nfat5 knockdown  (MedChemExpress)

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Increased expression of NFAT5 in microglia after OGD/R and MCAO modeling. (A) Schematic of three cell lines undergoing OGD/R modeling. BV2, mouse microglia cell line; MA, mouse astrocyte cell line; HT22, mouse hippocampal neuron cell line. (B) Western blots for NFAT5 in BV2, MA, and HT22 cell lines after OGD/R modeling ( n = 3). (C) The NFAT5 protein level in the nuclei and cytoplasm of BV2 cells was detected by western blotting ( n = 3). (D) Representative immunofluorescence images of NFAT5 in BV2 cells (bar = 25 μm). (E) The overlap coefficient of DAPI and NFAT5 in BV2 cells ( n = 3). (F, G) Immunofluorescence for NFAT5 in microglia from peri-infarct brain tissues of mice (bar = 50 μm). Iba-1 was marked in green for microglia, and NFAT5 was marked in red. The cells indicated by the white arrows were microglia. Scale bar in magnified view: 10 μm. The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Expressing, Western Blot, Immunofluorescence, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Rescue of MCAO-induced cerebral infarction and neurological deficits in mice through microglial NFAT5 interference. (A) Design of adeno-associated virus (AAV) to knock down microglial NFAT5. (B, C) Representative immunofluorescence images depicting Iba-1 (in pink) and NFAT5 (in red) (B) and quantification of NFAT5 fluorescence intensity in Iba-1 positive cells (C). The AAV expressed enhanced green fluorescent protein (EGFP). The infected microglia were delineated with circular and square annotations. The magnified view focused on the microglia within the square annotation. Scale bar: 50 μm. Scale bar in magnified view: 5 μm. The data were presented as mean with standard deviation ( n = 3). (D, G) Representative images and quantification of magnetic resonance imaging data. The data were presented as median ( n = 5). (E, F) Maximum and mean mouse limb grip strength ( n = 12). The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Virus, Knockdown, Immunofluorescence, Fluorescence, Infection, Standard Deviation, Magnetic Resonance Imaging

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Microglial NFAT5 knockdown mitigates MCAO-induced brain morphological damage and apoptosis. (A) Representative images of hematoxylin-eosin staining in the hippocampus and cortex of mice. Scale bar: 50 μm ( n = 3). (B – D) Representative images of Nissel staining (D) and quantification of Nissel-positive cells in the hippocampus (B) and cortex (C). Scale bar: 50 μm. The data were presented as mean with standard deviation ( n = 3). (E, F) Representative TUNEL assay images in cortical brain tissue regions (F) and quantification of TUNEL-positive cells per 0.1 mm 2 (E). TUNEL staining is shown in green, and nuclei are labeled in blue ( n = 3). Scale bar: 50 μm. (G – I) Western blotting analysis of Bcl-2 and Bax expression levels ( n = 3). Data presented as means with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Knockdown, Staining, Standard Deviation, TUNEL Assay, Labeling, Western Blot, Expressing

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Microglial NFAT5 silencing attenuates neuronal apoptosis in OGD/R model. (A) Schematic representation of BV2 (mouse microglia cell line) conditioned medium treatment on HT22 (mouse hippocampal neuron cell line). (B) A lactate dehydrogenase (LDH) assay was used to detect the released LDH in HT22 medium ( n = 8–12). ( C ) CCK-8 assay was used to measure the cell survival rate of HT22 ( n = 9). (D, E) Representative images and quantification of flow cytometry illustrating the percentage of annexin V-FITC and propidium iodide (PI)-labeled HT22 cells ( n = 3). (F–I) Representative immunofluorescence images of Bcl-2 (F) and Bax (H) and quantification of fluorescence intensity for Bcl-2 (G) and Bax (I) in HT22 cells ( n = 3). Scale bar: 25 μm. The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Lactate Dehydrogenase Assay, CCK-8 Assay, Flow Cytometry, Labeling, Immunofluorescence, Fluorescence, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 inhibition ameliorates microglia-mediated neuroinflammation and NLRP6 inflammasome activation in MCAO model. (A – E) Western blotting analysis of pro-inflammatory factor protein levels (IL-1β, TNF-α, and IL-6) in brain tissues (A), with protein levels normalized to the sham group (B–E) ( n = 3). (F, G) Representative immunofluorescence images of Iba-1 (F) and quantification of Iba-1-positive cells (G) in brain sections ( n = 3). Scale bar: 50 μm. (H, I) Representative immunofluorescence images of MPO (H) and quantification of MPO-positive cells (I) in brain sections ( n = 3). Scale bar: 40 μm. (J – N) Western blotting analysis was applied for NLRP6, ASC, pro-caspase-1, and cleaved-caspase-1 in brain tissue and the protein levels were normalized to the sham group ( n = 3). (O) Quantification of microglial morphology ( n = 3). The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 silencing suppresses inflammatory response and NLRP6 inflammasome activation in OGD/R Model. (A – F) Western blotting analysis of NFAT5, pro-IL-1β, IL-1β, TNF-α, and IL-6 protein levels in BV2 cells (A), with the protein levels normalized to the sh-NC control group (B–F) ( n = 3). (G – I) ELISA measurement of IL-1β, TNF-α, and IL-6 concentrations in BV2 cell culture medium ( n = 3). (K–N) Western blotting analysis was performed for NLRP6, pro-caspase-1, and cleaved-caspase-1 in BV2 cells, with the protein levels normalized to the sh-NC control group ( n = 3). (J) The Nlrp6 mRNA level in BV2 cells was detected by quantitative PCR ( n = 3). The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Activation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 is a transcription factor for the Nlrp6 promoter. (A) The diagram illustrating the predicted binding sites of NFAT5 on the Nlrp6 promoter. (B, C) Dual luciferase reporter assay of pGL4.10- Nlrp6 promoter after co-transfection with NFAT5 overexpressing (NFAT5) or control vector (TR) in N2A and 293T cell lines ( n = 8). (D) Construction of two Nlrp6 promoter fragments (P1 and P2) and a mutant of the Nlrp6 promoter. (E) Dual luciferase reporter assay of pGL4.10- Nlrp6 promoter (full length) or pGL4.10- Nlrp6 promoter fragments after co-transfection with NFAT5 overexpressing (NFAT5) or control vector (TR) into 293T cell line ( n = 4). (F) Dual luciferase reporter assay of pGL4.10- Nlrp6 promoter (full length) or pGL4.10- Nlrp6 promoter mutant after co-transfection with NFAT5-overexpressing (NFAT5) or control vector (TR) into 293T cell line ( n = 4). (G) The schematic depicting the positions of the Nlrp6 promoter probe for chromatin immunoprecipitation-quantitative PCR/PCR. (H, I) Chromatin immunoprecipitation with NFAT5 antibody in BV2 cells was analyzed by PCR ( n = 3) (H) and quantitative PCR ( n = 3) (I). H3 represents the positive control group, and IgG represents the negative control. The data were presented as mean with standard deviation. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Binding Assay, Luciferase, Reporter Assay, Cotransfection, Control, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 regulates NLRP6 mRNA stability through the Nlrp6 5′UTR. (A) Relative expression of Nlrp6 mRNA in wild-type BV2 cell line after OGD/R modeling and actinomycin D (Act D) treatment for 0, 1, 2, 4, and 6 h ( n = 3). ( B ) Relative expression of Nlrp6 mRNA in sh-NC and sh-NFAT5 BV2 cell lines after OGD/R modeling and Act D treatment for 0, 1, 2, 4, and 6 h ( n = 3). ( C ) Construction of pGL-promoter Nlrp6 5′UTR plasmid containing the mouse Nlrp6 5′UTR region for dual-luciferase reporter assay. (D – F) Relative luciferase activity of pGL-promoter or pGL-promoter Nlrp6 5′UTR after co-transfection with NFAT5-overexpressing (NFAT5) or control vector (TR) into the 293T cell line. Relative luciferase activity was determined and normalized to Renilla reference luciferase activity ( n = 3). (G) Construction of pGL-promoter Nlrp6 3′UTR plasmid containing the mouse Nlrp6 3′-UTR region for dual-luciferase reporter assay. (H – J) Relative luciferase activity of pGL-promoter or pGL-promoter Nlrp6 3′UTR after co-transfection with NFAT5 overexpressing (NFAT5) or control vector (TR) into the 293T cell line ( n = 3). The relative luciferase activity was determined and normalized to Renilla reference luciferase activity. The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, Cotransfection, Control, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: The schematic of microglial NFAT5 aggravating neuroinflammation and neuronal injury via mediating NLRP6 inflammasome following ischemic stroke.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques:

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: Expression of NFAT5 and NFAT5 target genes in CaKi-1 and HK-2 cells . CaKi-1 cells, as model for clear cell renal cell carcinoma, or HK-2 cells, as proximal tubular control cells, were kept in isoosmotic medium ( ; 300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H 2 O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for determination of NFAT5 transcription) or 16 h (for determination of S100A4 and AR transcription). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; # P < 0.05 vs. HK-2 isoosmotic medium; * P < 0.05 vs. HK-2 hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blots from 4 independent experiments are shown. (C) Cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After 24 h incubation in iso- or hyperosmotic medium, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; # P < 0.05 vs. HK-2 isoosmotic medium; * P < 0.05 vs. HK-2 hyperosmotic medium.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Expressing, Control, Incubation, Quantitative RT-PCR, Western Blot, Transfection, Construct, Activity Assay

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: NFAT5-knockdown attenuates S100A4 expression in CaKi-1 cells . CaKi-1 cells were transfected with siRNA constructs for NFAT5 (siNFAT5), S100A4 (siS100A4) or with non-targeting siRNA (siControl) as indicated. Cells were kept in isoosmotic medium ( ; 300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H 2 O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for NFAT5 determination) or 16 h (for S100A4 and AR determination). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; # P < 0.05 vs. siControl isoosmotic medium; * P < 0.05 vs. siControl hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blot from 4 independent experiments is shown.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Knockdown, Expressing, Transfection, Construct, Incubation, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: Effect of NFAT5 and S100A4 knockdown on proliferation, migration and survival of CaKi-1 cells. (A) Proliferation. CaKi-1 cells (10 4 per case) were transfected with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods and seeded into one well of a 96-well plate. After 96 h, the cell number in each well was determined by MTT assay. The number of viable cells treated with control siRNA was defined as 100%. Data are means ± s.e.m. for n = 6; * P < 0.05 vs. siControl. (B) Migration. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. After reaching confluency, the cell layer was scratched with a 10 μl pipette tip. Shown are representative phase-contrast images of cells migrating into the wounded area, immediately after scratching (0 h) and after an incubation time of 24 h. (C) Cell survival. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. Confluent cells were kept in isoosmotic medium ( ; 300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (■; 600 mosm/kg H 2 O) for 24 h. Thereafter, the cell number in each well was determined by MTT assay. Cell numbers in isosmotic controls ( ) were defined as 100%. Data are means ± s.e.m. for n = 6; * P < 0.05 vs. siControl hyperosmotic medium.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Knockdown, Migration, Transfection, Control, Construct, MTT Assay, Transferring, Incubation

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: Inhibition of FAK, Src, and MAP kinases attenuates NFAT5 activity in CaKi-1 cells . CaKi-1 cells were preincubated with the ERK1/2 inhibitor U0126 (U0; 10 μM), the p38 inhibitor SB202190 (SB; 10 μM), the JNK inhibitor SP600125 (SP; 10 μM), the FAK inhibitor PF-228 (PF; 5 μM), the Src inhibitor SrcI-1 (SrcI; 10 μM), or vehicle DMSO (Veh) for 30 min. Subsequently, cells were incubated at 300 or 500 mosm/kg H 2 O as indicated. (A,B) CaKi-1 cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After preincubation, the transfected cells were incubated for 24 h at 300 or 500 mosm/kg H 2 O, as indicated. Subsequently, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; * P < 0.05 vs. vehicle; # P < 0.05 vs. vehicle hyperosmotic medium. (C–F) After preincubation, CaKi-1 cells were incubated for 16 h at 300 or 500 mosm/kg H 2 O, as indicated. Subsequently, RNA was extracted and the abundance of NFAT5, S100A4 and β-actin mRNA transcripts was determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5 and S100A4 was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; * P < 0.05 vs. vehicle; # P < 0.05 vs. vehicle hyperosmotic medium.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Inhibition, Activity Assay, Incubation, Transfection, Construct, Control, Quantitative RT-PCR

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: Expression of NFAT5 and NFAT5 target genes in CaKi-1 and HK-2 cells . CaKi-1 cells, as model for clear cell renal cell carcinoma, or HK-2 cells, as proximal tubular control cells, were kept in isoosmotic medium ( ; 300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H 2 O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for determination of NFAT5 transcription) or 16 h (for determination of S100A4 and AR transcription). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; # P < 0.05 vs. HK-2 isoosmotic medium; * P < 0.05 vs. HK-2 hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blots from 4 independent experiments are shown. (C) Cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After 24 h incubation in iso- or hyperosmotic medium, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; # P < 0.05 vs. HK-2 isoosmotic medium; * P < 0.05 vs. HK-2 hyperosmotic medium.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Transfection, Construct, Activity Assay

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: NFAT5-knockdown attenuates S100A4 expression in CaKi-1 cells . CaKi-1 cells were transfected with siRNA constructs for NFAT5 (siNFAT5), S100A4 (siS100A4) or with non-targeting siRNA (siControl) as indicated. Cells were kept in isoosmotic medium ( ; 300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H 2 O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for NFAT5 determination) or 16 h (for S100A4 and AR determination). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; # P < 0.05 vs. siControl isoosmotic medium; * P < 0.05 vs. siControl hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blot from 4 independent experiments is shown.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Expressing, Transfection, Construct, Incubation, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: Effect of NFAT5 and S100A4 knockdown on proliferation, migration and survival of CaKi-1 cells. (A) Proliferation. CaKi-1 cells (10 4 per case) were transfected with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods and seeded into one well of a 96-well plate. After 96 h, the cell number in each well was determined by MTT assay. The number of viable cells treated with control siRNA was defined as 100%. Data are means ± s.e.m. for n = 6; * P < 0.05 vs. siControl. (B) Migration. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. After reaching confluency, the cell layer was scratched with a 10 μl pipette tip. Shown are representative phase-contrast images of cells migrating into the wounded area, immediately after scratching (0 h) and after an incubation time of 24 h. (C) Cell survival. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. Confluent cells were kept in isoosmotic medium ( ; 300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (■; 600 mosm/kg H 2 O) for 24 h. Thereafter, the cell number in each well was determined by MTT assay. Cell numbers in isosmotic controls ( ) were defined as 100%. Data are means ± s.e.m. for n = 6; * P < 0.05 vs. siControl hyperosmotic medium.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Migration, Transfection, Construct, MTT Assay, Transferring, Incubation

Journal: Frontiers in Physiology

Article Title: NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

doi: 10.3389/fphys.2014.00293

Figure Lengend Snippet: Inhibition of FAK, Src, and MAP kinases attenuates NFAT5 activity in CaKi-1 cells . CaKi-1 cells were preincubated with the ERK1/2 inhibitor U0126 (U0; 10 μM), the p38 inhibitor SB202190 (SB; 10 μM), the JNK inhibitor SP600125 (SP; 10 μM), the FAK inhibitor PF-228 (PF; 5 μM), the Src inhibitor SrcI-1 (SrcI; 10 μM), or vehicle DMSO (Veh) for 30 min. Subsequently, cells were incubated at 300 or 500 mosm/kg H 2 O as indicated. (A,B) CaKi-1 cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After preincubation, the transfected cells were incubated for 24 h at 300 or 500 mosm/kg H 2 O, as indicated. Subsequently, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; * P < 0.05 vs. vehicle; # P < 0.05 vs. vehicle hyperosmotic medium. (C–F) After preincubation, CaKi-1 cells were incubated for 16 h at 300 or 500 mosm/kg H 2 O, as indicated. Subsequently, RNA was extracted and the abundance of NFAT5, S100A4 and β-actin mRNA transcripts was determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5 and S100A4 was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; * P < 0.05 vs. vehicle; # P < 0.05 vs. vehicle hyperosmotic medium.

Article Snippet: Accell SMARTpool siRNA constructs for knockdown of NFAT5 or S100A4, and Accell non-targeting siRNA (#2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Inhibition, Activity Assay, Incubation, Transfection, Construct, Quantitative RT-PCR

Journal: Mediators of Inflammation

Article Title: NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells

doi: 10.1155/2012/513015

Figure Lengend Snippet: Expression and activation of NFAT5 in Met5A cells. Met5A cells were kept in isosmotic medium (300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (400 mosm/kg H 2 O). Medium osmolality was elevated by addition of glucose, NaCl, or mannitol as indicated. (a) Cells were incubated for 24 h and subsequently processed for immunoblotting as described in . To demonstrate comparable protein loading, the blots were also probed for actin. A representative blot from 3 independent experiments is shown. (b) Relative NFAT5 protein abundance was quantified by densitometric analysis of immunoblots and normalized to that of actin to correct for differences in protein loading. Means ± SEM for n = 3; * P < 0.05 versus isosmotic control. (c) Cells were incubated for 16 h. Thereafter, RNA was extracted and the abundance of MCP-1 mRNA transcript determined by qRT-PCR as described in . Relative MCP-1 mRNA abundance was normalized to that of β -actin to correct for differences in RNA input. Means ± SEM for n = 3 per point; * P < 0.05 versus isosmotic control. (d) Cells were incubated for 1 h, and subsequently cytoplasmic and nuclear extracts prepared and processed for immunoblotting as described in . To demonstrate purity of extracts and comparable protein loading, the blots were also probed for histone H1 and actin. A representative blot from 4 independent experiments is shown. (e) Relative NFAT5 nuclear versus cytoplasmic abundance was quantified by densitometric analysis of immunoblots. Means ± SEM for n = 3; * P < 0.05 versus isosmotic control. (f) Activity of the transactivation domain of NFAT5 during osmotic stress. Met5A cells were cotransfected with a vector encoding the fusion protein GAL4dbd-TonEBP-TAD (amino acids 548-1541 of NFAT5 fused to the yeast GAL4 DNA binding domain) together with the reporter vector pFR-SEAP. Cells were kept in isosmotic medium (300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (400 mosm/kg H 2 O). After 48 h, SEAP activity was measured as described in . Means ± SEM for n = 3; * P < 0.05 versus isosmotic control. (g) Met5A cells were transiently transfected with a reporter construct in which the SEAP gene is under control of two TonE sites. Cells were kept in isosmotic (300 mosm/kg H 2 O) medium or were exposed to hyperosmotic medium, with osmolalities between 325 and 550 mosm/kg H 2 O as indicated. After 24 h, SEAP activity was measured as described in . Means ± SEM for n = 4; * P < 0.05 versus isosmotic control.

Article Snippet: Accell SMARTpool siRNA construct for knockdown of NFAT5 or Accell nontargeting siRNA (no. 2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Expressing, Activation Assay, Incubation, Western Blot, Quantitative Proteomics, Control, Quantitative RT-PCR, Activity Assay, Plasmid Preparation, Binding Assay, Transfection, Construct

Journal: Mediators of Inflammation

Article Title: NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells

doi: 10.1155/2012/513015

Figure Lengend Snippet: NFAT5-knockdown attenuates osmolality-induced MCP-1 expression. Met5A cells were transfected with siRNA constructs for NFAT5 or with nontargeting siRNA as control as indicated. Cells were kept in isosmotic medium (gray column; 300 mosm/kg H 2 O) or were exposed to hyperosmotic medium (black column; 400 mosm/kg H 2 O). Medium osmolality was elevated by addition of glucose or NaCl as indicated, and cells were incubated for 24 h. (a) To demonstrate efficiency of NFAT5 knockdown, cells were processed for immunoblotting as described in . To demonstrate comparable protein loading, the blots were also probed for actin. (b) For determination of MCP-1 secretion, medium samples were collected and the concentration of MCP-1 in the cell culture supernatant was determined by ELISA as described in . Means ± SEM for n = 4 per point; * P < 0.05. (c) For determination of MCP-1 transcription, RNA was extracted from the cells and the abundance of MCP-1 mRNA transcript was determined by qRT-PCR as described in . Relative MCP-1 mRNA abundance was normalized to that of β -actin to correct for differences in RNA input. Means ± SEM for n = 4 per point; * P < 0.05.

Article Snippet: Accell SMARTpool siRNA construct for knockdown of NFAT5 or Accell nontargeting siRNA (no. 2) were obtained from Thermo Fisher Scientific (Epsom, UK).

Techniques: Knockdown, Expressing, Transfection, Construct, Control, Incubation, Western Blot, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR