pcmv5 flag smad3 s3  (Addgene inc)

Journal: Heliyon

Article Title: TGF-β/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells

doi: 10.1016/j.heliyon.2023.e21519

Figure Lengend Snippet: The expression of TGF-β ligands, SMADs, and TAZ are increased in human liver cancer. (A) Heat map for gene expression of TAZ/WWTR1, TGF-β pathway components (TGF-β1, TGF-β2, TGF-β3, SMAD2, SMAD3, and SMAD4), and EMT-markers (SNAI1, SNAI2, and VIMENTIN) was obtained analyzing LIHC dataset from TCGA, using UALCAN. The mRNA levels (TPM) were compared between normal liver samples (n = 50) versus tumor samples (n = 371). (B) Heat map for gene expression of TAZ/WWTR1, and TGF-β pathway components (TGF-β1, SMAD2, and SMAD3) was obtained by analyzing LIHC dataset from TCGA and GTEx, using GEPIA. The mRNA levels (TPM) were compared between normal liver samples (n = 160) versus tumor samples (n = 369). The heat maps were modified from the originals for better visualization. ( C) Correlation between the levels of TAZ mRNA and protein of 20 HCC cell lines, and ( D) TAZ protein levels of 20 HCC cell lines sorted by phenotype: epithelial (E) (circles), epithelial-mesenchymal (E/M) (squares) or mesenchymal (M) (triangles). Data were replotted from the original CCLE data for better visualization.

Article Snippet: HepG2 cells were transiently transfected with each pGL3-basic/hTAZ-Luc reporter plasmids with or without any of the following plasmids: pCMV5/TβRI(WT)-HA (ALK5 wild-type or ALK5(WT)), pCMV5/TβRI(T204D)-HA (constitutively activated ALK5 or ALK5(TD)), pCMV5/Flag-SMAD2 (S2), pCMV5/Flag-SMAD3 (S3), or pCMV5/HA-SMAD4 (S4), or in the presence of 3XFlag-pCMV5-TOPO-TAZ(S89A) (No. 24815) from Addgene [ ] or pBABE-puro-H-RasV12 (No. 9051) from Addgene.

Techniques: Expressing, Gene Expression, Modification

Journal: Heliyon

Article Title: TGF-β/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells

doi: 10.1016/j.heliyon.2023.e21519

Figure Lengend Snippet: Human TAZ/WWTR1 gene promoter is responsive to canonical TGF-β/SMAD pathway. ( A) The pGL3 constructs containing the different DNA fragments of the hTAZ promoter and one mutant are shown. ( B) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc along with ALK5(WT) or ALK5(TD) for 48 h, and then treated or not for 24 h with 0.2 nM TGF-β. At 72 h post-transfection, luciferase activity was measured. Data are representative of 2 independent experiments in triplicate. ( C) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc or hTAZ(407)-Luc reporters along with ALK5(WT) or ALK5(TD), and then luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. ( D) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc or hTAZ(1.13-mutTRE)-Luc constructs, each in co-transfection with ALK5(WT) or ALK5(TD), and luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. P < 0.05*. ( E) HepG2 cells were co-transfected as indicated, with pGL3-basic/hTAZ(1.13)-Luc along with plasmids bearing SMADs full-length cDNA: pCMV5/Flag-SMAD2 (S2), pCMV5/Flag-SMAD3 (S3) or pCMV5/HA-SMAD4 (S4), and ALK5(WT) or ALK5(TD). Then, cells were lysed after 72 h post-transfection, and luciferase activity was analyzed. Raw RLU (Relative Light Units) values are expressed as means ± SEM of 2 independent experiments in quadruplicate. ( F) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc alone or with ALK5 (TD); then, ChIP on plasmid assay was carried out using anti-SMAD2 for IP. PCR was performed using primers spanning the canonical SBE region (648 bp) and a part of the pGL3-basic vector (supplementary raw data). Data are representative of 3 independent experiments. ( G) HepG2 cells were transiently transfected without or with ALK5(TD) for indicated times, and then TAZ, pSMAD2, and SMAD2 protein levels were evaluated by immunoblot; β-ACTIN was used as a loading control (supplementary raw data). Data are representative of 2 independent experiments.

Article Snippet: HepG2 cells were transiently transfected with each pGL3-basic/hTAZ-Luc reporter plasmids with or without any of the following plasmids: pCMV5/TβRI(WT)-HA (ALK5 wild-type or ALK5(WT)), pCMV5/TβRI(T204D)-HA (constitutively activated ALK5 or ALK5(TD)), pCMV5/Flag-SMAD2 (S2), pCMV5/Flag-SMAD3 (S3), or pCMV5/HA-SMAD4 (S4), or in the presence of 3XFlag-pCMV5-TOPO-TAZ(S89A) (No. 24815) from Addgene [ ] or pBABE-puro-H-RasV12 (No. 9051) from Addgene.

Techniques: Construct, Mutagenesis, Transfection, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Western Blot, Control

Journal: The American journal of pathology

Article Title: ERK5 inhibition ameliorates pulmonary fibrosis via regulating Smad3 acetylation.

doi: 10.1016/j.ajpath.2013.08.014

Figure Lengend Snippet: Figure 3 ERK5 is not involved in Smad3 phosphorylation and nuclear translocation in response to TGF-b. A: A549 cells were treated with 5 mmol/L BIX02189 for 1 hour, before incubation with 2 ng/mL TGF-b1 for 30 or 60 minutes. The cell lysates were immunoblotted with antibodies to phospho-Smad3 and ERK5. B: The 10T1/2 fibroblast cells transfected with 100 pmol/L control (CON) or ERK5 siRNA were treated with 2 ng/mL TGF-b1 for 10, 30, or 60 minutes. Cell lysates were used for immunoblotting with phospho-Smad3 and ERK5 antibodies. C: A549 cells were incubated with 5 mmol/L BIX02189 for 1 hour before incubation with 2 ng/mL TGF-b1 for 30 minutes. Cells were stained with anti-Smad3 antibody, and nucleus was stained with DAPI. The images were visualized under immunofluorescence microscope. Original magnification, 200.

Article Snippet: The plasmid encoding human Smad3 (pCMV5-FlagSmad3) was obtained from the Addgene plasmid repository (Addgene plasmid 11742; a gift from Dr. Jeffrey Wrana, University of Toronto at Ontario, Toronto, ON, Canada). pXpress-ERK5 and pHA-CA-MEK5a were cloned as 1759 described previously.19 pcDNA3.1-p300 was obtained from the Addgene plasmid repository (Addgene plasmid 23252; a gift from Dr. Warner.

Techniques: Phospho-proteomics, Translocation Assay, Incubation, Transfection, Control, Western Blot, Staining, Microscopy

Journal: The American journal of pathology

Article Title: ERK5 inhibition ameliorates pulmonary fibrosis via regulating Smad3 acetylation.

doi: 10.1016/j.ajpath.2013.08.014

Figure Lengend Snippet: Figure 2 TGF-b signal to Smad3 and PAI-1 promoter activity is ERK5 dependent. A: A549 cells were transfected with 5 GAL4-Luc reporter gene and GAL4-Smad3 and then treated with 5 mmol/L BIX02189 in the presence or absence of 2 ng/mL TGF-b for 6 hours. The luciferase activity was measured by dual luciferase reporter assay kit and normalized with Renilla luciferase activity derived from pRL-tk construct. A549 and 10T1/2 fibro- blast cells were transfected with SBE-Luc (B) or p800-Luc (C) containing a fragment of the PAI-1 promoter. Transfected cells were incubated with 5 or 10 mmol/L BIX02189 before treatment with 2 ng/mL TGF-b1 for 6 hours. Luciferase activity in cell lysates was measured by the dual luciferase re- porter assay kit and Glomax 20/20 luminometer. Transfection efficiency was normalized by renilla luciferase activity derived from pRL-tk construct. Data are expressed as means SD from three independent experiments. *P < 0.05, **P < 0.01 versus control; yP < 0.05 versus TGF-b1 treated.

Article Snippet: The plasmid encoding human Smad3 (pCMV5-FlagSmad3) was obtained from the Addgene plasmid repository (Addgene plasmid 11742; a gift from Dr. Jeffrey Wrana, University of Toronto at Ontario, Toronto, ON, Canada). pXpress-ERK5 and pHA-CA-MEK5a were cloned as 1759 described previously.19 pcDNA3.1-p300 was obtained from the Addgene plasmid repository (Addgene plasmid 23252; a gift from Dr. Warner.

Techniques: Activity Assay, Transfection, Luciferase, Reporter Assay, Derivative Assay, Construct, Incubation, Control

Journal: The American journal of pathology

Article Title: ERK5 inhibition ameliorates pulmonary fibrosis via regulating Smad3 acetylation.

doi: 10.1016/j.ajpath.2013.08.014

Figure Lengend Snippet: Figure 4 ERK5 affects TGF-b signaling via Smad3 acetylation. A: A549 cells were transfected with SBE-Luc. Transfected cells were treated with 100 nmol/L TSA and 5 mmol/L BIX02189 for 1 hour before incubation with 2 ng/mL TGF-b1 for 24 hours. SBE transcriptional activity was measured by using a luciferase assay kit and normalized by Renilla luciferase activity derived from a pRL-tk construct. B: A549 cells were transfected with FLAG-Smad3. After 24 hours, transfected cells were incubated with BIX02189 for 1 hour before treatment with TGF-b1 for 30 minutes, 60 minutes, and 2 hours, respectively. The cell lysates were applied for immunoprecipitation analysis with anti-Flag antibody or control IgG, and then blotted with antibody-recognizing acetyl-lysine. The total amount of Smad3 from whole cell lysates was determined by immunoblotting. C: Cells were cotransfected with Smad3-WT and GAL4-Luc with p300 acetyl- transferase in the presence or absence of pERK5 and pCA-MEK5a, and acetylation-dependent transcriptional activation of Smad3 was measured by luciferase assay with or without BIX02189 treatment. Selectivity of acetylation in ERK5-induced Smad3 transcriptional activity was checked by transfecting HEK293 cells with the GAL4-fused constructs encoding full-length (D) and N-terminal deletion form of Smad3 (E) (Smad3 LC) for Smad3-WT (white bars) and acetylation- defective mutant form of Smad3 (Smad3-K378R; black bars), respectively. A reporter gene was cotransfected with pG5-Luc in the presence or absence of pERK5 and pCA-MEK5a, as indicated. Luciferase activity was measured by the dual luciferase reporter assay kit and Glomax 20/20 luminometer. F: Primary lung fibroblasts were transfected with Smad3-WT and Smad3-K378R. After 24 hours, transfected cells were incubated with TGF-b1 for 24 hours. Protein level was analyzed by immunoblotting with specific antibodies against fibronectin and tubulin. Data are expressed as means SD from three independent experiments. *P < 0.05 versus control vector; yP < 0.05, yyP < 0.01 versus Smad3 WT.

Article Snippet: The plasmid encoding human Smad3 (pCMV5-FlagSmad3) was obtained from the Addgene plasmid repository (Addgene plasmid 11742; a gift from Dr. Jeffrey Wrana, University of Toronto at Ontario, Toronto, ON, Canada). pXpress-ERK5 and pHA-CA-MEK5a were cloned as 1759 described previously.19 pcDNA3.1-p300 was obtained from the Addgene plasmid repository (Addgene plasmid 23252; a gift from Dr. Warner.

Techniques: Transfection, Incubation, Activity Assay, Luciferase, Derivative Assay, Construct, Immunoprecipitation, Control, Western Blot, Activation Assay, Mutagenesis, Reporter Assay, Plasmid Preparation