plasmid encoding human nfatc3  (Addgene inc)

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