Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: Knockdown of NFATc3-attenuated PMA/Io induced REDD1 expression in HT29 cells. (A) HT29 cells were transfected with control siRNA or siRNA specifically targeting NFATc1, c2, c3, or c4. After a 46-h incubation, transfected cells were treated with PMA (100 nM) plus Io (2.5 μM) for an additional 1.5 h. Cells were lysed, and Western blot analysis was performed using antibodies against REDD1 and β-actin. (B) HT29 cells were transfected with control siRNA or siRNA targeting NFATc1, c2, c3, or c4. After a 46-h incubation, total RNA was extracted, and real-time RT-PCR was performed for analysis of NFATc1, NFATc2, NFATc3, and NFATc4 mRNA expression. (Data represent mean ± SD; * p < 0.01 vs control as determined by two-sample t tests.) (C) HT29 cells were transfected with control siRNA or siRNA targeting NFATc1, c2, c3, or c4. After a 46-h incubation, total protein was extracted, and Western blotting was performed for analysis of NFATc1, NFATc2, NFATc3, and NFATc4 protein expression.

Article Snippet: The plasmid encoding human NFATc3 was from Addgene (Cambridge, MA).

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

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3 regulated REDD1 expression in HT29, Caco-2, HCT116, and SW480 cells. (A) HT29 cells were transfected with control vector or NFATc3 (left) or control siRNA or siRNA targeting NFATc3 (right). After a 48-h incubation, REDD1, NFATc3, β-actin, phospho-mTOR (pS2448), mTOR, phospho-S6 (pS235/236), and S6 expression was determined by Western blotting. (B and C) HT29 cells were transfected with control vector or NFATc3 plasmid (B) or transfected with control siRNA or siRNA targeting NFATc3 (C). After a 48-h incubation, total RNA was extracted and REDD1 and NFATc3 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; * p < 0.01 vs. control as determined by two-sample t tests.) (D and E) Caco-2, HCT116, and SW480 cells were transfected with control vector or NFATc3 (D) or transfected with control siRNA or siRNA targeting NFATc3 (E). After a 48-h incubation, REDD1, NFATc3, β-actin, phospho-S6 (pS235/236), and S6 expression was determined by Western blotting.

Article Snippet: The plasmid encoding human NFATc3 was from Addgene (Cambridge, MA).

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

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3 is a transcriptional activator of REDD1 in HT29 cells. (A and B) HT29 cells were transfected with a construct containing REDD1 promoter sequence (−2931/−97) alone (A) or together with either control plasmid or an NFATc3 plasmid (B). At 43-h posttransfection, cells were treated with or without PMA/Io for 5 h. Cells were harvested, and luciferase activity was assayed. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). (Data shown are mean ± SD; * p < 0.05, PMA/Io vs. control, or NFATc3 alone or PMA/Io plus vector vs. vector alone; #, p < 0.05, PMA/Io plus CsA vs. control or PMA/Io plus NFATc3 vs. NFATc3 alone, as determined by ANOVA.) (C) HT29 cells were subjected to ChIP assay; soluble chromatin was prepared from HT29 cells treated with PMA/Io for 1 h and immunoprecipitated with NFATc3 antibody or IgG. Total (Input) and immunoprecipitated DNAs or H 2 O (negative control) were then PCR-amplified using primer pairs covering −890/−97 within the human REDD1 promoter.

Article Snippet: The plasmid encoding human NFATc3 was from Addgene (Cambridge, MA).

Techniques: Transfection, Construct, Sequencing, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Negative Control, Amplification

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3/REDD1 regulation of c-Myc expression. (A) Total protein from HT29 cells stably transfected with control or REDD1 shRNA was extracted and analyzed by Western blotting using anti-REDD1, anti-S6, anti–phospho-S6 (pS235/236), and anti–β-actin antibodies. (B) HT29 cells were transfected with control siRNA or siRNA targeting NFATc3. After a 46.5-h incubation, transfected cells were treated with PMA (100 nM) plus Io (2.5 μM) for an additional 1.5 h. Cells were lysed, and Western blot analysis was performed using antibodies against c-Myc, REDD1, NFATc3, and β-actin. (C) Caco-2 and HCT116 cells were transfected with control vector or NFATc3 plasmid. After a 48-h incubation, c-Myc and NFATc3 expression was determined by Western blotting. β-actin was blotted to confirm equal loading.

Article Snippet: The plasmid encoding human NFATc3 was from Addgene (Cambridge, MA).

Techniques: Expressing, Stable Transfection, Transfection, shRNA, Western Blot, Incubation, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells

doi: 10.1091/mbc.E12-01-0037

Figure Lengend Snippet: NFATc3 and REDD1 regulation of MUC2 mRNA expression. (A) HT29 cells were pretreated with CsA for 30 min; this was followed by treatment with the combination of PMA/Io for 1.5 h. (B) HT29 cells were transfected with control siRNA or siRNA targeting NFATc3 (left panel) or transfected with control vector or NFATc3 plasmid (right panel). Transfected cells were incubated for 48 h. (C) HT29 cells were transfected with control siRNA or siRNA targeting NFATc3. After a 46-h incubation, transfected cells were treated with PMA/Io for an additional 1.5 h. (D) HT29 cells, stably transfected with control or REDD1 shRNA, were treated with PMA/Io for 1.5 h. (E) HT29 cells were transfected with control siRNA or siRNA targeting TSC2 and incubated for 48 h. Total RNA was extracted, and MUC2 mRNA levels were determined by real-time RT-PCR (A–E). (Data represent mean ± SD; * p < 0.01 vs. control or control siRNA or control shRNA or vector alone; # p < 0.01 vs. PMA/Io alone as determined by ANOVA.) Total protein was extracted and subjected to Western blotting using anti-TSC2, anti–phospho-S6 (pS235/236), anti-S6, and anti–β-actin antibodies (E).

Article Snippet: The plasmid encoding human NFATc3 was from Addgene (Cambridge, MA).

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Stable Transfection, shRNA, Quantitative RT-PCR, Western Blot

Journal: Neurology: Genetics

Article Title: Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D

doi: 10.1212/NXG.0000000000000318

Figure Lengend Snippet: (A) C2C12 WT and DNAJB6 KO myoblasts differentiated into myotubes for 6 days and stained for myosin. DNAJB6 KO myoblasts fused to form large myotubes with enhanced fusion index (B). Error bars represent the standard error of 3 independent experiments. (C) Western blot confirmed increased inactive GSK3β-P(ser-9) in KO myoblasts. GSK3β-P(ser-9)/GSK3β-total ratio is given below western blot. (D) Increased β-catenin transcriptional activity in DNAJB6 KO myoblasts compared with WT myoblasts measured by TOPFLASH luciferase assay. (E) Increased NFATc3 transcriptional activity in DNAJB6 KO myoblasts compared with WT myoblasts. Error bars represent the standard error of at least 2 independent experiments. GSK3β = glycogen synthase kinase-β; KO = knockout; NFATc3 = nuclear factor of activated T cells cytoplasmic 3; WT = wild type.

Article Snippet: For NFATc3 luciferase experiments, HeLa cells were transfected with 0.5 μg of plasmid encoding NFATc3 to stimulate transcriptional activity (pBS mNFATc3-EE, Addgene plasmid #17868).

Techniques: Staining, Western Blot, Activity Assay, Luciferase, Knock-Out

Journal: Neurology: Genetics

Article Title: Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D

doi: 10.1212/NXG.0000000000000318

Figure Lengend Snippet: (A) Western blot of skeletal muscle lysates from 3-month old LGMD1D mutant mice (F93Lb, n = 2) vs control (WTb, n = 2) demonstrating significantly reduced inactive GSK3β-P(ser-9) in mutant mice. Error bars represent the standard error. (B) Quantitation of western blot. Dual-luciferase assay demonstrating reduced β-catenin (C) and NFATc3 (D) transcriptional activity in HeLa cells transfected with mutant DNAJB6b constructs compared with unstimulated controls. β-catenin transcriptional activity was stimulated via treatment with 20 mM LiCl for 12 hours. NFATc3 transcriptional activity was stimulated by overexpression of NFATc3 via transient transection. Error bars in C and D represent the standard error from 3 separate experiments. (E) Proposed model for DNAJB6's spectrum of impact on GSK3β activation and myogenesis. GSK3β = glycogen synthase kinase-β; LGMD1D = limb-girdle muscular dystrophy 1D; NFATc3 = nuclear factor of activated T cells cytoplasmic 3; WT = wild type.

Article Snippet: For NFATc3 luciferase experiments, HeLa cells were transfected with 0.5 μg of plasmid encoding NFATc3 to stimulate transcriptional activity (pBS mNFATc3-EE, Addgene plasmid #17868).

Techniques: Western Blot, Mutagenesis, Control, Quantitation Assay, Luciferase, Activity Assay, Transfection, Construct, Over Expression, Activation Assay

Journal: Nature Communications

Article Title: Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration

doi: 10.1038/ncomms10871

Figure Lengend Snippet: ( a ) Luciferase assay in primary myoblasts transfected with the stabilin-2 promoter construct. At 24 h post-transfection, cells were incubated with vehicle, calcium ionophore A23187 (CI, 1 μM), or CI plus cyclosporine A (CsA, 1 μM) for 24 h. ( b ) Luciferase assay in primary myoblasts transfected with the stabilin-2 promoter construct along with plasmid encoding activated calcineurin (aCnA) and/or GFP-VIVIT. ( c ) Luciferase assay in primary myoblasts transfected with stabilin-2 promoter construct along with the indicated NFAT expression vector. ( d ) Luciferase assay in primary myoblasts transfected with the stabilin-2 promoter construct along with the indicated amount of plasmid encoding NFATc1. ( e ) Real-time PCR analysis of Stab2 mRNA in primary myoblasts infected with retrovirus encoding NFATc1 or retrovirus from pMXs IRES-puro vector. ( f ) Luciferase assay in primary myoblasts transfected the stabilin-2 promoter construct or a series of 5' deletion constructs along wth plasmid encoding NFATc1 or empty vector (Con). ( g ) Luciferase assay in primary myoblasts transfected with the stabilin-2 promoter construct (nt −482 to +205) or its NFAT mutant along with plasmid encoding NFATc1. ( h ) Soluble chromatin was prepared from human myoblasts (DM1) for ChIP assays and immunoprecipitated with anti-NFATc1 antibody. An isotype-matched control antibody was used as the negative control. Immunoprecipitates were subjected to PCR with primers specific to the NFAT-responsive element in stabilin-2 promoter. GAPDH primers were used as a negative control. The result shown is a representative of three independent experiments. ( i ) Representative immunoblot of NFATc1 in primary myoblasts infected with retrovirus encoding NFATc1 shRNA or retrovirus from pMXs-U6 vector. ( j – l ) Primary myoblasts infected with retrovirus encoding NFATc1 shRNA (shNFATc1) or retrovirus from pMXs-U6 vector (Control) were induced to differentiate for 2 days. Expression of stabilin-2 mRNA was analysed by quantitative real-time PCR ( j ). Representative images in DM2 ( k ) are shown. Scale bars, 100 μm. Fusion indices ( l ) were calculated. Relative luciferase activities were normalized as fold over that of the stabilin-2 promoter in the absence of NFATc1. Data are presented as mean±s.d. of three independent experiments. Asterisks indicate statistical significance (* P <0.05, Student's t -test).

Article Snippet: Cho (Seoul National University), and plasmids encoding NFATc1 and NFATc3 were purchased from Origene.

Techniques: Luciferase, Transfection, Construct, Incubation, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Infection, Mutagenesis, Immunoprecipitation, Negative Control, Western Blot, shRNA