Journal: PLOS ONE

Article Title: Genome-wide analysis of long noncoding RNAs as cis -acting regulators of transcription factor-encoding genes in IgA nephropathy

doi: 10.1371/journal.pone.0304301

Figure Lengend Snippet: The primer sequences used in this study.

Article Snippet: The primary antibodies rabbit anti-NFAT5 (ab3446, Abcam, England) and rabbit anti-vitamin D Receptor (VDR) (GB114918, Servicebio, China) were added dropwise and the sections were incubated overnight at 4°C in a wet box.

Techniques: Sequencing

Journal: PLOS ONE

Article Title: Genome-wide analysis of long noncoding RNAs as cis -acting regulators of transcription factor-encoding genes in IgA nephropathy

doi: 10.1371/journal.pone.0304301

Figure Lengend Snippet: A. The Heatmap showing the expression profile of the lncRNA and cis TF in .G B. The reads distribution showing lncRNA NQO1 − DT and its regulated cis TF NFAT5. Boxplot showing the expression of lncRNA, cis TF and target DEGs. * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001. C. The reads distribution showing lncRNA RP5 − 1057I20.6 and its regulated cis TF VDR. Boxplot showing the expression of lncRNA, its regulated cis TF and target DEGs. * P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001.

Article Snippet: The primary antibodies rabbit anti-NFAT5 (ab3446, Abcam, England) and rabbit anti-vitamin D Receptor (VDR) (GB114918, Servicebio, China) were added dropwise and the sections were incubated overnight at 4°C in a wet box.

Techniques: Expressing

Journal: PLOS ONE

Article Title: Genome-wide analysis of long noncoding RNAs as cis -acting regulators of transcription factor-encoding genes in IgA nephropathy

doi: 10.1371/journal.pone.0304301

Figure Lengend Snippet: A . Immunofluorescence analysis showing that VDR expression level in healthy control (HC) was higher than in IgAN . B. Immunofluorescence analysis showing that NFAT5 expression level in IgAN was higher than in healthy control.

Article Snippet: The primary antibodies rabbit anti-NFAT5 (ab3446, Abcam, England) and rabbit anti-vitamin D Receptor (VDR) (GB114918, Servicebio, China) were added dropwise and the sections were incubated overnight at 4°C in a wet box.

Techniques: Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. Rat primary IMCD cells in isotonic (300 mOsM) or hypertonic (640 mOsM) medium were exposed to anoxia (replacement of O 2 by N 2 ) for 0, 8, or 16 hrs. We prepared total protein homogenates and determined NFAT5 protein abundance by Western blot. A representative picture is shown in the upper section and the graph shows mean ± SEM. * or & P≤0.05; n = 5. (* vs. 300 mosmol/normoxia and & vs. 640 mosmol/normoxia). B. NFAT5 cellular distribution after 2 hrs of anoxia evaluated in primary IMCD cells by immunofluorescence. Green = NFAT5 labelling (Alexa488); blue = nuclei (Hoechst 33258). C. HEK293 cells stably expressing ORE-X cultured at 300 mosmol or 500 mosmol by 16 hrs; during this time the cells were exposed for 0, 8 or 16 hrs to anoxia, and luciferase reporter assay was used to evaluate transcription activity; Bar graph represents Mean ± SEM. (* or &, P<0.05; n = 5). D. HEK293 cells cultured at 300 mosmol were exposed by 2 hrs to anoxia (a) or normoxia (n). Nuclear and cytoplasmatic fractions were separated by NE-PER and NFAT5 abundance was determined by Western blot. Bar graph represents Mean ± SEM. (* P<0.05; n = 5).

Article Snippet: In brief, after blocking nonspecific binding, membranes were incubated with rabbit anti-NFAT5 (NFAT5) (Affinity Bio Reagents, Golden, CO), goat anti-AR (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-tubulin (Cell Signalling), rabbit anti-cleaved Caspase-3 (Asp175) (Cell Signalling), mouse anti-M30 CytoDEATH (ROCHE), rabbit anti-ATM (cell signalling), rabbit anti-phospho ATM (cell signalling), rabbit anti-p110α (Cell signalling), rabbit anti-AKT (Cell signalling), rabbit anti-phospho-AKT-308 (Cell signalling) or mouse anti-HIF-1α (Abcam) antibody overnight at 4°C.

Techniques: Western Blot, Immunofluorescence, Stable Transfection, Expressing, Cell Culture, Luciferase, Reporter Assay, Activity Assay

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. Kidney sections of sham and I/R animals (72 hrs) were incubated with rabbit anti-NFAT5. Representative pictures of medulla from sham and I/R animals are shown. Preimmune serum did not stain significantly (data not shown). Scale bar = 100 µm. The arrows indicate the localization of the corresponding marker for NFAT5. B. NFAT5 mRNA abundance in cortex and medulla of kidneys were determined by qRT-PCR. C. NFAT5 protein abundance in cortex and medulla of kidneys were determined by Western blot D. HIF-1α (protein abundance in cortex and medulla of kidneys was determined by Western blot. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. * or & indicates P<0.05; n = 5 (* vs sham medulla and & vs sham cortex).

Article Snippet: In brief, after blocking nonspecific binding, membranes were incubated with rabbit anti-NFAT5 (NFAT5) (Affinity Bio Reagents, Golden, CO), goat anti-AR (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-tubulin (Cell Signalling), rabbit anti-cleaved Caspase-3 (Asp175) (Cell Signalling), mouse anti-M30 CytoDEATH (ROCHE), rabbit anti-ATM (cell signalling), rabbit anti-phospho ATM (cell signalling), rabbit anti-p110α (Cell signalling), rabbit anti-AKT (Cell signalling), rabbit anti-phospho-AKT-308 (Cell signalling) or mouse anti-HIF-1α (Abcam) antibody overnight at 4°C.

Techniques: Incubation, Staining, Marker, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. HEK293 cells cultured at 300 mosmol were subjected to dose-response curve of hypoxia (21, 5, 2.5 and 1% O 2 ). NFAT5 (A1) and HIF-1α (A2) protein abundance was determinate by Western blot. B. Using 2.5% of PO 2 , cells were exposed for 0, 4, 8, and 16 hrs to analyse the HIF-1α gene expression by qRT-PCR (B1) and Western blot (B3). NFAT5 gene expression was also determined by qRT-PCR (B2) and Western blot (B4). Protein abundance and mRNA were normalized by tubulin (Tub) and 18S, respectively. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: In brief, after blocking nonspecific binding, membranes were incubated with rabbit anti-NFAT5 (NFAT5) (Affinity Bio Reagents, Golden, CO), goat anti-AR (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-tubulin (Cell Signalling), rabbit anti-cleaved Caspase-3 (Asp175) (Cell Signalling), mouse anti-M30 CytoDEATH (ROCHE), rabbit anti-ATM (cell signalling), rabbit anti-phospho ATM (cell signalling), rabbit anti-p110α (Cell signalling), rabbit anti-AKT (Cell signalling), rabbit anti-phospho-AKT-308 (Cell signalling) or mouse anti-HIF-1α (Abcam) antibody overnight at 4°C.

Techniques: Cell Culture, Western Blot, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: HEK293 cells cultured at 300 mosmol were transfected with control (C), NFAT5 or HIF-1α siRNA. 48 hrs after transfection the cells were cultured in normoxia (N) or 8 hrs of hypoxia (H). A. Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against NFAT5. B. Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against HIF-1α. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: In brief, after blocking nonspecific binding, membranes were incubated with rabbit anti-NFAT5 (NFAT5) (Affinity Bio Reagents, Golden, CO), goat anti-AR (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-tubulin (Cell Signalling), rabbit anti-cleaved Caspase-3 (Asp175) (Cell Signalling), mouse anti-M30 CytoDEATH (ROCHE), rabbit anti-ATM (cell signalling), rabbit anti-phospho ATM (cell signalling), rabbit anti-p110α (Cell signalling), rabbit anti-AKT (Cell signalling), rabbit anti-phospho-AKT-308 (Cell signalling) or mouse anti-HIF-1α (Abcam) antibody overnight at 4°C.

Techniques: Cell Culture, Transfection, Western Blot

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. Cultures at 300 mosmol were incubated with DMSO (D) or Wortmannin (W) by 1 hour. Then the cells were cultured in normoxia (N) or 8 hrs of hypoxia (H) and NFAT5 abundance was studied by Western blot. B. Cells cultured at 300 mosmol were transfected with HRE-Luciferase and 24 hrs after transfection the cells were incubated with DMSO (D) or Wortmannin (W) by 1 hour. After this treatment, the cells were cultured in normoxia (300 or 500 mOsM) or 8 hrs of hypoxia (300 or 500 mOsM) and the luciferase activity was assayed. C. Cells cultured at 300 mosmol were cotransfected with HRE-Luciferase and siRNA (control or NFAT5). 48 hrs after transfection the cells were cultured in normoxia or 8 hrs of hypoxia (300 or 500 mOsM) and the luciferase activity was assayed. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: In brief, after blocking nonspecific binding, membranes were incubated with rabbit anti-NFAT5 (NFAT5) (Affinity Bio Reagents, Golden, CO), goat anti-AR (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-tubulin (Cell Signalling), rabbit anti-cleaved Caspase-3 (Asp175) (Cell Signalling), mouse anti-M30 CytoDEATH (ROCHE), rabbit anti-ATM (cell signalling), rabbit anti-phospho ATM (cell signalling), rabbit anti-p110α (Cell signalling), rabbit anti-AKT (Cell signalling), rabbit anti-phospho-AKT-308 (Cell signalling) or mouse anti-HIF-1α (Abcam) antibody overnight at 4°C.

Techniques: Incubation, Cell Culture, Western Blot, Transfection, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: HEK293 cells cultured at 300 mosmol were transfected with control and NFAT5 siRNA. 48 hrs after transfection the cells were exposed for 8 hrs to 2.5% PO 2 . A. Western blot of NFAT5. B. LDH activity was assayed in cell culture media and cell lysate by spectrometric determination of NADH. C. Western blot of M30. D. Western blot of Cleaved caspase-3. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: In brief, after blocking nonspecific binding, membranes were incubated with rabbit anti-NFAT5 (NFAT5) (Affinity Bio Reagents, Golden, CO), goat anti-AR (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-tubulin (Cell Signalling), rabbit anti-cleaved Caspase-3 (Asp175) (Cell Signalling), mouse anti-M30 CytoDEATH (ROCHE), rabbit anti-ATM (cell signalling), rabbit anti-phospho ATM (cell signalling), rabbit anti-p110α (Cell signalling), rabbit anti-AKT (Cell signalling), rabbit anti-phospho-AKT-308 (Cell signalling) or mouse anti-HIF-1α (Abcam) antibody overnight at 4°C.

Techniques: Cell Culture, Transfection, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. Rat primary IMCD cells in isotonic (300 mOsM) or hypertonic (640 mOsM) medium were exposed to anoxia (replacement of O 2 by N 2 ) for 0, 8, or 16 hrs. We prepared total protein homogenates and determined NFAT5 protein abundance by Western blot. A representative picture is shown in the upper section and the graph shows mean ± SEM. * or & P≤0.05; n = 5. (* vs. 300 mosmol/normoxia and & vs. 640 mosmol/normoxia). B. NFAT5 cellular distribution after 2 hrs of anoxia evaluated in primary IMCD cells by immunofluorescence. Green = NFAT5 labelling (Alexa488); blue = nuclei (Hoechst 33258). C. HEK293 cells stably expressing ORE-X cultured at 300 mosmol or 500 mosmol by 16 hrs; during this time the cells were exposed for 0, 8 or 16 hrs to anoxia, and luciferase reporter assay was used to evaluate transcription activity; Bar graph represents Mean ± SEM. (* or &, P<0.05; n = 5). D. HEK293 cells cultured at 300 mosmol were exposed by 2 hrs to anoxia (a) or normoxia (n). Nuclear and cytoplasmatic fractions were separated by NE-PER and NFAT5 abundance was determined by Western blot. Bar graph represents Mean ± SEM. (* P<0.05; n = 5).

Article Snippet: Briefly, tissue sections were dewaxed, rehydrated, rinsed in 0.05 M tris-phosphate-saline (TPS) buffer (pH 7.6) and incubated with rabbit anti-NFAT5 antibody (Affinity BioReagents, Golden, CO) overnight at 22°C.

Techniques: Western Blot, Immunofluorescence, Stable Transfection, Expressing, Cell Culture, Luciferase, Reporter Assay, Activity Assay

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. Kidney sections of sham and I/R animals (72 hrs) were incubated with rabbit anti-NFAT5. Representative pictures of medulla from sham and I/R animals are shown. Preimmune serum did not stain significantly (data not shown). Scale bar = 100 µm. The arrows indicate the localization of the corresponding marker for NFAT5. B. NFAT5 mRNA abundance in cortex and medulla of kidneys were determined by qRT-PCR. C. NFAT5 protein abundance in cortex and medulla of kidneys were determined by Western blot D. HIF-1α (protein abundance in cortex and medulla of kidneys was determined by Western blot. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. * or & indicates P<0.05; n = 5 (* vs sham medulla and & vs sham cortex).

Article Snippet: Briefly, tissue sections were dewaxed, rehydrated, rinsed in 0.05 M tris-phosphate-saline (TPS) buffer (pH 7.6) and incubated with rabbit anti-NFAT5 antibody (Affinity BioReagents, Golden, CO) overnight at 22°C.

Techniques: Incubation, Staining, Marker, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. HEK293 cells cultured at 300 mosmol were subjected to dose-response curve of hypoxia (21, 5, 2.5 and 1% O 2 ). NFAT5 (A1) and HIF-1α (A2) protein abundance was determinate by Western blot. B. Using 2.5% of PO 2 , cells were exposed for 0, 4, 8, and 16 hrs to analyse the HIF-1α gene expression by qRT-PCR (B1) and Western blot (B3). NFAT5 gene expression was also determined by qRT-PCR (B2) and Western blot (B4). Protein abundance and mRNA were normalized by tubulin (Tub) and 18S, respectively. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: Briefly, tissue sections were dewaxed, rehydrated, rinsed in 0.05 M tris-phosphate-saline (TPS) buffer (pH 7.6) and incubated with rabbit anti-NFAT5 antibody (Affinity BioReagents, Golden, CO) overnight at 22°C.

Techniques: Cell Culture, Western Blot, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: HEK293 cells cultured at 300 mosmol were transfected with control (C), NFAT5 or HIF-1α siRNA. 48 hrs after transfection the cells were cultured in normoxia (N) or 8 hrs of hypoxia (H). A. Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against NFAT5. B. Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against HIF-1α. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: Briefly, tissue sections were dewaxed, rehydrated, rinsed in 0.05 M tris-phosphate-saline (TPS) buffer (pH 7.6) and incubated with rabbit anti-NFAT5 antibody (Affinity BioReagents, Golden, CO) overnight at 22°C.

Techniques: Cell Culture, Transfection, Western Blot

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: A. Cultures at 300 mosmol were incubated with DMSO (D) or Wortmannin (W) by 1 hour. Then the cells were cultured in normoxia (N) or 8 hrs of hypoxia (H) and NFAT5 abundance was studied by Western blot. B. Cells cultured at 300 mosmol were transfected with HRE-Luciferase and 24 hrs after transfection the cells were incubated with DMSO (D) or Wortmannin (W) by 1 hour. After this treatment, the cells were cultured in normoxia (300 or 500 mOsM) or 8 hrs of hypoxia (300 or 500 mOsM) and the luciferase activity was assayed. C. Cells cultured at 300 mosmol were cotransfected with HRE-Luciferase and siRNA (control or NFAT5). 48 hrs after transfection the cells were cultured in normoxia or 8 hrs of hypoxia (300 or 500 mOsM) and the luciferase activity was assayed. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: Briefly, tissue sections were dewaxed, rehydrated, rinsed in 0.05 M tris-phosphate-saline (TPS) buffer (pH 7.6) and incubated with rabbit anti-NFAT5 antibody (Affinity BioReagents, Golden, CO) overnight at 22°C.

Techniques: Incubation, Cell Culture, Western Blot, Transfection, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

doi: 10.1371/journal.pone.0039665

Figure Lengend Snippet: HEK293 cells cultured at 300 mosmol were transfected with control and NFAT5 siRNA. 48 hrs after transfection the cells were exposed for 8 hrs to 2.5% PO 2 . A. Western blot of NFAT5. B. LDH activity was assayed in cell culture media and cell lysate by spectrometric determination of NADH. C. Western blot of M30. D. Western blot of Cleaved caspase-3. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Article Snippet: Briefly, tissue sections were dewaxed, rehydrated, rinsed in 0.05 M tris-phosphate-saline (TPS) buffer (pH 7.6) and incubated with rabbit anti-NFAT5 antibody (Affinity BioReagents, Golden, CO) overnight at 22°C.

Techniques: Cell Culture, Transfection, Western Blot, Activity Assay

Journal: Molecular Vision

Article Title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

doi:

Figure Lengend Snippet: Primer pairs used in PCR experiments. s, sense. as, anti-sense.

Article Snippet: The following antibodies were used: rabbit anti-human extracellular signal-regulated kinases 1 and 2 (ERK1/2, p44/p42; 19 ng/ml; Cell Signaling, Frankfurt/M., Germany), a rabbit anti-phosphorylated ERK1/2 (95 ng/ml; Cell Signaling), a rabbit anti-human p38 mitogen-activated protein kinase (p38 MAPK; 8 ng/ml; Cell Signaling), a rabbit anti-human phosphorylated p38 MAPK (27 ng/ml; Cell Signaling), a rabbit anti-human NFAT5 (1 µg/ml; Santa Cruz), a rabbit anti-human nuclear factor (NF)-κB p65 (144 ng/ml; Cell Signaling), a rabbit anti-human signal transducer and activator of transcription 3 (STAT3; 89 ng/ml; Cell Signaling), a rabbit anti-human histone H3 (160 ng/ml; Cell Signaling), a rabbit anti-GAPDH (12 ng/ml; Cell Signaling), an anti-rabbit IgG conjugated with alkaline phosphatase (35 ng/ml; Cell Signaling), a rabbit anti-AQP5 (2 µg/ml; Santa Cruz), a mouse anti-vimentin (V9; 1.5 µg/ml; DakoCytomation, Hamburg, Germany), a Cy2-coupled goat anti-mouse IgG (3.5 µg/ml; Jackson ImmunoResearch, Suffolk, UK), and a Cy3-coupled goat anti-rabbit IgG (3.7 µg/ml; Jackson).

Techniques: Sequencing, Amplification

Journal: Molecular Vision

Article Title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

doi:

Figure Lengend Snippet: Hyperosmolarity induces NFAT5 gene and protein expression, and the DNA binding of NFAT5, in RPE cells. The mRNA level ( A, B ) was determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The protein levels ( C-E ) were determined by western blot analysis of cytosolic ( C ) and nuclear extracts ( D, E ) of cells stimulated for 6 and 24 h, respectively. A. Effects of osmolarity changes, CoCl 2 (150 µM; n=5), and cell culture in 1% O 2 (n=4) on the level of NFAT5 mRNA. The hyperosmotic media were made up by adding 100 mM NaCl (n=5) and 100 mM sucrose (n=3), respectively. The hypoosmotic medium contained 60% of control osmolarity (n=5). Inset: Expression of β-actin and NFAT5 genes in RPE cells from different donors (1, 2) determined by RT–PCR. Negative controls (-) were done by adding double-distilled water instead of cDNA as a template. B. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. C. Effects of CoCl 2 (150 µM), as well as of hyperosmotic (+ 100 mM NaCl) and hypoosmotic (Hypo) media, on the cellular level of the NFAT5 protein. Similar results were obtained in three independent experiments using cells from different donors. D. Hyperosmolarity (+ 50 and + 100 mM NaCl, respectively) increased dose-dependently the nuclear level of the NFAT5 protein. E. Hyperosmolarity (+ 100 mM NaCl) increased the nuclear levels of NFAT5 and p65/NF-κB proteins of RPE cells, while the nuclear level of STAT3 protein remained unchanged. As a control, the nuclear level of the histone H3 (HisH3) protein was determined. F. Hyperosmolarity (+ 100 mM NaCl) induces the DNA binding of NFAT5, as indicated by the appearance of complexes of NFAT5 protein and labeled oligonucleotides in EMSA that were not observed under isoosmotic conditions. An excess of unlabeled oligonucleotides (Competitor) abrogated the binding of NFAT5 protein to labeled oligonucleotides. Similar results were obtained in three independent experiments using cells from different donors. Bars represent means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.

Article Snippet: The following antibodies were used: rabbit anti-human extracellular signal-regulated kinases 1 and 2 (ERK1/2, p44/p42; 19 ng/ml; Cell Signaling, Frankfurt/M., Germany), a rabbit anti-phosphorylated ERK1/2 (95 ng/ml; Cell Signaling), a rabbit anti-human p38 mitogen-activated protein kinase (p38 MAPK; 8 ng/ml; Cell Signaling), a rabbit anti-human phosphorylated p38 MAPK (27 ng/ml; Cell Signaling), a rabbit anti-human NFAT5 (1 µg/ml; Santa Cruz), a rabbit anti-human nuclear factor (NF)-κB p65 (144 ng/ml; Cell Signaling), a rabbit anti-human signal transducer and activator of transcription 3 (STAT3; 89 ng/ml; Cell Signaling), a rabbit anti-human histone H3 (160 ng/ml; Cell Signaling), a rabbit anti-GAPDH (12 ng/ml; Cell Signaling), an anti-rabbit IgG conjugated with alkaline phosphatase (35 ng/ml; Cell Signaling), a rabbit anti-AQP5 (2 µg/ml; Santa Cruz), a mouse anti-vimentin (V9; 1.5 µg/ml; DakoCytomation, Hamburg, Germany), a Cy2-coupled goat anti-mouse IgG (3.5 µg/ml; Jackson ImmunoResearch, Suffolk, UK), and a Cy3-coupled goat anti-rabbit IgG (3.7 µg/ml; Jackson).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Western Blot, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Labeling

Journal: Molecular Vision

Article Title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

doi:

Figure Lengend Snippet: The hyperosmotic induction of AQP5 and VEGF depends on the activity of NFAT5. The mRNA levels ( A, B, D-G ) were determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control ( A, B, D ) and NaCl control ( E-G ), respectively. The level of VEGF-A 165 protein ( C, H ) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 429.5±71.4 pg/ml [ C ] and 324.2±35.1 pg/ml VEGF [ H ], respectively). A-C. The NFAT5 inhibitor rottlerin (2 and 10 µM, respectively) inhibits the hyperosmotic gene expression of AQP5 ( A ; n=7) and VEGF ( B ; n=3), as well as the hyperosmotic secretion of VEGF ( C ; n=5) from RPE cells. Hyperosmolarity was achieved by the addition of 100 mM NaCl to the culture medium. Vehicle control was made with DMSO (1%). D, E. Transfection of RPE cells with NFAT5 siRNA (siNFAT5; 5 nM) results in a reduction of the NFAT5 mRNA level in RPE cells cultured in isoosmotic ( D ; n=5) and hyperosmotic (+ 100 mM NaCl) media ( E ; n=3). Data shown in ( D ) were obtained 48 h after siRNA transfection. Thereafter, the cells were stimulated with a hyperosmotic medium for 2, 6, and 24 h, respectively ( E ). As negative controls, nontargeted siRNA (siNon; 5 nM) and a transfection reagent (TR) without siRNA were used. F, G. Knocking down the gene expression of NFAT5 with siRNA (siNFAT5; 5 nM) reduced the levels of AQP5 ( F ; n=6) and VEGF mRNAs ( G ; n=6) in cells cultured for 24 ( F ) and 6 h ( G ), respectively, in hyperosmotic (+ 100 mM NaCl) medium. As a negative control, nontargeted siRNA (siNon; 5 nM) was used (n=6 each). H. NFAT5 siRNA also reduced the secretion of VEGF induced by hyperosmotic (+ 100 mM NaCl) medium (n=6). Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.

Article Snippet: The following antibodies were used: rabbit anti-human extracellular signal-regulated kinases 1 and 2 (ERK1/2, p44/p42; 19 ng/ml; Cell Signaling, Frankfurt/M., Germany), a rabbit anti-phosphorylated ERK1/2 (95 ng/ml; Cell Signaling), a rabbit anti-human p38 mitogen-activated protein kinase (p38 MAPK; 8 ng/ml; Cell Signaling), a rabbit anti-human phosphorylated p38 MAPK (27 ng/ml; Cell Signaling), a rabbit anti-human NFAT5 (1 µg/ml; Santa Cruz), a rabbit anti-human nuclear factor (NF)-κB p65 (144 ng/ml; Cell Signaling), a rabbit anti-human signal transducer and activator of transcription 3 (STAT3; 89 ng/ml; Cell Signaling), a rabbit anti-human histone H3 (160 ng/ml; Cell Signaling), a rabbit anti-GAPDH (12 ng/ml; Cell Signaling), an anti-rabbit IgG conjugated with alkaline phosphatase (35 ng/ml; Cell Signaling), a rabbit anti-AQP5 (2 µg/ml; Santa Cruz), a mouse anti-vimentin (V9; 1.5 µg/ml; DakoCytomation, Hamburg, Germany), a Cy2-coupled goat anti-mouse IgG (3.5 µg/ml; Jackson ImmunoResearch, Suffolk, UK), and a Cy3-coupled goat anti-rabbit IgG (3.7 µg/ml; Jackson).

Techniques: Activity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Transfection, Negative Control

Journal: PLoS ONE

Article Title: Mediator of DNA Damage Checkpoint 1 (MDC1) Contributes to High NaCl-Induced Activation of the Osmoprotective Transcription Factor TonEBP/OREBP

doi: 10.1371/journal.pone.0012108

Figure Lengend Snippet: “Input” is the protein extract before immunoprecipitation. A. Osmolality bathing HEK 293 cells stably transfected with TonEBP/OREBP-1-547-V5 or empty vector-V5 (EV) was raised from 300 to 500 mosmol/kg by adding NaCl for 1, 3 and 6 h. Proteins were immunoprecipitated with rabbit IgG or anti-V5 antibodies and immunoblotted with anti-V5 or anti-MDC1 antibody. B . Osmolality bathing HEK 293 cells stably transfected with TonEBP/OREBP-1-547-V5 or empty vector-V5 was raised from 300 to 500 mosmol/kg by adding NaCl for 2 h. Proteins were immunoprecipitated from nuclear lysates with rabbit IgG or anti-MDC1 and immunoblotted with anti-MDC1 or anti-V5 antibody. C . 100 µg/ml ethidium bromide was added during immunoprecipitation with anti-MDC1 from nuclear extracts of the stably transfected HEK293 cells expressing TonEBP/OREBP-1-547-V5.

Article Snippet: Membranes then were incubated with rabbit anti-NFAT5 (TonEBP/OREBP) (Affinity BioReagents, Golden, CO), mouse anti-V5 (Invitrogen, Carlsbad, CA), rabbit anti-MDC1 (Bethyl Laboratories, Montgomery, TX), mouse anti-ATM (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-aldose reductase (Santa Cruz), mouse anti-BRG1 (Santa Cruz) or rabbit anti-P-ATM (Rockland Immunochemicals, Gilbertsville, PA) at 4°C overnight.

Techniques: Immunoprecipitation, Stable Transfection, Transfection, Plasmid Preparation, Expressing

Journal: PLoS ONE

Article Title: Mediator of DNA Damage Checkpoint 1 (MDC1) Contributes to High NaCl-Induced Activation of the Osmoprotective Transcription Factor TonEBP/OREBP

doi: 10.1371/journal.pone.0012108

Figure Lengend Snippet: A. HEK293 cells stably expressing an ORE-X reporter were transiently transfected with 25 nM of siRNA against MDC1 or control siRNA for 48 hours. Then, the osmolality of medium was changed to 500 mosm/kg by adding NaCl or kept at 300 mosmol/kg, and reporter activity was measured 24 h later. Knock down of MDC1 is shown in the upper panel. ORE-X reporter activity is relative to “control” at 300 mosmol/kg. B. Control for specificity for ORE. Same as (A) except using an IL2 min reporter (no ORE-X DNA element). C. Same as (A) except using HEK293 stably expressing the binary GAL4dbd TAD reporter and measuring luciferase activity 16 hours after adjusting osmolality. D. Control for specificity for TonEBP/OREBP TAD. Same as (C) except using HEK293 cells stably transfected with Gal4 DBD (no TAD). Mean ±SEM, *, P<0.01, n = 3.

Article Snippet: Membranes then were incubated with rabbit anti-NFAT5 (TonEBP/OREBP) (Affinity BioReagents, Golden, CO), mouse anti-V5 (Invitrogen, Carlsbad, CA), rabbit anti-MDC1 (Bethyl Laboratories, Montgomery, TX), mouse anti-ATM (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-aldose reductase (Santa Cruz), mouse anti-BRG1 (Santa Cruz) or rabbit anti-P-ATM (Rockland Immunochemicals, Gilbertsville, PA) at 4°C overnight.

Techniques: Stable Transfection, Expressing, Transfection, Activity Assay, Luciferase

Journal: PLoS ONE

Article Title: Mediator of DNA Damage Checkpoint 1 (MDC1) Contributes to High NaCl-Induced Activation of the Osmoprotective Transcription Factor TonEBP/OREBP

doi: 10.1371/journal.pone.0012108

Figure Lengend Snippet: A. Lack of effect of siRNA-mediated knockdown of MDC1 on nuclear localization of TonEBP/OREBP. HEK 293 cells were transiently transfected with 25 nM MDC1 siRNA or control siRNA. 48 h later the osmolality of medium was changed to 200 or 500 mosmol/kg or kept at 300 mosmol/kg for 1 h. Nuclear and cytoplasmic extracts were prepared. TonEBP/OREBP nuclear/cytoplasmic ratio was calculated from its abundance in nuclear and cytoplasmic extracts. Antibodies to BRG1 and aldose reductase serve as controls for nuclear and cytoplasmic fractionation, respectively. B . High NaCl decreases the nuclear localization of MDC1. Osmolality was changed to 200 or 500 mosmol/kg or kept at 300 mosmol/kg for 1 h. Nuclear and cytoplasmic extracts were prepared. MDC1 nuclear/cytoplasmic ratio was calculated from its abundance in nuclear and cytoplasmic extracts. C. Lack of effect of siRNA-mediated knockdown of MDC1 on phosphorylation of ATM on Ser1981. As in (A), except that abundance of ATM was measured in whole cell extracts by Western analysis using a non-phosphospecific antibody and its phosphorylation on Ser1981 was measured using phosphospecific anti-ATM 1981S-P antibody. Results are presented as the ratio of phosphorylated to non phosphorylated ATM. (* P<.05, compared to 300 mosmol).

Article Snippet: Membranes then were incubated with rabbit anti-NFAT5 (TonEBP/OREBP) (Affinity BioReagents, Golden, CO), mouse anti-V5 (Invitrogen, Carlsbad, CA), rabbit anti-MDC1 (Bethyl Laboratories, Montgomery, TX), mouse anti-ATM (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-aldose reductase (Santa Cruz), mouse anti-BRG1 (Santa Cruz) or rabbit anti-P-ATM (Rockland Immunochemicals, Gilbertsville, PA) at 4°C overnight.

Techniques: Transfection, Fractionation, Western Blot