Journal: Experimental and Therapeutic Medicine

Article Title: KDM6A suppresses hepatocellular carcinoma cell proliferation by negatively regulating the TGF-β/SMAD signaling pathway

doi: 10.3892/etm.2020.9000

Figure Lengend Snippet: KDM6A negatively regulates the TGF-β/SMAD signaling pathway. (A) The effect of KDM6A overexpression on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in Huh7 and LM3 cells was (A) determined by western blotting and (B) semi-quantified. The effect of KDM6A knockdown on the expression of TGF-β, p-smad2, p-smad4, Ki67 and PCNA in YY-8103 and SNU-398 was (C) determined by western blotting and (D) semi-quantified. ** P<0.01 and *** P<0.001 vs. vector or SCR. KDM6A, lysine demethylase 6A; TGF-β, transforming growth factor-β; p, phosphorylated; PCNA, proliferating cell nuclear antigen; SCR, scrambled; sh, short hairpin RNA; ns, not significant.

Article Snippet: Subsequently, the membranes were incubated at 4˚C overnight with the following primary antibodies: Anti-KDM6A (1:1,000; cat. no. orb333886; Biorbyt Ltd.), anti-transforming growth factor (TGF)-β (1:1,000; cat. no. 3711; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-smad2 (1:1,000; cat. no. 18338; Cell Signaling Technology, Inc.), anti-Smad2 (1:1,000; cat. no. 12570-1-AP; ProteinTech Group, Inc.), anti-p-smad4 (1:1,000; cat. no. 10231-8-AP; ProteinTech Group, Inc.), anti-smad4 (1:1,000; cat. no. 10231-1-AP; ProteinTech Group, Inc.), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. 10205-2-AP; ProteinTech Group, Inc.), anti-Ki67 (1:1,000; cat. no. 27309-1-AP; ProteinTech Group, Inc.), anti-GAPDH (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) and anti-Flag (1:4,000; cat. no. F7425; Sigma-Aldrich; Merck KGaA).

Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation, shRNA

Journal: Disease Models & Mechanisms

Article Title: Smad4 restricts injury-provoked biliary proliferation and carcinogenesis

doi: 10.1242/dmm.050358

Figure Lengend Snippet: Smad4 suppresses cholangiocyte proliferation in response to injury. (A) Generation of compound mutant mice carrying Alb-Cre and Arid1a fl/fl ( Arid1a del ), Kras LSL-G12D ( Kras G12D ), Smad4 fl/fl ( Smad4 del ) or Tp53 fl/fl ( Tp53 del ). Experimental and control cohorts [no Alb-Cre , ( Ctl )] were put on a DDC diet for 2 weeks and their livers were harvested, n =3-4 mice per cohort. (B) Quantification of the ductular reaction area of the experimental cohorts compared to the control cohort as outlined in A, showing Smad4 del with a significant expansion of the biliary compartment. (C) Representative images of ductular reactions (surrounded by dashed lines), magnified 200×. (D) Quantification of reactive cholangiocytes (identified as PanCK+) in Smad4 del and Ctl livers demonstrating more reactive cholangiocytes in the DDC-injured Smad4del livers compared to that of Ctl . Three random images per mouse (magnified 100×) were used for quantification. Representative ductular reactions are shown on the right. (E) Quantification of proliferating cholangiocytes (KRT19+ and Ki67+) in Smad4 del and Ctl livers revealing more proliferative cholangiocytes in DDC-injured Smad4del livers compared to that of Ctl . For quantification, three random portal fields per mouse were aggregated by genotype. Representative images (magnified 100×) are shown; boxed areas are shown enlarged on the right. * P <0.05, ** P <0.01, *** P <0.0001; scale bars: 50 µm (C) and 100 µm (D,E).

Article Snippet: The lentiviral construct pLenti-C-Myc-DDK-P2A-Puro expressing murine Smad4 was generated by Origene (catalog #MR208755L3).

Techniques: Mutagenesis, Control

Journal: Disease Models & Mechanisms

Article Title: Smad4 restricts injury-provoked biliary proliferation and carcinogenesis

doi: 10.1242/dmm.050358

Figure Lengend Snippet: Smad4 is involved in the regulation of pathways associated with proliferation, metabolism and inflammation. (A) Schematic outlining the strategy to isolate and sequence reactive cholangiocytes. Livers are enzymatically digested and flow-sorted to isolate the EPCAM-positive/CD45-negative/Ter119-negative (EPCAM+/CD45-/Ter119-) population from which RNA had been prepared for RNA sequencing. Representative FACS plots are shown on the left. Boxed areas enclose cells of interest (P5 = CD45 − /Ter119 − cells, EpCAM = EpCAM + cells). n =5 per cohort. (B) Heat map showing the top 50 most significantly upregulated and downregulated genes in Smad4 del -perturbed reactive cholangiocytes. (C) Gene set enrichment analysis (GSEA) of genes differentially expressed in Smad4 del -perturbed reactive cholangiocytes. Enrichment plots from the Hallmarks Gene Set Collection are shown for the top five gene sets most positively correlated (top row) in genes upregulated in Smad4 del cells or most negatively correlated (bottom row) in genes downregulated in Smad4 del cells. Ox-Phos, oxidative phosphorylation; EMT, epithelial mesenchymal transition; NES, normalized enrichment score.

Article Snippet: The lentiviral construct pLenti-C-Myc-DDK-P2A-Puro expressing murine Smad4 was generated by Origene (catalog #MR208755L3).

Techniques: Sequencing, RNA Sequencing, Phospho-proteomics

Journal: Disease Models & Mechanisms

Article Title: Smad4 restricts injury-provoked biliary proliferation and carcinogenesis

doi: 10.1242/dmm.050358

Figure Lengend Snippet: Smad4 suppresses cancer progression in the AKP and AP hepatobiliary cancer models. (A) mRNA expression levels assessed by qPCR of TGFβ family-related genes in (A) AK mouse model-derived HCC (AK HCC) or (B) AKP mouse model-derived CCA (AKP CCA) tissue compared to normal liver tissue showing increased expression of TGFβ ligands, indicating activation of the TGFβ pathway. Expression was normalized to Rhoa , n =3 mice per cohort. (C) Immunohistochemistry image of phosphorylated (phospho)-SMAD2 in AKP CCA tissue and adjacent normal (Norm) tissue (magnified 100×). Boxed areas are shown enlarged on the right, with CCA tissue at the top and normal tissue at the bottom. Arrowhead shows positive phospho-SMAD2 nuclei in malignant epithelium. (D) Compound mutant mice carrying Alb-Cre ; Kras LSL-G12D ; Tp53 fl/+ and either Smad4 fl/fl or Smad4 wt/wt alleles (AKPS or AKP mouse models, respectively) were followed for survival ( n =5–10 mice per cohort). (E) Kaplan–Meier plot comparing survival curves of AKPS (red) and AKP (black) mice, showing decreased overall survival of the AKPS compared to the AKP cohort ( P <0.05). (F) Representative histology and immunohistochemistry images of AKPS CCA, high-grade BilIN and HCC (magnified 100×). Boxed areas are shown enlarged to the right. Staining of reactive cholangiocytes (PanCK) highlights malignant biliary epithelium. (G) Compound mutant mice carrying Alb-Cre ; Tp53 fl/fl and Smad4 fl/fl or Smad4 wt/wt alleles (APS or AP mouse models, respectively) were followed and sacrificed at one year of age, n =8–15 mice per cohort. (H) Representative histology images of APS mixed CCA/HCC and hamartomas (magnified 100×). Boxed areas are shown enlarged to the right. Staining of PanCK highlights biliary differentiation. * P <0.05, ** P <0.01, Scale bars: 50 µm.

Article Snippet: The lentiviral construct pLenti-C-Myc-DDK-P2A-Puro expressing murine Smad4 was generated by Origene (catalog #MR208755L3).

Techniques: Expressing, Derivative Assay, Activation Assay, Immunohistochemistry, Mutagenesis, Staining

Journal: Disease Models & Mechanisms

Article Title: Smad4 restricts injury-provoked biliary proliferation and carcinogenesis

doi: 10.1242/dmm.050358

Figure Lengend Snippet: Pathway analyses, suggesting conserved functions of Smad4 in CCA and reactive cholangiocytes. (A) Plotted are the growth curves of two CCA cell lines (#1 and #2) derived from liver tumors from mice in the AKPS cohort expressing only Smad4 (red) or empty vector ( EV ; black), and Smad4 or EV plus treatment with TGFβ (gray or orange, respectively), showing growth arrest/inhibition with TGFβ treatment following reintroduction of Smad4 to both AKPS #1 and #2. (B) Western blot confirming restoration of Smad4 (S4) protein levels in the two AKPS lines assessed in A. (C) Heat map showing the top 50 most significantly up- and downregulated genes in Smad4 -restored CCA cell lines. (D) Individual and shared gene sets enriched with loss of Smad4 signaling (top) and with intact Smad4 signaling (bottom) in AKPS CCA cell lines (right) compared to those enriched in reactive cholangiocytes (left).

Article Snippet: The lentiviral construct pLenti-C-Myc-DDK-P2A-Puro expressing murine Smad4 was generated by Origene (catalog #MR208755L3).

Techniques: Derivative Assay, Expressing, Plasmid Preparation, Inhibition, Western Blot

Journal: Disease Models & Mechanisms

Article Title: Smad4 restricts injury-provoked biliary proliferation and carcinogenesis

doi: 10.1242/dmm.050358

Figure Lengend Snippet: Smad4 is a determinant of DNA methylation. (A) Histograms indicating the frequency of differentially methylated regions binned according to changes in DNA methylation (DNAme; x -axis) in the two AKPS mouse model-derived CCA lines described in Fig.4. Increases (red) and decreases (blue) of DNAme were scored for AKPS # 2 versus AKPS # 1 (top) and for Smad4 re-expression empty vector ( EV ) (bottom). (B) Boxplots indicate the distance (in kb) of differentially methylated regions relative to the nearest transcriptional start site (TSS). Regions from ‘Decreased DNAme Smad4 vs. EV ’ were found at a greater distance from the nearest TSS, suggesting a differential effect of Smad4 on DNAme distal to promoter regions. (C) Differentially methylated regions were analyzed based on sample type (AKPS #2 vs AKPS #1; two pie charts on left) or Smad4 re-expression [ Smad4 vs. EV ; two pie charts on right) and further determined based on increased or decreased DNAme. Regions were then annotated and color-coded based on genetic context.

Article Snippet: The lentiviral construct pLenti-C-Myc-DDK-P2A-Puro expressing murine Smad4 was generated by Origene (catalog #MR208755L3).

Techniques: DNA Methylation Assay, Methylation, Derivative Assay, Expressing, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin

doi: 10.3389/fcell.2018.00105

Figure Lengend Snippet: MyD88 expression levels regulate endogenous SMAD4 expression in Huh7 hepatoma cells. (A,B) MyD88 overexpression enhances endogenous SMAD4 expression. (A) Huh7 cells transiently transfected with an empty vector (pCMV) or HA-tagged MyD88 plasmid (pMyD88). Total cell lysates were analyzed by western blotting for endogenous SMAD4 expression. Expression of the β-actin protein was used as a loading control. (B) Densitometric quantification of SMAD4 levels in western blots from three independent experiments. (C–E) MyD88 repression lowers endogenous SMAD4 expression. (C) Representative western blot of MyD88 and SMAD4 expression in Huh7 cells transiently transfected with control psiRNA-LucGL3 (si-Ctrl) or psiRNA-hMyD88 (si-MyD88). Expression of the β-actin protein was used as a loading control. (D,E) Densitometric quantification of (D) MyD88 and (E) SMAD4 protein levels from three independent experiments. Results are presented as mean ± SEM. Statistical analyses were performed with Student’s t -test.

Article Snippet: Plasmid pCMV-HA-MyD88, also referred as pMyD88, contained full length MyD88 with hemagglutinin (HA) tag and was a gift from B. Beutler (Addgene plasmid #12287); pRK-DPC4-Flag contained SMAD4 with a Flag tag and was a gift from R. Derynck (Addgene plasmid #12627); pCI-His-hUbi contained ubiquitin with a histidine tag and was a gift from A. Winoto (Addgene plasmid #31815); pCS2-HA-SMAD6 contained SMAD6 with a HA tag (pSMAD6) and was a gift of J. Massague (Addgene plasmid #14962); and pRK-Myc-SMURF1 contained SMURF1 with a Myc tag (pSMURF1) and was a gift from Y. Zhang (Addgene plasmid #13676).

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin

doi: 10.3389/fcell.2018.00105

Figure Lengend Snippet: MyD88 directly interacts with SMAD4 through the Toll/Interleukin-1 receptor (TIR) domain of MyD88. (A–C) Co-immunoprecipitation of MyD88 with SMAD4. (A) MyD88 KO HEK293 cells (HEK293-I3A) were transfected transiently with HA-tagged MyD88 together with Flag-tagged SMAD4. Cell lysates were subjected to immunoprecipitation (IP) with the anti-Flag, anti-HA or normal IgG antibody (as a control) and analyzed by immunoblotting (IB) with an anti-HA antibody to detect MyD88 and an anti-Flag antibody to detect SMAD4. (B) Co-immunoprecipitation of endogenous MyD88 with Flag-tagged SMAD4 and, reciprocally, of endogenous SMAD4 with HA-tagged MyD88. (C) Co-immunoprecipitation of endogenous MyD88 with endogenous SMAD4 in Huh7 cells treated with BMP6 (+BMP6) for 24 h. (D,E) The TIR domain of MyD88 is necessary for its interaction with SMAD4. HEK293-I3A cells were transiently transfected with pCMV-HA-MyD88 or one of the three MyD88-deletion plasmids as shown in (E) . (D) Total cellular lysates (TCL) were extracted, incubated with GST-SMAD4, and analyzed by western blot with the indicated antibodies. (E) Schematic representation of plasmids encoding different truncated forms of MyD88. FL, full length; DD, death domain; TIR, Toll/Interleukin-1 receptor domain; ID, intermediate domain. (F–H) Defective MyD88 mutant (ΔTIR domain) abolishes the induction of endogenous SMAD4 and HAMP promoter activation by MyD88 overexpression. Huh7 cells were transfected with HAMP-Luc and pCMV or HA-tagged MyD88 vector (pMyD88) or the MyD88 vector lacking the TIR domain (pMyD88ΔTIR). (F) Expression of endogenous SMAD4 and transfected HA-tagged MyD88 was analyzed by western blotting. β-actin protein was used as a loading control. (G) Densitometric quantification of SMAD4 levels in western blots from three independent experiments. (H) Luciferase activity assessed 24 h after transfection. Results are presented as mean ± SEM of the relative activity ( Firefly / Renilla ratio). Data are representative of a minimum of three experiments. Statistical analysis was performed with one-way ANOVA; n.s., not significant compared to pCMV.

Article Snippet: Plasmid pCMV-HA-MyD88, also referred as pMyD88, contained full length MyD88 with hemagglutinin (HA) tag and was a gift from B. Beutler (Addgene plasmid #12287); pRK-DPC4-Flag contained SMAD4 with a Flag tag and was a gift from R. Derynck (Addgene plasmid #12627); pCI-His-hUbi contained ubiquitin with a histidine tag and was a gift from A. Winoto (Addgene plasmid #31815); pCS2-HA-SMAD6 contained SMAD6 with a HA tag (pSMAD6) and was a gift of J. Massague (Addgene plasmid #14962); and pRK-Myc-SMURF1 contained SMURF1 with a Myc tag (pSMURF1) and was a gift from Y. Zhang (Addgene plasmid #13676).

Techniques: Immunoprecipitation, Transfection, Control, Western Blot, Incubation, Mutagenesis, Activation Assay, Over Expression, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin

doi: 10.3389/fcell.2018.00105

Figure Lengend Snippet: MyD88 L265P mutation enhances hepcidin expression and intracellular iron accumulation in B cell lines. (A) MyD88 L265P mutant interacts with SMAD4. MyD88 KO HEK293T cells (HEK293-I3A) were transfected transiently with either pCMV (control) or HA-tagged MyD88 (as Wt) or HA-MyD88L265P (as L265P), together with Flag-tagged SMAD4. Cell lysates were subjected to immunoprecipitation (IP) with the anti-Flag antibody and analyzed by immunoblotting (IB) with an anti-HA antibody to detect MyD88 and an anti-Flag antibody to detect SMAD4. Total cell lysates (Input) before IP were immunoblotted with anti-HA, anti-Flag, and anti-β-actin antibodies. (B,C) MyD88 L265P mutant upregulates hepcidin expression in B cell lines. (B) Namalwa and (C) Raji B cell lines were co-transfected with HAMP-MetLuc2 and control (pCMV) or MyD88 plasmids (pMyD88): wild-type (Wt) or mutated MyD88 (L265P). Luciferase activity was assessed 24 hr after transfection. Data are from a minimum of three independent experiments. (D,E) MyD88 L265P mutant increases intracellular iron content in B cell lines. (D) Namalwa and (E) Raji B cell lines were co-transfected with HAMP-MetLuc2 and control (pCMV) or MyD88 plasmids (pMyD88): wild-type (Wt) or mutated MyD88 (L265P). Intracellular iron concentrations were determined using the QuantiChrom Iron Assay Kit. Concentrations of iron are given in μg iron per 10 6 live cells and data are from a minimum of three independent experiments. (F,G) MyD88 L265P mutant decreases Ferroportin 1 expression. (F) Namalwa cells were co-transfected with HAMP-MetLuc2 and control (pCMV) or MyD88 plasmids (pMyD88): wild-type (Wt) or mutated MyD88 (L265P). Total cell lysates were analyzed by western blotting for Ferroportin 1 and β-actin as loading control. (G) Densitometric quantification of Ferroportin 1 levels in western blots from three independent experiments. Statistical analysis was performed with one-way ANOVA. Results are presented as mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.001, ∗∗∗ P < 0.0001, and n.s., not significant compared with empty plasmid (pCMV).

Article Snippet: Plasmid pCMV-HA-MyD88, also referred as pMyD88, contained full length MyD88 with hemagglutinin (HA) tag and was a gift from B. Beutler (Addgene plasmid #12287); pRK-DPC4-Flag contained SMAD4 with a Flag tag and was a gift from R. Derynck (Addgene plasmid #12627); pCI-His-hUbi contained ubiquitin with a histidine tag and was a gift from A. Winoto (Addgene plasmid #31815); pCS2-HA-SMAD6 contained SMAD6 with a HA tag (pSMAD6) and was a gift of J. Massague (Addgene plasmid #14962); and pRK-Myc-SMURF1 contained SMURF1 with a Myc tag (pSMURF1) and was a gift from Y. Zhang (Addgene plasmid #13676).

Techniques: Mutagenesis, Expressing, Transfection, Control, Immunoprecipitation, Western Blot, Luciferase, Activity Assay, Iron Assay, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: TGF-β-Induced PAUF Plays a Pivotal Role in the Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cell Line Panc-1

doi: 10.3390/ijms252111420

Figure Lengend Snippet: TGF-β-induced expression of pancreatic adenocarcinoma upregulated factor (PAUF) is mediated by activating the Smad signaling pathway. ( A ) Different PDAC cell lines were treated with vehicle or TGF-β (10 ng/mL) for 1 h. Phosphorylation and protein levels of TGF-β-mediated Smad signaling molecules were examined using immunoblotting. Densitometry was performed using the ImageJ software ( n = 3). Panc-1 cells were pretreated with SB-431542 (10 μM) for 1 h and stimulated with vehicle or TGF-β for ( B ) 1 or ( C , D ) 24 h. ( B ) Phosphorylation and protein levels of Smads were determined using immunoblotting. ( C ) mRNA and protein levels of PAUF were determined using RT-PCR and immunoblotting, respectively. ( D ) Intracellular PAUF expression was confirmed through immunofluorescence staining with an Alexa 488-labeled anti-PAUF antibody and nuclei were stained with DAPI. Scale bar, 50 μm. Relative PAUF-expressing cells were quantitated in four fields per sample using the ImageJ software ( n = 4). Panc-1 cells were transfected with 100 nM scrambled (Ctrl), Smad2/3, or Smad4 siRNAs alone or in combination, followed by treatment with vehicle or TGF-β for ( E ) 1 or ( F , G ) 24 h. ( E ) Phosphorylation and expression of Smad proteins were measured using immunoblotting. ( F ) mRNA and protein levels of PAUF were determined using RT-PCR and immunoblotting, respectively, and ( G ) secreted PAUF levels were measured in culture supernatants using ELISA ( n = 4). BxPC-3 cells were transfected with control vector pCMV6 (Mock) or Smad4 expression vector pCMV-Smad4 (pcSmad4), followed by treatment with vehicle or TGF-β (10 ng/mL) for ( H ) 1 or ( I , J ) 24 h. ( H ) Smad2/3 phosphorylation and Smad4 expression were determined using immunoblotting. ( I ) PAUF expression was assessed using RT-PCR and immunoblotting. ( J ) Intracellular PAUF levels were visualized using immunofluorescence staining with an Alexa 488-labeled anti-PAUF antibody and nuclei were stained with DAPI. Scale bar, 50 μm. Relative PAUF-expressing cells were quantitated in four fields per sample in a randomized manner using the ImageJ software ( n = 4). Statistical significance was calculated using two-way ANOVA followed by post-hoc multiple comparisons test with Bonferroni correction. Data are presented as the mean ± standard deviation (SD). ns, not statistically significant, ** p < 0.01, *** p < 0.001.

Article Snippet: Panc-1 or BxPC-3 cells (4 × 10 5 ) were cotransfected with 100 nM Smad-targeting siRNAs or 1 μg of Smad4 expression vector (pCMV-Smad4; OriGene Technologies, Inc.) along with 1 μg of PAUF promoter-Luc vectors (SBE WT , SBE Mut , and SBE ∆ mu t ) and 1 μg of the β-gal expression vector (pCMV-LacZ; Clontech, Palo Alto, CA, USA) using the Lipofectamine 3000 reagent (Invitrogen).

Techniques: Expressing, Western Blot, Software, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Labeling, Transfection, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: TGF-β-Induced PAUF Plays a Pivotal Role in the Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cell Line Panc-1

doi: 10.3390/ijms252111420

Figure Lengend Snippet: Smad-binding element (SBE) is essential for TGF-β-inducibility of the pancreatic adenocarcinoma upregulated factor (PAUF) promoter. ( A ) A schematic representation of the PAUF promoter (−1.7 Kb), highlighting the putative SBE used to analyze the PAUF promoter activity. ( B ) Panc-1 cells were pretreated with SB-431542 (10 μM) for 1 h and subsequently with vehicle or TGF-β (10 ng/mL) for 1 h. Binding of pSamd3 to SBE within the PAUF promoter was determined using the chromatin immunoprecipitation (ChIP) assay. ( C ) Panc-1 cells transfected with the PAUF promoter-Luc vector (pLuc-PAUF) were stimulated with vehicle or TGF-β for 24 h after pretreatment with SB-431542 (10 μM) for 1 h. The promoter activity was determined in cell lysates with a luminometer ( n = 3). ( D ) Panc-1 cells were transfected with scrambled-, Smad2/3-, or Smad4-targeting siRNAs alone or in combination, followed by stimulation with or without TGF-β for 1 h to assess the binding of pSmad3 to SBE within the PAUF promoter using the ChIP analysis. ( E ) Panc-1 cells were transfected with the indicated siRNA alone or in combination with the pLuc-PAUF vector, and then treated with vehicle or TGF-β for 24 h to evaluate the PAUF promoter activity in cell lysates with a luminometer ( n = 3). ( F ) BxPC-3 cells transfected with pCMV6 (Mock) or pCMV-Smad4 (pcSmad4) were stimulated with TGF-β for 24 h. Binding activity of pSmad3 to SBE within the PAUF promoter was analyzed using the ChIP assay. The fold intensity of ChIP enrichment for the SBE motif at −804/−801 was quantified and normalized against the input ( n = 3). ( G ) BxPC-3 cells were transfected with mock or pcSmad4 in combination with the PAUF promoter-Luc vector, and then treated with vehicle or TGF-β for 24 h. The PAUF promoter activity was measured in cell lysates ( n = 3). ( H ) Comparison of the putative SBE motif within the PAUF promoter (−1.7 Kb) with the SBE mutants. ( I ) Wild-type (SBE WT ) and SBE site mutants (SBE Mut and SBE ∆ mut ) of PAUF promoter-Luc vectors were transiently transfected into Panc-1 cells. After 24 h of transfection, the cells were stimulated with vehicle or TGF-β for 24 h and promoter activities were measured in cell lysates ( n = 3). ( J ) BxPC-3 cells were transfected with mock or pcSmad4 in combination with PAUF promoter-Luc vectors (SBE WT , SBE Mut , or SBE ∆ mut ), and then treated with vehicle or TGF-β for 24 h. The promoter activity was determined in cell lysates ( n = 3). Statistical significance was determined using two-way ANOVA followed by post-hoc multiple comparisons test with Bonferroni correction. Data are displayed as the mean ± standard deviation (SD). ns, not statistically significant, ** p < 0.01, *** p < 0.001.

Article Snippet: Panc-1 or BxPC-3 cells (4 × 10 5 ) were cotransfected with 100 nM Smad-targeting siRNAs or 1 μg of Smad4 expression vector (pCMV-Smad4; OriGene Technologies, Inc.) along with 1 μg of PAUF promoter-Luc vectors (SBE WT , SBE Mut , and SBE ∆ mu t ) and 1 μg of the β-gal expression vector (pCMV-LacZ; Clontech, Palo Alto, CA, USA) using the Lipofectamine 3000 reagent (Invitrogen).

Techniques: Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Comparison, Standard Deviation