encoding uPAR) in a dose-dependent manner (p<0.05). b, c Flow cytometry of uPAR expression showed an ionomycin-induced uPAR cell surface expression (p<0.01). d–f Same as ionomycin, LPS induced uPAR expression in podocytes. In contrast, 11R-VIVIT (NFAT inhibitor, 10–1,000 nM) inhibited the LPS-induced expression of Plaur mRNA (encoding uPAR (d); p<0.05 and p<0.01, respectively) and uPAR protein (e, f) (p<0.01) in a dose-dependent manner. NFATc1 was silenced using NFATc1-siRNA. Differentiated podocytes were identified by synaptopodin, a marker of this cell type (g). h, i Western blotting and quantitative real-time RT-PCR analysis showed that NFATc1-siRNA inhibited the expression of the NFATc1 protein (h) and mRNA (i) (p<0.01). j–l NFATc1-siRNA-treated podocytes showed a substantial reduction of Plaur mRNA (encoding uPAR (j); p<0.01) and uPAR protein (k, l) (p<0.01). m Schematic of constructs used for the generation of podocyte-specific, Dox-inducible NFATc1-^nuc-transgenic mice. rTetR reverse Tet repressor, rtTA reverse tetracycline-controlled transactivator, VP16 herpes simplex VP16 protein, tetO tet operator sequences, pCMV cytomegalovirus promoter. Western blot analysis of isolated glomeruli from double-transgenic NFATc1^nuc mice treated with Dox for 4 days revealed an increase in uPAR protein levels when compared to Dox-treated single transgenic rtTA mice used as the negative control. The protein levels of synaptopodin were not affected by NFATc1^nuc transgene expression. The intensity of immunoblot signals was quantified by densitometry and the uPAR protein levels are depicted relative to the GAPDH levels in arbitrary units. n ChIP analysis in podocytes using antibody to NFATc1, followed by qPCR using the Plaur gene promoter-specific primer. The Amplified promoter sequence is designed at the region 455–572 bp upstream of TSS (chr7:25246946+25247063). Fold enrichment=[%(ChIP/Input)]/[%(Negative control/Input)]. All values are expressed as the means±SD. ^§p<0.01, ^§§p<0.05 versus control podocytes; *p<0.01, **p<0.05 versus LPS-treated podocytes