Journal: Oncogene

Article Title: CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment

doi: 10.1038/onc.2012.24

Figure Lengend Snippet: CXCL13 transcriptional control of RANKL expression in human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. (A) SAKA-T and (B) MC3T3-E1 cells were transiently transfected with hRANKL-luc P#3 plasmid by lipofectamine and cultured with CXCL13 (0–15 ng/ml) for 6 h. Cells mock-transfected with empty vector (EV) served as control. Total cell lysates prepared were assayed for luciferase activity. (C) MC3T3-E1 cells were treated with different concentrations of recombinant CXCL13 (0–20 ng/ml) for 6 h. Total cell lysates obtained were analyzed for c-Myc, NFATc3 and RANKL expression by Western blot. (D) CXCL13 activation of c-Myc in SAKA-T and MC3T3-E1 cells. Cells were treated with CXCL13 (15 ng/ml) for 0–60 min and total cell lysates obtained were analyzed for p-c-Myc by Western blot. β-actin expression served as control. Data represent three replicate studies and mean ± SD (*p<0.05).

Article Snippet: Anti- NFATc3, anti-c-Myc, anti-ERK, anti-p-ERK, peroxidase-conjugated secondary antibodies and siRNA against NFATc3 and c-Myc were purchased from Santa Cruz Biotechnology, CA.

Techniques: Control, Expressing, Derivative Assay, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Activity Assay, Recombinant, Western Blot, Activation Assay

Journal: Oncogene

Article Title: CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment

doi: 10.1038/onc.2012.24

Figure Lengend Snippet: RANKL, p-c-Myc, NFATc3 expression in OSCC tumor-bone microenvironment. (A) OSCC tumor progression in athymic mice. NCr-nu/nu athymic mice were subcutaneously injected with SCC-11B cells (7×10 6 ) with or without stable RANKL-Luc reporter gene construct in PBS over calvaria. Tumors were allowed to grow for five weeks and imaged the calvarial region of mice facing up towards the CCD (charge-coupled device) camera using IVIS imaging system at 20 min after luciferin (150 mg/kg, i.p). (B) Immuno-histochemical analysis of RANKL, NFATc3, p-c-Myc expression and alkaline phosphatase (ALP) activity at the tumor-bone interface in athymic mice. (C) Immunohistochemical analysis of p-c-Myc and NFATc3 expression in primary human OSCC tumor specimens and adjacent normal tissues. Data shown are representative of three replicate studies.

Article Snippet: Anti- NFATc3, anti-c-Myc, anti-ERK, anti-p-ERK, peroxidase-conjugated secondary antibodies and siRNA against NFATc3 and c-Myc were purchased from Santa Cruz Biotechnology, CA.

Techniques: Expressing, Injection, Construct, Imaging, Activity Assay, Immunohistochemical staining

Journal: Oncogene

Article Title: CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment

doi: 10.1038/onc.2012.24

Figure Lengend Snippet: CXCL13 activation of c-Myc expression induces RANKL expression. (A) CXCL13 activation of ERK1/2 in human bone marrow stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. Cells were stimulated with CXCL13 (15 ng/ml) for different time points (0–60 min) and total cell lysates were subjected to Western blot analysis for ERK1/2 and pERK1/2 expression. (B) siRNA suppression of NFATc3 inhibits RANKL but not c-Myc expression and (C) siRNA suppression of c-Myc inhibits RANKL and NFATc3 expression in MC3T3-E1 cells. Cells were transiently transfected with double stranded siRNA against c-Myc or NFATc3 using oligofectamine. After 48 h, cells were treated with CXCL13 (15 ng/ml) for 6 h and total cell lysates were analyzed by Western blot for RANKL, c-Myc and NFATc3 expression. β-actin expression serves as control. Data shown are representative of three independent experiments.

Article Snippet: Anti- NFATc3, anti-c-Myc, anti-ERK, anti-p-ERK, peroxidase-conjugated secondary antibodies and siRNA against NFATc3 and c-Myc were purchased from Santa Cruz Biotechnology, CA.

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Control

Journal: Oncogene

Article Title: CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment

doi: 10.1038/onc.2012.24

Figure Lengend Snippet: CXCL13 transcriptional control of RANKL expression in human bone marrow derived stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. (A) SAKA-T and (B) MC3T3-E1 cells were transiently transfected with hRANKL-luc P#3 plasmid by lipofectamine and cultured with CXCL13 (0–15 ng/ml) for 6 h. Cells mock-transfected with empty vector (EV) served as control. Total cell lysates prepared were assayed for luciferase activity. (C) MC3T3-E1 cells were treated with different concentrations of recombinant CXCL13 (0–20 ng/ml) for 6 h. Total cell lysates obtained were analyzed for c-Myc, NFATc3 and RANKL expression by Western blot. (D) CXCL13 activation of c-Myc in SAKA-T and MC3T3-E1 cells. Cells were treated with CXCL13 (15 ng/ml) for 0–60 min and total cell lysates obtained were analyzed for p-c-Myc by Western blot. β-actin expression served as control. Data represent three replicate studies and mean ± SD (*p<0.05).

Article Snippet: Anti- NFATc3, anti-c-Myc, anti-ERK, anti-p-ERK, peroxidase-conjugated secondary antibodies and siRNA against NFATc3 and c-Myc were purchased from Santa Cruz Biotechnology, CA.

Techniques: Control, Expressing, Derivative Assay, Transfection, Plasmid Preparation, Cell Culture, Luciferase, Activity Assay, Recombinant, Western Blot, Activation Assay

Journal: Oncogene

Article Title: CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment

doi: 10.1038/onc.2012.24

Figure Lengend Snippet: RANKL, p-c-Myc, NFATc3 expression in OSCC tumor-bone microenvironment. (A) OSCC tumor progression in athymic mice. NCr-nu/nu athymic mice were subcutaneously injected with SCC-11B cells (7×10 6 ) with or without stable RANKL-Luc reporter gene construct in PBS over calvaria. Tumors were allowed to grow for five weeks and imaged the calvarial region of mice facing up towards the CCD (charge-coupled device) camera using IVIS imaging system at 20 min after luciferin (150 mg/kg, i.p). (B) Immuno-histochemical analysis of RANKL, NFATc3, p-c-Myc expression and alkaline phosphatase (ALP) activity at the tumor-bone interface in athymic mice. (C) Immunohistochemical analysis of p-c-Myc and NFATc3 expression in primary human OSCC tumor specimens and adjacent normal tissues. Data shown are representative of three replicate studies.

Article Snippet: Anti- NFATc3, anti-c-Myc, anti-ERK, anti-p-ERK, peroxidase-conjugated secondary antibodies and siRNA against NFATc3 and c-Myc were purchased from Santa Cruz Biotechnology, CA.

Techniques: Expressing, Injection, Construct, Imaging, Activity Assay, Immunohistochemical staining

Journal: Oncogene

Article Title: CXCL13 Activation of c-Myc Induce RANK Ligand Expression in Stromal/Preosteoblast Cells in the Oral Squamous Cell Carcinoma Tumor-Bone Microenvironment

doi: 10.1038/onc.2012.24

Figure Lengend Snippet: CXCL13 activation of c-Myc expression induces RANKL expression. (A) CXCL13 activation of ERK1/2 in human bone marrow stromal cells (SAKA-T) and murine preosteoblast (MC3T3-E1) cells. Cells were stimulated with CXCL13 (15 ng/ml) for different time points (0–60 min) and total cell lysates were subjected to Western blot analysis for ERK1/2 and pERK1/2 expression. (B) siRNA suppression of NFATc3 inhibits RANKL but not c-Myc expression and (C) siRNA suppression of c-Myc inhibits RANKL and NFATc3 expression in MC3T3-E1 cells. Cells were transiently transfected with double stranded siRNA against c-Myc or NFATc3 using oligofectamine. After 48 h, cells were treated with CXCL13 (15 ng/ml) for 6 h and total cell lysates were analyzed by Western blot for RANKL, c-Myc and NFATc3 expression. β-actin expression serves as control. Data shown are representative of three independent experiments.

Article Snippet: Anti- NFATc3, anti-c-Myc, anti-ERK, anti-p-ERK, peroxidase-conjugated secondary antibodies and siRNA against NFATc3 and c-Myc were purchased from Santa Cruz Biotechnology, CA.

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Control

Journal: Oncotarget

Article Title: Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

doi: 10.18632/oncotarget.24320

Figure Lengend Snippet: ( A ) Mouse total lung macrophages were stimulated with LPS (100 ng/mL for 0.5 and 4.0 h). The cytoplasmic and nuclear proteins were isolated using Peirce Nuclear and Cytoplasmic protein isolation kit and equal amount of proteins were immunoblotted for NFATc1, NFATc2, NFATc3 and NFATc4 to determine their cytoplasmic vs. nuclear distribution. ( B ) Mouse PMVEC were treated with LPS (100 ng/mL for 0.5 and 4.0 h). The cytoplasmic and nuclear proteins were isolated and analyzed for NFAT1-NFAT4 nuclear translocation. ( C ) Mouse AEC type II cells were treated with LPS (100 ng/mL for 0.5 and 4.0 h) and analyzed for NFAT1-NFAT4 nuclear translocation. Purity of cytoplasmic and nuclear proteins is determined by immunoblotting with β-Actin and p53.

Article Snippet: Myc-DDK tagged human NFATc3 expression vector (catalog# RC212523) was purchased from Origene (Rockville, MD).

Techniques: Isolation, Translocation Assay, Western Blot

Journal: Oncotarget

Article Title: Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

doi: 10.18632/oncotarget.24320

Figure Lengend Snippet: BMDMs from WT and NFATc3 −/− mice were stimulated with LPS for 24 h and analyzed for ( A ). Expression of CCR2 by flow cytometry ( B ) Extracellular medium for TNFα release by ELISA. ( C ) Binding of NAFTc3 to the CCR2 promoter was determined by Electrophoretic mobility shift assay. ( D ) Binding of NAFTc3 to the TNFα promoter was determined by Chromatin ImmunoPrecipitation using QIAGEN One day ChIP kit. ( E ) NFATc3 was overexpressed in NFATc3 −/− BMDMs by electroporation with a pCMV-NFATc3 plasmid and stimulated with LPS for 24 h. LPS induced TNFα release in to the culture medium was measured using R&D systems Quantikine TNFα ELISA kit. B-D-E, * P ≤ 0.01 LPS vs. control; ** p ≤ 0.001 NFATc3 −/− vs. WT or pCMV-NFATc3 vs pCMV-Control.

Article Snippet: Myc-DDK tagged human NFATc3 expression vector (catalog# RC212523) was purchased from Origene (Rockville, MD).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Electroporation, Plasmid Preparation

Journal: Oncotarget

Article Title: Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

doi: 10.18632/oncotarget.24320

Figure Lengend Snippet: Mouse BMDM stimulated with LPS (100 ng/mL) for 24 h were co-cultured with mouse PMVEC monolayer grown on cell culture inserts, added with FITC-Dextran to the trans wells at the time of initiating co-culture. ( A ) Flux of FITC-Dextran in WT and NFATc3 −/− macrophage co-culture was determined by measuring fluorescence of the medium in the bottom wells as described in methods. Similarly, ( B ) FITC-Dextran flux in neutralizing TNFα pre- treated WT BMDM cells is quantitated as described in methods. ( C ) Homogeneity of NFATc3 −/− & WT BMDM was determined by staining with anti-APC-NFATc3 and F480. ( D ) Homogeneity of PMVEC is confirmed by staining with anti-PE-CD31. A–B * p ≤ 0.001 LPS vs PBS; † p ≤ 0.01 NFATc3 −/− /NeutralizingTNFα vs NFATc3 +/+ (WT)/Normal IgG.

Article Snippet: Myc-DDK tagged human NFATc3 expression vector (catalog# RC212523) was purchased from Origene (Rockville, MD).

Techniques: Cell Culture, Co-Culture Assay, Fluorescence, Staining

Journal: Oncotarget

Article Title: Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

doi: 10.18632/oncotarget.24320

Figure Lengend Snippet: NFATc3 −/− and WT mice were subjected to CLP or sham surgery and analyzed after 24 h to determine ( A ) protein leakage in to BALF, ( B) . TNFα released in to BALF was measured by ELISA ( C–D ) Hema 3.0 Stained Neutrophils and macrophages in BALF. ( E ) NFATc3 −/− and WT mice were subjected to LPS (i.p 15 mg/kg) sepsis for 16 h. Lungs were fixed, embedded in paraffin, sectioned and H&E stained. Lung histology showing neutrophilic infiltration in to NFATc3 −/− and wild type mice. (A–D) * p ≤ 0.001 WT-CLP vs WT- Sham; ** p ≤ 0.01 NFATc3 −/− CLP vs WT-CLP; † p ≤ 0.05 macrophages in NFATc3 −/− CLP vs WT-CLP.

Article Snippet: Myc-DDK tagged human NFATc3 expression vector (catalog# RC212523) was purchased from Origene (Rockville, MD).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Oncotarget

Article Title: Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

doi: 10.18632/oncotarget.24320

Figure Lengend Snippet: ( A ) NFATc3 −/− and wild type mice ( N = 5) were injected with 15 mg/kg LPS i.p and the arterial oxygen saturation was measured using STARR Life Sciences Corp. Pulse Oximeter. ( B – C ) NFATc3 −/− and wild type mice ( N = 5 sham control group; N = 10 CLP group) were subjected to severe CLP, maintained on regular or imipenem monohydrate supplemented water and the survival was monitored over a period of 96 h. A * p ≤ 0.001 WT-LPS vs. WT-Control; ** p ≤ 0.01 NFATc3 −/− LPS vs. WT-LPS.

Article Snippet: Myc-DDK tagged human NFATc3 expression vector (catalog# RC212523) was purchased from Origene (Rockville, MD).

Techniques: Injection

Journal: Oncotarget

Article Title: Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

doi: 10.18632/oncotarget.24320

Figure Lengend Snippet: WT or NFATc3 −/− mice were pretreated with codronate liposomes for 72 h to deplete lung resident macrophages. The macrophage deleted mice were adoptively transferred with lung macrophages from reciprocal NFATc3 background and administered with i.p LPS (15 mg/kg). Pulmonary inflammation and edema was measured by ( A ) TNFα in BALF ( B ) BALF protein content ( C ) lung wet to dry ratios. ( D ) Depletion of lung resident macrophage population after clodronate liposome administration compared to PBS-liposomes was determined by flow sorting cells for CD45 and CD11b/F4/80 positive population. * p ≤ 0.01 WT-WT or WT-KO/LPS vs WT-WT or WT-KO/PBS; † p ≤ 0.01 KO-KO-LPS or KO-WT-LPS vs WT-WT-LPS or WT-KO-LPS.

Article Snippet: Myc-DDK tagged human NFATc3 expression vector (catalog# RC212523) was purchased from Origene (Rockville, MD).

Techniques:

Journal: Oncotarget

Article Title: Inhibition of nuclear factor of activated T cells (NFAT) c3 activation attenuates acute lung injury and pulmonary edema in murine models of sepsis

doi: 10.18632/oncotarget.24320

Figure Lengend Snippet: Mouse BMDM were pretreated with 1 μM of ( A ) control CP9-VAVAA or ( B ) CP9-ZIZIT inhibitor peptides and stimulated with PBS or LPS (100 ng/mL) for 1 hour. Localization of NFATc3 (green) and peptides (CP9-VAVAA or CP9-ZIZIT in red) with PBS/LPS stimulation is determined using Zeiss LSM 510 Meta microscope (Carl Zeiss MicroImaging, Inc.) equipped with 488 nm and 543 nm excitation lasers. Single confocal optical sections (pinhole set to achieve 1 Airy unit) are shown ( n = 7–10 cells/group). Bars correspond to 10 μm.

Article Snippet: Myc-DDK tagged human NFATc3 expression vector (catalog# RC212523) was purchased from Origene (Rockville, MD).

Techniques: Microscopy

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: NFATc3 is upregulated during human and mouse AAD. (A) t -Distributed stochastic neighbor embedding ( t -SNE) visualization of cell types isolated from human ascending aortic wall tissue via scRNA-seq ( GSE155468 ). (B) Mean expression values scaled via mean-centering and transformed to a scale from −2 to 2. (C) Boxplot of NFATc3 expression level across major cell types. (D) Relative NFATc3 mRNA in the ascending aortas of patients undergoing coronary bypass, but without AAD (Con), and patients with TAAD. n = 36. (E) Relative NFATc3 protein levels in ascending aortas of patients with or without TAAD, and in abdominal aortas of patients with AAA ( n = 8). (F) Western blot analysis of NFATc3 levels in the cytoplasm and nucleus of aortas from patients without TAAD and those with TAAD or AAA ( n = 4). (G) NFATc3 and ACTA2 immunofluorescence in aortas of patients without TAAD or with TAAD or AAA ( n = 8). (H) Relative NFATc3 protein levels in mice aortas treated with saline, BAPN/AngII, or AAV- Pcsk9 DY /AngII ( n = 8). (I) NFATc3 and ACTA2 immunofluorescence in mice aortas treated with saline, BAPN/AngII, or AAV- Pcsk9 DY /AngII ( n = 6). Data are presented as mean ± SD. (C, D) Mann–Whitney U-test with the exact method; two-tailed P -values; (E–I) One-way ANOVA followed by Dunnett’s correction; adjusted P -values.

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Isolation, Expressing, Transformation Assay, Western Blot, Immunofluorescence, Saline, MANN-WHITNEY, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: VSMC–NFATc3 deficiency mitigates AAD in mice. (A–H) Five-week-old Nfatc3 fl/fl or Nfatc3 smcKO male mice administered BAPN (0.3 g/kg/day) for 28 days, followed by AngII infusion (1000 ng/kg/min) for 3 days ( n = 22). (A) Mouse aorta macrographs. (B) Thoracic aorta ultrasound images. (C) Survival curves analyzed via Kaplan–Meier and compared using log-rank tests. (D) AAD incidence per group. (E) Maximal aortic diameter ex vivo ( n = 15 for Nfatc3 fl/fl ; n = 22 for Nfatc3 smcKO ). (F) H&E, van Geison (elastin), and Masson staining of mouse ascending aorta. (G, H) Elastin degradation grade (G) and collagen content (H) in the aortic wall. (I–K) Eight-week-old Nfatc3 fl/fl or Nfatc3 smcKO mice were injected AAV- Pcsk9 DY via the tail vein and fed a Western-type diet. The mice were infused with AngII (1500 ng/kg/min) after 2 weeks for another 4 weeks. (I) Mouse aorta macrographs. (J) AAA incidence per group. (K) Maximal aortic diameter ex vivo ( n = 17 for Nfatc3 fl/fl ; n = 21 for Nfatc3 smcKO ). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (E, H, K) Unpaired Student’s t -test; two-tailed P -values. (G) Mann–Whitney U-test with the exact method; two-tailed P -values.

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Ex Vivo, Staining, Injection, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: VSMC–NFATc3 overexpression aggravates AAD in mice. (A–H) Five-week-old Nfatc3 -KI fl/fl or Nfatc3 smcKI male mice were administered BAPN (0.3 g/kg/day) for 28 d before infusion with AngII (1000 ng/kg/min) for 3 d ( n = 19 for Nfatc3 -KI fl/fl ; n = 20 for Nfatc3 smcKI ). (A) Mouse aorta macrographs. (B) Thoracic aorta ultrasound images. (C) Survival curves per group analyzed using Kaplan–Meier and compared using log-rank tests. (D) AAD incidence. (E) Maximal aortic diameter ex vivo ( n = 13 for Nfatc3 -KI fl/fl ; n = 7 for Nfatc3 smcKI ). (F) H&E, van Geison (elastin), and Masson staining of the mouse ascending aorta. (G, H) Elastin degradation grade (G) and collagen content (H) in the aortic wall. (I–K) Eight-week-old Nfatc3 -KI fl/fl or Nfatc3 smcKI mice injected AAV- Pcsk9 DY via tail vein and fed a Western-type diet. Two weeks later, the mice were infused with AngII (1500 ng/kg/min) for 4 weeks. (I) Mouse aorta macrographs. (J) AAA incidence. (K) Maximal aortic diameter ex vivo ( n = 13 for Nfatc3 -KI fl/fl ; n = 10 for Nfatc3 smcKI ). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (E, H) Unpaired Student’s t -test; two-tailed P -values. (G, K) Mann–Whitney U-test with the exact method; two-tailed P -values.

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Over Expression, Ex Vivo, Staining, Injection, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: VSMC–NFATc3 facilitates AAD progression in the BAPN model. (A) Relative NFATc3 mRNA levels in ascending aortas of patients without TAAD undergoing coronary bypass (Control, n = 36) and TAAD with and without hypertension ( n = 25; 11, respectively). (B) NFATc3 Western blot in mouse aortas with different AngII treatment durations ( n = 4). (C–I) Five-week-old male Nfatc3 fl/fl ( n = 15), Nfatc3 smcKO ( n = 15), Nfatc3 -KI fl/fl ( n = 17), and Nfatc3 smcKI ( n = 18) mice were administered BAPN (0.6 g/kg/day) for 28 days. (C) Aorta macrographs. (D) TAAD incidence. (E) Thoracic aorta ultrasound images. (F) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (G) Maximum aortic diameter. (H) H&E, elastin, and Masson aorta staining. (I, J) Phenylephrine (PE)-induced vascular contraction (I) and acetylcholine (Ach)-induced endothelium-dependent vasodilation (J) of aortic rings from Nfatc3 fl/fl and Nfatc3 smcKO mice treated with vehicle or BAPN for 28 days ( n = 8). (K) Western blot and ACTA2, CNN1, SM22 α , and OPN quantification in aortas ( n = 6). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (A, B, G) One-way ANOVA with Tukey’s correction; adjusted P -values. (I–K) Two-way ANOVA with Tukey’s correction; adjusted P -values. NS, no significance ( P > 0.05).

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Control, Western Blot, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: NFATc3 promotes ECM degradation by transcriptionally upregulating MMP9 and MMP2 expression in VSMCs. (A) Gene Ontology term enrichment analysis of the biological process influenced by NFATc3 deficiency based on the RNA-seq dataset. Downregulated processes in the aortas of Nfatc3 smcKO mice compared to those in Nfatc3 fl/fl mice treated with BAPN (0.6 g/kg/day) for 28 days ( n = 3). (B) Gene expression profiles were compared between Nfatc3 smcKO to Nfatc3 fl/fl mice treated with BAPN, and heatmaps were generated based on the expression of significantly different genes related to ECM organization (blue, downregulated; red, upregulated). Genes with a corresponding adjusted P < 0.05 were considered statistically significant. (C) Relative mRNA levels of genes related to ECM organization in the aortas of Nfatc3 smcKO and Nfatc3 fl/fl mice treated with BAPN ( n = 6). (D) MMP2 and MMP9 Western blot in the aortas of Nfatc3 smcKO and Nfatc3 fl/fl mice treated with vehicle or BAPN ( n = 6). (E) MMP2 and MMP9 protein levels in HASMCs infected with Ad- LacZ and Ad- NFATc3 ( n = 6). (F) Immunofluorescence of in situ zymography and immunostaining for ACTA2 in Nfatc3 fl/fl and Nfatc3 smcKO ascending aortas after vehicle or BAPN treatment ( n = 6). (G) Gelatin zymography for detecting pro-MMP9, active-MMP9, pro-MMP2, and active-MMP2 in the ascending aortas of Nfatc3 fl/fl and Nfatc3 smcKO mice after vehicle or BAPN treatment. (H, I) Correlation analysis of NFATc3 mRNA with MMP9 (H) or MMP2 (I) levels in the ascending aortas of patients without AAD (Con) ( n = 36) and with TAAD ( n = 36). (J) NFATc3-binding motifs. (K) Luciferase reporter analysis in 293T cells ( n = 6). (L) ChIP-qPCR validation of H3K27ac and H3K4me3 enrichment on the Mmp9 and Mmp2 promoter ( n = 6). Data are presented as mean ± SD. (C, E, L) Unpaired Student’s t -test; two-tailed P -values. (D, F, K) Two-way ANOVA with Tukey’s correction; adjusted P -values. (H, I) Spearman’s or Pearson’s correlation coefficient test; regression coefficients. NS, no significance ( P > 0.05).

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Expressing, RNA Sequencing, Gene Expression, Generated, Western Blot, Infection, Immunofluorescence, In Situ, Zymography, Immunostaining, Binding Assay, Luciferase, ChIP-qPCR, Biomarker Discovery, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: Cytoplasmic NFATc3 facilitates VSMC contractile-to-synthetic phenotype switching by promoting global protein synthesis. (A) Representative images and quantification of NFATc3 and ACTA2 expression using immunofluorescence staining in MASMCs isolated from mice treated with vehicle and BAPN for 28 days ( n = 6). (B) NFATc3-associated protein subcellular localization based on LC–MS/MS. (C) Gene Ontology analysis of NFATc3-associated proteins identified using LC–MS/MS. (D) Polysome profiling shows NFATc3 distribution in MASMCs using sucrose density gradient centrifugation ( n = 3). Western blot of NFATc3 in the elution profiles. (E) Representative Western blot images and quantification of puromycin incorporation assays from MASMCs isolated from Nfatc3 -KI fl/fl and Nfatc3 smcKI mice ( n = 4). (F) HPG incorporation assay in VSMCs isolated from the aortas of Nfatc3 -KI fl/fl and Nfatc3 smcKI mice treated with BAPN for 28 days ( n = 4). (G) Western blot and quantification of ACTA2, CNN1, SM22 α , and OPN in MASMCs treated with PDGF-BB for 24 h before a 12-h CHX treatment ( n = 4). (H) Representative Western blot images and quantification of ACTA2, CNN1, SM22 α , and OPN in MASMCs isolated from aortas of Nfatc3 -KI fl/fl and Nfatc3 smcKI mice and treated with PDGF-BB (20 μg/L) for 24 h before a 12-h CHX (50 μmol/L) treatment ( n = 4). (I) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. Western blot of RPL28 and RPS6 levels. Data are presented as mean ± SD. (E) Unpaired Student’s t -test; two-tailed P -values. (G) Two-way ANOVA with Tukey’s correction; adjusted P -values. (H) One-way ANOVA with Tukey’s correction; adjusted P -values.

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Expressing, Immunofluorescence, Staining, Isolation, Liquid Chromatography with Mass Spectroscopy, Gradient Centrifugation, Western Blot, Immunoprecipitation, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: NFATc3 facilitates global protein synthesis by suppressing eEF2 phosphorylation. (A) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. The eEF2 level was determined using Western blot. (B) HEK293T cells were co-transfected with His-eEF2 and Flag-NFATc3 plasmids. Cell lysates were immunoprecipitated with anti-Flag antibodies, and the precipitates were analyzed using immunoblotting with anti-His antibodies. (C) Immunofluorescence for eEF2 and NFATc3 detection in MASMCs. (D) Nfatc3 fl/fl and Nfatc3 smcKO mice were treated with or without BAPN for 28 days. Western blot and quantification of p-eEF2 and eEF2 in aortic tissues ( n = 4). (E) Western blots of binding assays show the binding preference of NFATc3. HEK293T cells were transfected with eEF2-T56D and eEF2-T56A plasmids for 48 h, and lysates were immunoprecipitated with anti-NFATc3 antibodies ( n = 4). (F) Schematic illustration of NFATc3 structures and the presence/absence of binding between NFATc3 and eEF2 are indicated by + or – (top panel). Full-length His-eEF2 and various truncated Flag-NFATc3 forms were co-expressed in 293T cells, and immunoprecipitation with anti-Flag antibodies followed by Western blot (bottom panel) was performed. (G) Western blots and quantification of p-eEF2 and eEF2 in MASMCs transfected with vector, Plasmid-Nfatc3, or Plasmid-Nfatc3-Mutant before a 24-h PDGF-BB treatment ( n = 4). (H) Representative images and quantification of eEF2 and ACTA2, p-eEF2, and ACTA2 expression using immunofluorescence staining in the ascending aortas of patients without TAAD and patients with TAAD ( n = 8). (I) Relative eEF2 and p-eEF2 protein levels in the aortas of mice administered BAPN/AngII and AAV- Pcsk9 DY /AngII ( n = 6). (J) Western blot of puromycin incorporation assays and p-eEF2/eEF2 levels from MASMCs isolated from Nfatc3 fl/fl and Nfatc3 smcKO mice and treated with A484954 (10 μmol/L, 10 min) ( n = 4). Data are presented as mean ± SD. (D) Two-way ANOVA with Tukey’s correction; adjusted P -values. (E, H) Unpaired Student’s t -test; two-tailed P -values. (G, I, J) One-way ANOVA followed by Tukey’s correction; adjusted P -values. NS, no significance ( P > 0.05).

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Transfection, Immunofluorescence, Binding Assay, Plasmid Preparation, Mutagenesis, Expressing, Staining, Isolation, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: Supplementation with the eEF2 inhibitor cabamiquine mitigates the deleterious effects of NFATc3 overexpression on AAD development. (A) BAPN model establishment. (B–D) Four-week-old male Nfatc3 smcKO mice were treated with Lenti-Con or Lenti eEF2; 7 days after transfection, five-week-old mice were treated with vehicle or BAPN (0.6 g/kg/day) for 28 days ( n = 13). (B) aorta macrographs. (C) AAD incidence. (D)The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (E–K) Five-week-old male Nfatc3 smcKI mice were treated with BAPN (0.6 g/kg/day) for 28 days. Saline and cabamiquine (3 mg/kg/day) were administered orally for 28 days ( n = 15). (E) Aorta macrographs. (F) AAD incidence. (G) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (H) Maximum aortic diameter. (I) H&E, van Geison (elastin), and Masson aorta staining. (J, K) Elastin degradation grade (J) and collagen content (K) in the aortic wall. (H–K) n = 5 for saline, n = 11 for cabamiquine. (L–O) Five-week-old Nfatc3 smcKI male mice were administered BAPN (0.3 g/kg/day) for 28 days before being infused with AngII (1000 ng/kg/min) for 3 days; saline and cabamiquine (3 mg/kg/day) were administered orally for 31 days ( n = 17 for saline, n = 18 for cabamiquine). (L) Aorta macrographs. (M) AAD incidence. (N) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (O) Maximum aortic diameter (saline: n = 6; cabamiquine: n = 12). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (H, K, O) Unpaired Student’s t -test; two-tailed P -values. (J) Mann–Whitney U -test with the exact method; two-tailed P -values.

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Over Expression, Transfection, Saline, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Carcinogenesis

Article Title: Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression

doi: 10.4103/1477-3163.112268

Figure Lengend Snippet: Influence of Oleandrin (I, 100nM) and digitoxin (II, 500 nM) on recombinant nuclear factor of activated T-cells 1-4 driven NFAT-luciferase - fusion expression. (a) Inhibition of nuclear factor of activated T-cells (NFAT)-driven luciferase expression by cardiac glycosides. Cells were treated with oleandrin (I, 100 nM) or digitoxin (II, 500 nM) in the presence of NFAT luciferase fusion construct and expression plasmids for NFAT1, 2, 3 or 4 for 24 h. Both drugs are effective inhibitors at these maximal concentrations. The luciferase fusion construct contains multimers of the NFAT binding site from IL-2. Data are the average of triplicate assays. Differences from respective controls are significant at (* P < 0.05). (b) Expression levels of recombinant nuclear factor of activated T-cells (NFAT) (1-4) following transfection. HeLa cells were transfected overnight with empty vector, or vector expressing NFAT1, NFAT2, NFAT3 or NFAT4. Cells were then harvested, and cultures individually analysed by western blot with antibodies against NFAT1, NFAT2, NFAT3 and NFAT4, respectively. Each cell culture listed under “Transfection” contains various amount of endogenous NFAT (1-4). Therefore, the different amounts of NFAT (1-4) detected in each culture, respectively, are indicated along the respective NFAT (1-4)-specific rows. Levels of recombinant NFAT (1-4) expression clearly depend on transfection efficiency, promoter efficiency and conditions of the cells. However, in all cases levels of recombinant protein are greater than endogenous levels

Article Snippet: Antibodies were as follows: (i) NFAT1 (NF-ATc2, NF-ATp: Mouse monoclonal antibody from BD Pharmingen); (ii) NFAT2 (NF-ATc1, NF-ATc: Mouse monoclonal antibody from BD Pharmingen); (iii) NFAT3 (NFATc4: Rabbit monoclonal antibody from Cell Signaling); (iv) NFAT4 (NFATc3, NFATx: Rabbit monoclonal antibody from Cell Signaling).

Techniques: Recombinant, Luciferase, Expressing, Inhibition, Construct, Binding Assay, Transfection, Plasmid Preparation, Western Blot, Cell Culture

Journal: Journal of Carcinogenesis

Article Title: Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression

doi: 10.4103/1477-3163.112268

Figure Lengend Snippet: Titration of cardiac glycoside effects on nuclear factor of activated T-cells (NFAT)-driven luciferase expression, in the presence of recombinant NFAT isoforms (1-4). (a) Influence of cardiac glycosides on nuclear factor of activated T-cells (NFAT) 1 and NFAT3-driven transcription of NFAT-luciferase expression. Cells were treated for 10h with oleandrin (I: 10, 30, 50, 70, 100 nM) or digitoxin (II: 25, 50, 100, 300, 500 nM), in the presence of an NFAT luciferase fusion construct and expression plasmids for NFAT1 and NFAT3, for 24 h. The fusion construct contained multimers of the NFAT binding site from IL-2. Luciferase levels were measured and plotted. Data are the averages of triplicate assays. Differences from control for all doses of either oleandrin (I) or digitoxin (II) are significant at * P < 0.05. (b) Influence of cardiac glycosides on NFAT2 and NFAT4-driven transcription of NFAT-luciferase expression. Cells were treated for 10 h with oleandrin (I: 10, 30, 50, 70, 100 nM) or digitoxin (II: 25, 50, 100, 300, 500 nM), in the presence of an NFAT luciferase fusion construct, and expression plasmids for NFAT2 and NFAT4, for 24 h. Luciferase levels were measured and plotted. Data are the averages of triplicate assays. Differences from control for all doses of either oleandrin (I) or digitoxin (II) are significant at * P < 0.05. (c) IC50 values for high affinity cardiac glycoside inhibition of nuclear factor of activated T-cells NFAT (1-4) driven transcription of NFAT-luciferase expression. Summary data computed from are displayed

Article Snippet: Antibodies were as follows: (i) NFAT1 (NF-ATc2, NF-ATp: Mouse monoclonal antibody from BD Pharmingen); (ii) NFAT2 (NF-ATc1, NF-ATc: Mouse monoclonal antibody from BD Pharmingen); (iii) NFAT3 (NFATc4: Rabbit monoclonal antibody from Cell Signaling); (iv) NFAT4 (NFATc3, NFATx: Rabbit monoclonal antibody from Cell Signaling).

Techniques: Titration, Luciferase, Expressing, Recombinant, Construct, Binding Assay, Control, Inhibition

Journal:

Article Title: Functional antagonism between Helicobacter pylori CagA and vacuolating toxin VacA in control of the NFAT signaling pathway in gastric epithelial cells

doi: 10.1073/pnas.0502529102

Figure Lengend Snippet: CagA activates NFAT-dependent genes. (A) AGS cells were cotransfected with pNFAT-luc and WT-CagA or PR-CagA expression vector. Cell lysates were subjected to luciferase assay (Left) or immunoblotting (Right). The promoter activation was shown as a ratio of the luciferase activities in cells transfected with control empty vector (mean ± SD, n = 3 experiments). (B) AGS cells were transfected with WT-CagA expression vector for 18 h or infected with cagA-positive H. pylori strain NCTC11637 or its cagA-negative isogenic strain for 5 h at a multiplicity of infection of 50. Total cell lysates were subjected to immunoblotting. (C) AGS cells were cotransfected with pGL3-p21-luc and WT-CagA or PR-CagA expression vector. The promoter activation was shown as a ratio of the luciferase activities in cells transfected with control vector (mean ± SD, n = 3 experiments). (D) AGS cells were transfected with WT-CagA, PR-CagA, or control empty vector. Total cell lysates were subjected to immunoblotting. (E) AGS cells were transfected with EGFP, EGFP-tagged WT-CagA, or EGFP-tagged PR-CagA expression vector. After 36 h, cells were harvested, and GFP-positive cells were subjected to the cell-cycle analysis.

Article Snippet: Anti-hemagglutinin-epitope polyclonal antibody (Y-11), anti-SHP-2 polyclonal antibody (C-18), anti-p21 WAF1/Cip1 polyclonal antibody (C-19), and anti-NFATc3 monoclonal antibody (F-1) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Plasmid Preparation, Luciferase, Western Blot, Activation Assay, Transfection, Control, Infection, Cell Cycle Assay

Journal:

Article Title: Functional antagonism between Helicobacter pylori CagA and vacuolating toxin VacA in control of the NFAT signaling pathway in gastric epithelial cells

doi: 10.1073/pnas.0502529102

Figure Lengend Snippet: Effect of VacA on CagA-dependent NFAT activation. (A) AGS cells were treated with PMA (33 nM) plus calcium ionophore A23187 (Io) (0.5 μM) for 30 min in the presence or absence of 2.5 μg/ml VacA. Cell lysates were immunoblotted with anti-NFATc3 antibody. p-NFATc3 indicates the phosphorylated form of NFATc3. (B) AGS cells cotransfected with WT-CagA expression vector and pNFAT-luc (Left) or pGL3-p21-luc (Right) were cultured in the presence or absence of VacA. The promoter activation was shown as a ratio of the luciferase activities in cells transfected with control vector (mean ± SD, n = 3 experiments). (C) AGS cells transfected with WT-CagA or control empty vector were cultured in the presence or absence of VacA. Cells were stained with anti-NFATc3. (D) AGS cells transfected with WT-CagA expression vector or control empty vector were cultured in the presence or absence of VacA. Cell lysates were subjected to immunoblotting. (E) AGS cells were transfected with WT-CagA expression vector in the presence or absence of VacA. After 18 h, cell morphology was examined by microscopy. Relative hummingbird induction was shown as the percentage ratio of the number of hummingbird cells induced by WT-CagA in the absence of VacA (mean ± SD, n = 3 experiments). (F) AGS cells were mock-infected or infected with H. pylori strain F29 or F57 at a multiplicity of infection of 50. After 5 h, cells were stained with anti-NFATc3 antibody and DAPI.

Article Snippet: Anti-hemagglutinin-epitope polyclonal antibody (Y-11), anti-SHP-2 polyclonal antibody (C-18), anti-p21 WAF1/Cip1 polyclonal antibody (C-19), and anti-NFATc3 monoclonal antibody (F-1) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Cell Culture, Luciferase, Transfection, Control, Staining, Western Blot, Microscopy, Infection

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo–activated ERK1/2 is critical for the promyogenic function of Stim1

doi: 10.1091/mbc.e11-07-0634

Figure Lengend Snippet: FIGURE 1: Stim1 promotes myotube formation, and its expression is impaired in Cdo−/− developing muscles and differentiating myoblasts. (A) Lysates of C2C12 cells cultured at near confluence in growth medium (D0) or in differentiation medium (DM) for indicated times were immunoblotted with indicated antibodies. (B) C2C12 cells were stably transfected with the control or Stim1 shRNA expression vector, and cell lysates were Western blotted with the indicated antibodies. Depleted protein and β-tubulin loading control signals were quantified by densitometry; ratio is reported under each lane in arbitrary units, with control transfectants set to 1. (C) Control and Stim1 shRNA2-expressing cells were induced to differentiate for 3 d, followed by immunostaining with an antibody to MHC to analyze myotube formation. (D) Quantification of myotube formation shown in C. Values represent means ± SEM from three independent experiments with triple determinations (n = 3). *p < 0.01. (E) C2C12 myoblasts were stably transfected with pcDNA or Stim1 expression vectors and analyzed by immunoblotting. Overexpressed protein and β-tubulin loading control signals were quantified by densitometry; ratio is reported under each lane in arbitrary units, with control transfectants set to 1. (F) Cells shown in E were cultured in DM for 48 h and immunostained with MHC antibodies. (G) Quantification of myotube formation in F. Values represent means ± SEM from three independent experiments with triple determinations (n = 3). *p < 0.005. (H) Extracts of hindlimb muscles from postnatal day 1 (P1) and P5 mice were immunoblotted with indicated antibodies. Stim1 and β-tubulin loading control signals were quantified by densitometry; ratio is reported under each lane at the bottom in arbitrary units, with extracts from wild-type hindlimb muscles at P1 set to 1. (I) Lysates of Cdo+/+ and Cdo−/− primary myoblasts at D0 or cultured under differentiation conditions for 1 or 2 d were immunoblotted with indicated antibodies. (J) Lysates of Cdo+/+ and Cdo−/− primary myoblasts cultured under similar conditions were immunoblotted with antibodies to phosphorylated NFATc3 (an inactive form, p-NFATc3), NFATc3, and β-tubulin as a loading control. p-NFATc3 and NFATc3 signals, as well as p-NFATc3 and β-tubulin loading control signals, were quantified by densitometry; ratio is reported arbitrary units, with signals from Cdo+/+ myoblasts at D0 set to 1. (K) RNAs isolated from Cdo+/+ and Cdo−/− myoblasts at various differentiation time points were analyzed by RT-PCR. Stim1 and control glyceraldehyde-3-phosphate dehydrogenase signals were quantified by densitometry, and signals from Cdo+/+ myoblasts at D0 set to 1.

Article Snippet: Primary antibodies used were as follows: Cdo (R&D Systems, Minneapolis, MN, and Zymed, San Francisco, CA), Stim1 (ProSci, Poway, CA, and Sigma-Aldrich, St. Louis, MO), Cadherin (Sigma-Aldrich), β-tubulin (Zymed), MHC (MF-20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), myogenin (Santa Cruz Biotechnology, Santa Cruz, CA), EB1 (Santa Cruz Biotechnology), TRPC1 (Santa Cruz Biotechnology), ERK1/2, pERK1/2 (Sigma-Aldrich), p-NFATc3, NFATc3 (Sigma-Aldrich), and SRT (Lee et al., 2001).

Techniques: Expressing, Muscles, Cell Culture, Stable Transfection, Transfection, Control, shRNA, Plasmid Preparation, Western Blot, Immunostaining, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo–activated ERK1/2 is critical for the promyogenic function of Stim1

doi: 10.1091/mbc.e11-07-0634

Figure Lengend Snippet: FIGURE 2: Forced expression of calcineurin restores differentiation of Cdo-depleted C2C12 cells. (A) Lysates of C2C12 cells transfected with a constitutively active form of Calcineurin (Calcineurin*) or control (−) expression vector were subjected to immunoblotting. (B) Lysates of C2C12 cells stably transfected with pSuper or Cdo shRNA were subjected to immunoblotting with antibodies to Cdo and β-tubulin. Cdo and the loading control β-tubulin signals were quantified by densitometry. (C) C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or calcineurin* expression vectors plus a GFP expression vector to mark transfectants. Cells at DM3 were immunostained for MHC (red) and visualized for GFP expression, followed by DAPI staining (blue). (D) Quantification of myotube formation of three independent experiments shown in C. Cultures were scored as MHC negative or MHC positive, with MHC-positive cells further scored as having a single nucleus, two to five nuclei, or six or more nuclei. Values represent means ± SEM from three independent experiments with triple determinations (n = 3). *p < 0.005. (E) Lysates of C2C12/ pSuper and C2C12/Cdo shRNA transfected with pcDNA or calcineurin* expression vectors at DM2 were subjected to immunoblotting with antibodies to MHC, Stim1, and β-tubulin.

Article Snippet: Primary antibodies used were as follows: Cdo (R&D Systems, Minneapolis, MN, and Zymed, San Francisco, CA), Stim1 (ProSci, Poway, CA, and Sigma-Aldrich, St. Louis, MO), Cadherin (Sigma-Aldrich), β-tubulin (Zymed), MHC (MF-20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), myogenin (Santa Cruz Biotechnology, Santa Cruz, CA), EB1 (Santa Cruz Biotechnology), TRPC1 (Santa Cruz Biotechnology), ERK1/2, pERK1/2 (Sigma-Aldrich), p-NFATc3, NFATc3 (Sigma-Aldrich), and SRT (Lee et al., 2001).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Stable Transfection, shRNA, Staining

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo–activated ERK1/2 is critical for the promyogenic function of Stim1

doi: 10.1091/mbc.e11-07-0634

Figure Lengend Snippet: FIGURE 3: Cdo and Stim1 form complexes in 293T cells and differentiating C2C12 cells, and netrin-2 enhances this interaction. (A) 293T cells were transfected with SRT-tagged Stim1, Cdo, or control (−) expression vectors, and 48 h later, lysates were immunoprecipitated with Cdo antibodies and immunoblotted with antibodies for SRT or Cdo. (B) Lysates of 293T cells transfected with indicated vectors were subjected to immunoprecipitation with an antibody to a SRT tag and immunoblotting with antibodies for SRT or Cdo. (C) Lysates of C2C12 cell cultures from G, DM1, or DM2 were immunoprecipitated with Stim1 antibodies and immunoblotted with antibodies for Cdo or Stim1 (left). Lysates of C2C12 cells at DM1 were immunoprecipitated with control rabbit IgG or Stim1 antibodies and immunoblotted with Cdo and Stim1 (right). (D) Lysates of C2C12 cell cultures from G, DM0, or DM1 were immunoprecipitated with Cdo antibodies and immunoblotted with antibodies for Cdo or Stim1 (left). As control, lysates of C2C12 cells at DM1 were immunoprecipitated with control rabbit IgG or Cdo antibodies and immunoblotted with Cdo and Stim1 (right). (E) Confluent C2C12 myoblasts were incubated for 8 h in DM and treated with netrin-2 for indicated times. Lysates were subjected to immunoprecipitation with Cdo antibodies and immunoblotting with indicated antibodies. p-NFATc3 and NFATc3 signals were quantified by densitometry. The signal ratio of C2C12 cells without netrin-2 was set to 1 (left). As control, lysates of C2C12 cells treated with netrin-2 for 60 min were immunoprecipitated with control rabbit IgG or Cdo antibodies and immunoblotted with Cdo and Stim1 (right). (F) Control (top; the red boxed areas in a–c are shown in a′–c′) and Cdo-depleted (bottom; the red boxed areas in d–e are shown in d′–e′) C2C12 cells transfected with Stim1-RFP were treated with netrin-2 for the indicated times, and live confocal images were taken. The TG (1 μM) treatment of the Cdo-depleted cell serves as the positive control.

Article Snippet: Primary antibodies used were as follows: Cdo (R&D Systems, Minneapolis, MN, and Zymed, San Francisco, CA), Stim1 (ProSci, Poway, CA, and Sigma-Aldrich, St. Louis, MO), Cadherin (Sigma-Aldrich), β-tubulin (Zymed), MHC (MF-20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), myogenin (Santa Cruz Biotechnology, Santa Cruz, CA), EB1 (Santa Cruz Biotechnology), TRPC1 (Santa Cruz Biotechnology), ERK1/2, pERK1/2 (Sigma-Aldrich), p-NFATc3, NFATc3 (Sigma-Aldrich), and SRT (Lee et al., 2001).

Techniques: Transfection, Control, Expressing, Immunoprecipitation, Western Blot, Incubation, Positive Control

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo–activated ERK1/2 is critical for the promyogenic function of Stim1

doi: 10.1091/mbc.e11-07-0634

Figure Lengend Snippet: FIGURE 4: Netrin-2 induces phosphorylation of Stim1 at serine 575 via ERK activation. (A) The consensus ERK phosphorylation motif and a potential phosphorylation site at serine 575. (B) In vitro phosphorylation of the putative ERK phosphorylation site of Stim1 by ERK. The wild-type GST-Stim1 encompassing amino acids 542–607 or the alanine mutant form of GST-Stim1 at serine 575 (S575A) was used as substrate, and phosphorylation was measured by 32P incorporation. GST serves as a negative control. The substrate levels were determined using fast green staining. p-GST-STIM1 and GST-STIM1 signals were quantified by densitometry. The signal ratio of GST-STIM1WT was set to 1. (C) 293T cells transfected with Stim1 were treated with PD98059 for 1 h and analyzed by immunoblotting. Signals of p-NFATc3, NFATc3, p575-Stim1, Stim1, and β-tubulin were quantified by densitometry, and the ratio from control transfectants was set to 1. (D) Lysates of 293T cells cotransfected with control or Stim1 expression vector plus a scrambled siRNA or ERK2 siRNA were subjected to immunoblotting with antibodies to p-NFATc3, NFATc3, pS575-Stim1, Stim1, ERK2, and β-tubulin as a loading control. Signals of p-NFATc3, NFATc3, ERK2, and β-tubulin were quantified by densitometry; ratios are reported under each lane at the bottom in arbitrary units, with control transfectants set to 1. (E) Lysates from C2C12 cells treated with netrin-2 for indicated times were immunoblotted with antibodies to p-ERK1/2 or ERK1/2. (F) C2C12 cells were incubated for 8 h in DM and treated with netrin-2 for 1 h. For the treatment with PD98059, cells were pretreated with PD98059 for 1 h, prior to the netrin treatment. Lysates were immunoblotted with indicated antibodies. Signals of p-NFATc3, NFATc3, and β-tubulin were quantified by densitometry, and the ratio from the control transfectant was set to 1. (G) 293T cells transfected with Stim1 were treated with SB203580 for 1 h and analyzed by immunoblotting with antibodies to pS575-Stim1, Stim1, and β-tubulin as a loading control. Signals of pS575-Stim1 and Stim1 were quantified by densitometry, and the ratio from the control transfectant was set to 1.

Article Snippet: Primary antibodies used were as follows: Cdo (R&D Systems, Minneapolis, MN, and Zymed, San Francisco, CA), Stim1 (ProSci, Poway, CA, and Sigma-Aldrich, St. Louis, MO), Cadherin (Sigma-Aldrich), β-tubulin (Zymed), MHC (MF-20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), myogenin (Santa Cruz Biotechnology, Santa Cruz, CA), EB1 (Santa Cruz Biotechnology), TRPC1 (Santa Cruz Biotechnology), ERK1/2, pERK1/2 (Sigma-Aldrich), p-NFATc3, NFATc3 (Sigma-Aldrich), and SRT (Lee et al., 2001).

Techniques: Phospho-proteomics, Activation Assay, In Vitro, Mutagenesis, Negative Control, Staining, Transfection, Western Blot, Control, Expressing, Plasmid Preparation, Incubation

Journal: Molecular Biology of the Cell

Article Title: Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo–activated ERK1/2 is critical for the promyogenic function of Stim1

doi: 10.1091/mbc.e11-07-0634

Figure Lengend Snippet: FIGURE 5: Phosphorylation of Stim1 at serine 575 is required for the full promyogenic activity of Stim1. (A) Lysates of C2C12 cells treated with netrin-2 for indicated times were immunoprecipitated with a Stim1 antibody followed by immunoblotting with antibodies to pS575-Stim1 or Stim1. (B) Lysates of C2C12 cells at DM0 and DM2 are subjected to immunoprecipitation with Stim1 antibodies and immunoblotted with antibodies to ERK1/2 and Stim1 (left). As control, lysates of C2C12 cells at DM2 are immunoprecipitated with the control rabbit IgG or Stim1 antibodies, followed by immunoblotting (right). (C) 293T cells were transiently transfected with indicated expression vectors, followed by immunoprecipitation and immunoblotting. (D) Lysates from C2C12 cells at DM0, DM1, or DM2 were analyzed for the phosphorylation status of Stim1 at serine 575 and ERK1/2 by immunoblotting. Signals of p-Stim1 and Stim1 were quantified by densitometry, and the ratio from C2C12 cells at D0 was set to 1. (E) C2C12/pSuper and C2C12/Stim1 shRNA cells were transiently cotransfected with indicated expression vectors, and 48 h later, cells were analyzed by immunoblotting with antibodies to Stim1 and β-tubulin as a loading control. Stim1 and β-tubulin signals were quantified by densitometry; ratios are reported under each lane in arbitrary units, with control transfectants set to 1. (F) C2C12/ Stim1 shRNA cells in E were induced to differentiate for 3 d and immunostained with GFP antibodies, followed by DAPI staining. (G) Quantification of myotube formation of three independent experiments shown in F. Cultures were scored as GFP-positive cells having one or two nuclei, three to six nuclei, or seven or more nuclei. Values represent means ± SEM from three independent experiments with triple determinations (n = 3). *p < 0.01.

Article Snippet: Primary antibodies used were as follows: Cdo (R&D Systems, Minneapolis, MN, and Zymed, San Francisco, CA), Stim1 (ProSci, Poway, CA, and Sigma-Aldrich, St. Louis, MO), Cadherin (Sigma-Aldrich), β-tubulin (Zymed), MHC (MF-20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), myogenin (Santa Cruz Biotechnology, Santa Cruz, CA), EB1 (Santa Cruz Biotechnology), TRPC1 (Santa Cruz Biotechnology), ERK1/2, pERK1/2 (Sigma-Aldrich), p-NFATc3, NFATc3 (Sigma-Aldrich), and SRT (Lee et al., 2001).

Techniques: Phospho-proteomics, Activity Assay, Immunoprecipitation, Western Blot, Control, Transfection, Expressing, shRNA, Staining