Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: TGF-β1 represses inflammation-induced PPARβ expression. (A) RNase protection assay (RPA) of PPARβ, keratin 5 (K5) and 6 (K6) after treatment of the primary keratinocytes with TNF-α (5 ng/ml), IFN-γ (5 ng/ml) or LPS (1 ng/ml) in the presence of increasing amounts of TGF-β1 (1 and 5 ng/ml). (B) Regulation of PPARβ expression by TNF-α (5 ng/ml) and/or TGF-β1 (5 ng/ml) in the presence (+) or absence (−) of cycloheximide (CH; 5 μg/ml) as analyzed by RPA. (C) Control of CH efficiency by Western blot analysis of PPARβ, JunB and PAI-1, from keratinocytes treated as in (B). One representative RPA result out of four independent experiments is shown. RPA values were normalized using the ribosomal protein L27 mRNA levels. Values from unstimulated keratinocytes were arbitrarily assigned a value of 1. Values of PPARβ, JunB and PAI-1 were normalized using tubulin expression. The figures obtained in the absence of TGF-β1, TNF-α and CH were given a value of 1. The other values indicated represent relative fold increase (decrease) as compared to unstimulated keratinocytes.

Article Snippet: Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, Rnase Protection Assay, Western Blot

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: TGF-β1 modulates PPARβ expression via Smad3. (A) The activity of the full-length (PPARβ(−1880)) PPARβ promoter carrying various deletions (PPARβ(−587), (−445) and (−277)) and site-directed mutations was quantified in transactivation assays in primary keratinocytes. The PPARβ(−445) promoter construct contains three putative AP-1 sites (solid bar). The mutations of the AP-1 sites and their position relative to the transcription start site are indicated by the hatched bars and associated italic numbers. The PPARβ(Δ308/Δ197) promoter construct is similar to PPARβ(−445), but has two putative AP-1-binding sites (at positions −308 and −197) mutated, while the −414 was left intact. The PPARβ(Δ414) promoter construct contains a mutated AP-1 at −414. The AP-1(−414) site is the only site critical for both TNF-α (5 ng/ml) responsiveness and TGF-β1-mediated (5 ng/ml) repression. The means of at least six independent experiments are shown. (B) A dominant-negative c-JUN (TAM-67) or wild-type c-JUN was cotransfected in increasing amounts with either PPARβ(−445), PPARβ(Δ308/Δ197) or PPARβ(Δ414) promoter constructs. TNF-α-induced PPARβ promoter activity was repressed upon overexpression of TAM-67 (left panel). Overexpression of c-JUN overcomes TGF-β1-mediated repression (right panel). The means of at least six independent experiments are shown. (C) Cotransfections of PPARβ(−445) reporter construct with Smad1, 2, 3 or 4 (left panel) or with dominant-negative Smad 3(Δ3) or Smad 4(Δ4) (middle panel). As positive control, cotransfections were performed using Smad3, 4, SmadΔ3 and Δ4 with a luciferase reporter promoter construct containing three copies of the Smad-binding elements (3SBE-luc) (right panel). The means of at least three independent experiments are shown. Smad3 and Smad4 as well as dominant-negative Smad 3(Δ3) or Smad 4(Δ4) are equally expressed and protein integrity equally well preserved when transfected in primary keratinocytes. All Smad expression constructs contain an N-terminal FLAG tag. Integrity and expression levels of Smad3, 4, SmadΔ3 and Δ4 were verified by Western blot analysis using anti-FLAG antibody (see the right of the third panel). As a control of the FLAG antibody, the unrelated Flag-PPARγ2 protein was in vitro transcribed and translated. (D) Smad3+/+ and Smad3−/− keratinocytes were transfected with the PPARβ(−445) construct and stimulated with TNF-α in the presence or absence of TGF-β1. Smad3 deficiency blocked TGF-β1 repression of TNF-α-induced PPARβ promoter activity. Cotransfection with increasing Smad3, in Smad3−/− keratinocytes, restored responsiveness to TGF-β1 inhibition. The means of at least six independent experiments are shown. V: empty expression vector.

Article Snippet: Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Construct, Binding Assay, Dominant Negative Mutation, Over Expression, Positive Control, Luciferase, Transfection, FLAG-tag, Western Blot, In Vitro, Cotransfection, Inhibition, Plasmid Preparation

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Differential regulation of PPARβ expression of in vitro model mimicking different stages of wound repair. (A, B) RPA analyses of PPARβ, keratin 5 (K5) and 6 (K6) after exposure of the keratinocytes to various treatments, as indicated. NM: necrosis-derived conditioned medium mimicking early inflammation, initiated by using either minced skin (MS) or freezed/thawed fibroblasts (FT); AM: apoptotic-derived conditioned medium mimicking the context of late remodeling stages of wound healing, initiated by using either UV (UV)- or dexamethasone (Dex)-treated fibroblasts. The importance of TGF-β1 in AM was assessed by preincubation of AM with anti-TGF-β1 antibody (50 μg/ml). TGF-β1 (5 ng/ml), TNF-α (5 ng/ml) and LPS (1 ng/ml) were used. In all RPA analyses, data were normalized using the ribosomal protein L27 mRNA levels. Values represent the mean of at least three independent experiments. (C) ChIP performed on primary keratinocytes treated as in (B) with AM or NM in the presence of TNF-α or TGF-β1, as indicated. The results show a PCR amplification of the AP-1(−414) site (right panel) or control sequence (left panel) after chIP with anti-c-JUN antibody. Preimmune serum serves as a control for chIP. (D) JunB is not binding to the AP-1 site of the PPARβ promoter. ChIP assays were performed with the anti-JunB antibody. Keratinocytes exposed to NM, in the absence or presence of TGF-β1, were used for chIP. The AP-1(−414) site of the PPARβ promoter could not be amplified, indicating the lack of JunB binding to this site (top panel). As a positive control, the AP-1 site at position −783 of the mouse p16INK4a gene was amplified using senescent (passages 6 and 12) primary fibroblasts (bottom panel). For (C) and (D), the figure shows a representative result out of four independent experiments.

Article Snippet: Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, In Vitro, Derivative Assay, Amplification, Sequencing, Binding Assay, Positive Control

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Effects of Smad3–c-JUN interaction are promoter context dependent. (A) Cotransfection of the PPARβ(−445) promoter construct with increasing amounts of Smad3, Smad3(4A) (left panel), SmadΔ3-NLS (middle panel) and Smad3ΔC (right panel). Similar expression levels of Smad3 and Smad3(4A) were verified by immunoblotting with anti-FLAG antibody, whereas SmadΔ3-NLS and Smad3ΔC were verified by anti-myc antibody (see top of each panel); V: empty expression vector. The means of at least six independent experiments are shown. (B) Smad3–c-JUN interaction inhibits PPARβ expression. Protein–protein crosslink and chIP with anti-c-JUN (top panel) and anti-p300 (middle panel) antibodies, followed by Western blot (WB) with anti-Smad3, anti-Smad4 antibody or PCR amplification of the AP-1(−414) site on the PPARβ promoter are shown. In re-chIP experiment (bottom panel), first chIP was performed with anti-c-JUN antibody. After dissociation from the anti-c-JUN antibody, a second chIP (or re-chIP) was performed with anti-p300 antibody. Prior chIP, equal input was verified by immunoblotting with anti-tubulin antibody. A representative result out of three independent experiments is shown. (C) p300–c-Jun and p300–Smad3 complexes stimulate human PAI expression. ChIPs were performed on keratinocytes transfected with a 800 bp hPAI luciferase promoter construct and coincubated with TNF-α, AM, NM or TGF-β1 as indicated. ChIP assays with anti-c-JUN (top panel), chIP/re-chIP with anti-c-JUN/anti-p300 (middle panel) and anti-FLAG/anti-p300 (bottom panel) antibodies are shown. Our results also suggest that both transcription factors can recruit p300 coactivator to the hPAI promoter in response to either TNF-α/NM or TGF-β1/AM. One representative result out of three independent experiments is shown.

Article Snippet: Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Cotransfection, Construct, Expressing, Western Blot, Plasmid Preparation, Amplification, Transfection, Luciferase

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Smad3 inhibits AP-1-mediated transactivation of PPARβ expression. (A) Primary keratinocytes were transfected with the PPARβ(−445) reporter construct and promoter activity was measured upon induction by TNF-α (5 ng/ml). Inhibition of TNF-α-induced PPARβ promoter activities by TGF-β1 was obtained either by additional treatment with TGF-β1 (5 ng/ml) (left panel) or cotransfection with a Smad3 expression plasmid (right panel). This repression was partially overcome by overexpression of increasing amounts of p300. V: empty expression vector. Values represent the mean of four independent experiments. Top panel: Control by Western blot of the amounts of p300 expression in the corresponding keratinocyte whole-cell extracts, using baculovirus-produced His-tagged p300 as positive control. (B) ChIP and re-chIP of the AP-1(−414) site with indicated antibodies on keratinocytes treated with TNF-α and/or TGF-β1 as indicated, and transfected with increasing amounts of the c-JUN expression plasmid (top panel) or p300 expression plasmid (middle panel). TGF-β1 signaling was mimicked by overexpressing Smad3 (FLAG-Smad3) and chIP performed with an anti-FLAG antibody, since the commercially available anti-Smad3 antibodies were not suitable for chIP of endogenous Smad3 (bottom panel). A representative result out of three independent experiments is shown.

Article Snippet: Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Construct, Activity Assay, Inhibition, Cotransfection, Plasmid Preparation, Over Expression, Western Blot, Produced, Positive Control

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Smad3 is essential for inhibition of inflammation-induced PPARβ expression and downregulation of its target genes. (A) RPA analysis of PPARβ mRNA expression after treatment of Smad3+/+ and Smad3−/− primary keratinocytes with TNF-α (5 ng/ml) in the presence or absence of TGF-β1 (5 ng/ml). Ribosomal protein (L27) mRNA was used as internal control. Vehicle (PBS)-treated keratinocytes served as control. (B) RPA analyses of PPARβ, ILK and PDK1 mRNA expression in keratinocytes exposed to day 1 wound fluid (WFD1; 0.1 and 0.5% v/v) in the presence or absence of day 7 wound fluid (WFD7; 0.1 and 0.5% v/v). The importance of TGF-β1 in WFD7 was assessed by preincubation of WFD7 with anti-TGF-β1 antibody (10 and 50 μg/ml). ChIP analysis with anti-c-JUN antibody confirmed reduced c-JUN binding to AP-1(−414) of the PPARβ promoter in the presence of untreated or control Ig-treated (10 and 50 μg/ml) WFD7. The means of at least four independent experiments are shown. (C) RPA analyses of expression levels of PPARβ, ILK and PDK1 in day 1 (inflammation phase) and 7 (re-epithelialization phase) wound biopsies from Smad3+/+ and Smad3−/− mice, normalized against ribosomal protein (L27) mRNA levels. The experimental value, from unwounded wild-type skin, used as normalization unit was arbitrarily assigned a value of 1. The other values indicate relative fold increase (decrease) as compared to unwounded wild-type skin. Values represent the mean of three independent experiments. (D) Model for the role of Smad3 in PPARβ regulation. Upon injury, early inflammation signals, like TNF-α, predominate at the wound site. TNF-α stimulates PPARβ expression and activity via the SAPK signaling cascade resulting in the activation of AP-1 complex that binds at the AP-1(−414) site and the production of PPARβ ligands, respectively (Tan et al, 2001). Activated PPARβ upregulates the expression of ILK and PDK1, which activate Akt1 by phosphorylation (Di Poi et al, 2002). Phosphorylated Akt1 interacts with Smad3, resulting in a decrease in Smad3-mediated transcription (Conery et al, 2004; Remy et al, 2004). Hence, during early phases of wound repair, the growth–survival Akt1 pathway is dominant. As wound repair proceeds into the re-epithelialization/remodeling phase, TGF-β1 produced by both the infiltrating immune cells and the wound fibroblasts leads to strong activation of the TGF-β1/Smad3 pathway. Phosphorylation of Smad3 prevents the formation of Akt1–Smad3 complex, forms a complex with Smad4, which translocates to the nucleus and regulates gene expression. Smad3 either interacts with c-JUN or sequesters the p300 coactivator, which inhibits inflammation-induced PPARβ expression. Reduced Akt1 phosphorylation amplifies the effect of Smad3. Importantly, this mechanism allows for a shift in the control of PPARβ and downstream effects, from the prevalence of growth–survival pathway to the prevalence of TGF-β1-mediated growth arrest.

Article Snippet: Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Binding Assay, Activity Assay, Activation Assay, Produced

Journal: JCI insight

Article Title: TGF-β1 induces PD-1 expression in macrophages through SMAD3/STAT3 cooperative signaling in chronic inflammation.

doi: 10.1172/jci.insight.165544

Figure Lengend Snippet: Figure 2. TGF-β1 is involved in PD-1 expression on macrophages in chronic inflammatory tissues. (A) Correlations between PDCD1 and TGFB1 gene expression in various human tumors by TIMER web server (TIMER2.0; https://cistrome.shinyapps.io/timer/). Abbreviations for various human tumors are given according to the database. COAD, colon adenocarcinoma. (B) Correlation plot between PDCD1 and TGFB1 gene levels in COAD (n = 458; Spearman correlation coefficient = 0.593, P = 9.38 × 10–45). (C and D) Immunoblot analysis of TGF-β1 expression levels in the MC38 tumors and normal colon tissues of tumor-bearing mice (C) or in the livers of normal uninfected and S. japonicum–infected mice (8 weeks postinfection; D). TGF-β1 expression levels were normalized to GAPDH. (E and F) Recombinant TGF-β1, PBS, anti–TGF-β1 neutralizing antibody, or isotype control antibody was administrated intraperito- neally into MC38 tumor–bearing (E) or S. japonicum–infected mice (F). PD-1+ macrophages in the tumor tissue or liver were analyzed using flow cytometry. Representative histograms and quantification of PD-1+ macrophages are shown. Spearman’s rank correlation coefficient (A and B) or an unpaired 2-tailed t test (C–F) was used for statistical analysis. The data are expressed as the mean ± SD of 3–5 mice per group and are representative of 2 independent experiments. **P < 0.01, ***P < 0.001. TPM, transcripts per million.

Article Snippet: Anti–TGF-β1 neutralizing antibody (BE0057; BioXCell; 250 μg per mouse) was administrated intraperitoneally into mice daily every 3 days, starting at 6 weeks postinfection until 8 weeks postinfection or starting at 7 days after tumor implantation until 21 days postimplantation.

Techniques: Expressing, Gene Expression, Western Blot, Infection, Recombinant, Control, Flow Cytometry

Journal:

Article Title: Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members

doi:

Figure Lengend Snippet: Regulation of the p21 promoter by TGF-β1 and Smad proteins. (A and B) Effect of TGF-β1 on p21 and 3TP promoter activity. HepG2 cells were cotransfected with the −2,300/+8 p21 (A) or the p3TP-lux (B) reporter constructs alone (−) or in the presence (+) of TGF-β1 with (+) or without (−) a neutralizing anti-TGF-β1 antibody or a sheep IgG. Relative CAT (A) or luciferase (B) activity is reported. The activity of the −2,300/+8 p21 promoter in the absence of TGF-β1 and antibodies was set arbitrarily to 100%. (C) Dose-dependent repression of the −2,300/+8 p21 promoter activity by Smad4(1–514). HepG2 cells were cotransfected with the −2,300/+8 p21 reporter plasmid and increasing amounts of Smad4(1–514) in the absence (−, striped bars) or presence (+, solid bars) of TGF-β1. The ratio of Smad4(1–514) to −2,300/+8 p21 reporter plasmid input is indicated. (D) Mapping of the p21 promoter region that mediates transactivation by Smad3/4. HepG2 cells were cotransfected with the indicated p21 reporter plasmids without (−, striped bars) or with (+, solid bars) expression vectors for Smad3 and Smad4. The activity of the −2,300/+8 p21 promoter in the absence of Smads was set arbitrarily to 100%.

Article Snippet: Reagents were purchased from the following vendors: Restriction and modifying enzymes and DNA polymerases, from Minotech, New England Biolabs, or GIBCO/BRL; the Sequenase version 2 kit, from Amersham/United States Biochemicals; acetyl-CoA and dNTPs, from Pharmacia; cell culture reagents, from GIBCO/BRL; o -nitrophenyl galactoside, from Sigma; TGF-β1, from R & D Systems; rabbit polyclonal anti-TGF-β1 neutralizing antibody, from Celtrix; sheep total IgG, from Jackson ImmunoResearch; fluorescein isothiocyanate-conjugated goat anti-mouse IgG, from Chemicon; mouse monoclonal anti-FLAG M2 antibody, from IBI/Kodak; mouse monoclonal anti-myc (9E10) antibody, a gift from L. LeGallic (Univ.

Techniques: Activity Assay, Construct, Luciferase, Plasmid Preparation, Expressing

Journal:

Article Title: Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members

doi:

Figure Lengend Snippet: Transactivation of the proximal p21 promoter by Smad family members. (A and B) Transactivation of the −143/+8 p21 promoter by Smad family members. HepG2 cells were cotransfected with the −143/+8 p21 promoter construct in the absence (−) or presence of the indicated Smad proteins. Cells were grown in the absence (A and B, as indicated by −) or in the presence (B, as indicated by +) of TGF-β1. The activity of the −143/+8 p21 promoter in the absence of Smads or TGF-β1 was set arbitrarily to 100%. (C) Subcellular localization of Smad proteins in transfected HepG2 cells. Human Smad proteins were transfected into HepG2 cells and their localization was monitored by indirect immunofluorescence with an antibody against their unique epitope tag [C-terminal FLAG, Smad2 and Smad3; N-terminal FLAG: Smad3C, Smad4, and Smad4(1–514); C-terminal myc, Smad3N]. (D) Western blot analysis of Smad proteins in transfected HepG2 cells. Human Smad proteins 2 (lane 1) and 3 (lane 2) were transfected into HepG2 cells and cell extracts were subjected to SDS/PAGE and Western blot analysis. The resulting chemiluminogram is shown. Arrows indicate the relative migration of the two Smad proteins. Molecular mass markers are in kDa.

Article Snippet: Reagents were purchased from the following vendors: Restriction and modifying enzymes and DNA polymerases, from Minotech, New England Biolabs, or GIBCO/BRL; the Sequenase version 2 kit, from Amersham/United States Biochemicals; acetyl-CoA and dNTPs, from Pharmacia; cell culture reagents, from GIBCO/BRL; o -nitrophenyl galactoside, from Sigma; TGF-β1, from R & D Systems; rabbit polyclonal anti-TGF-β1 neutralizing antibody, from Celtrix; sheep total IgG, from Jackson ImmunoResearch; fluorescein isothiocyanate-conjugated goat anti-mouse IgG, from Chemicon; mouse monoclonal anti-FLAG M2 antibody, from IBI/Kodak; mouse monoclonal anti-myc (9E10) antibody, a gift from L. LeGallic (Univ.

Techniques: Construct, Activity Assay, Transfection, Immunofluorescence, Western Blot, SDS Page, Migration

Journal: International Journal of Molecular Sciences

Article Title: Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

doi: 10.3390/ijms23094714

Figure Lengend Snippet: Immunofluorescence microscopy visualization of β1-integrin receptors under resveratrol challenge on the surface of HCT116 and SW480 cells in the TME. β1-integrin immunolabeled (white arrows) and DAPI-stained nuclei from untreated basal control ( A , E ); TME-grown ( B , F ); resveratrol-treated (5 µM) ( C , G ), and further addition of anti-β1-integrin (2 µg/mL) ( D , H ) HCT116 and SW480 cells. Yellow arrows = change in distribution pattern of β1-integrin receptors. Microscope: Leica DM 2000. Magnification ×600; scale bar = 30 µm. Insets: magnification: ×1200; scale bar = 15 µm.

Article Snippet: Neutralizing monoclonal antibodies against β1-integrin (#sc-374429), β5-integrin (#sc-374429), and normal mouse IgGs (#sc-2025) were from Santa Cruz (#sc-398214) (CA, USA).

Techniques: Immunofluorescence, Microscopy, Immunolabeling, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

doi: 10.3390/ijms23094714

Figure Lengend Snippet: The impact of resveratrol on TME-triggered expression of β1- and β5-integrin. HCT116 cells in alginate matrix in basal control (B. Co.) or in TME were untreated or TME treated with diverse dosages of resveratrol (1, 5, 10, 20 μM) for 10–14 days as outlined in Materials and Methods. Immunolabeling of cell lysates was conducted by Western blotting with antibodies against β1- and β5-integrin. Data are originated from three separate assays, and β-actin was used as a reference. Y -axis: densitometric units confirming Western blot results. * p < 0.05, ** p < 0.01 compared with TME control.

Article Snippet: Neutralizing monoclonal antibodies against β1-integrin (#sc-374429), β5-integrin (#sc-374429), and normal mouse IgGs (#sc-2025) were from Santa Cruz (#sc-398214) (CA, USA).

Techniques: Expressing, Control, Immunolabeling, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

doi: 10.3390/ijms23094714

Figure Lengend Snippet: The importance of β1-integrin for resveratrol-mediated down-modulation of tumor cell viability: ( A , B ) CRC cells (HCT116, SW480) from basal control (Co.) or TME control were not treated or treated with resveratrol (1, 5 µM) in the presence or absence of IgG (1, 2, 5 µg/mL), the blocking anti-β1-integrin antibody (1, 2, 5 µg/mL); ( C , D ) bacitracin (1, 5 µM); ( E , F ) the blocking anti-β1-integrin antibody (1, 2, 5 µg/mL), anti-β5-integrin (5 µg/mL); ( G , H ) RGD peptide (1, 2, 5 µM), RGE peptide (5 µM) for 10–14 days. Tumor cell survival and, indirectly, proliferation were assessed by MTT assay. Relative to TME control, * p < 0.05 and ** p < 0.01.

Article Snippet: Neutralizing monoclonal antibodies against β1-integrin (#sc-374429), β5-integrin (#sc-374429), and normal mouse IgGs (#sc-2025) were from Santa Cruz (#sc-398214) (CA, USA).

Techniques: Control, Blocking Assay, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

doi: 10.3390/ijms23094714

Figure Lengend Snippet: The implication of β1-integrin in resveratrol-mediated down-modulation of tumor cell colony assembly in TME: ( A ) HCT116 cells from basal control (Basal Co.) or untreated TME or TME were treated with different concentrations of resveratrol (1, 5 µM) in the presence or absence of IgG (1, 2 µg/mL) or blocking anti-β1-integrin antibody (1, 2 µg/mL) for 10–14 days as indicated in Materials and Methods; ( B ) Colonies (black arrows) were quantified by scoring 20 different microscopic patches. Microscope: Zeiss Axiovert 40 CFL. Magnification (A): ×24, bar = 0.2 mm. “star” = Alginate. * p < 0.05, ** p < 0.01 compared with TME control.

Article Snippet: Neutralizing monoclonal antibodies against β1-integrin (#sc-374429), β5-integrin (#sc-374429), and normal mouse IgGs (#sc-2025) were from Santa Cruz (#sc-398214) (CA, USA).

Techniques: Control, Blocking Assay, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

doi: 10.3390/ijms23094714

Figure Lengend Snippet: β1-integrin is required for anti-tumor effects of resveratrol. TME-HCT116 cells were treated with resveratrol (5 µM) in the presence or absence of anti-β1- (1, 2, 5 µg/mL), or anti-β5- integrin (5 µg/mL) ( A ) or of RGD (1, 2, 5 µM), or RGE (5 µM) peptide ( B ) as mentioned in Materials and Methods. Western blot samples were probed with antibodies against FAK, p-FAK, p-NF-kB, cyclin D1, and activated caspase-3. In addition, anti-β-actin was used as a loading control. Y -axis: densitometric units confirming Western blot results. * p < 0.05, ** p < 0.01 compared with TME control.

Article Snippet: Neutralizing monoclonal antibodies against β1-integrin (#sc-374429), β5-integrin (#sc-374429), and normal mouse IgGs (#sc-2025) were from Santa Cruz (#sc-398214) (CA, USA).

Techniques: Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

doi: 10.3390/ijms23094714

Figure Lengend Snippet: Working model demonstrating resveratrol-mediated anti-proliferative and anti-viability activity through modulation of the β1-integrin receptor in CRC cells in the pro-inflammatory TME.

Article Snippet: Neutralizing monoclonal antibodies against β1-integrin (#sc-374429), β5-integrin (#sc-374429), and normal mouse IgGs (#sc-2025) were from Santa Cruz (#sc-398214) (CA, USA).

Techniques: Activity Assay

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: TGF-β1 represses inflammation-induced PPARβ expression. (A) RNase protection assay (RPA) of PPARβ, keratin 5 (K5) and 6 (K6) after treatment of the primary keratinocytes with TNF-α (5 ng/ml), IFN-γ (5 ng/ml) or LPS (1 ng/ml) in the presence of increasing amounts of TGF-β1 (1 and 5 ng/ml). (B) Regulation of PPARβ expression by TNF-α (5 ng/ml) and/or TGF-β1 (5 ng/ml) in the presence (+) or absence (−) of cycloheximide (CH; 5 μg/ml) as analyzed by RPA. (C) Control of CH efficiency by Western blot analysis of PPARβ, JunB and PAI-1, from keratinocytes treated as in (B). One representative RPA result out of four independent experiments is shown. RPA values were normalized using the ribosomal protein L27 mRNA levels. Values from unstimulated keratinocytes were arbitrarily assigned a value of 1. Values of PPARβ, JunB and PAI-1 were normalized using tubulin expression. The figures obtained in the absence of TGF-β1, TNF-α and CH were given a value of 1. The other values indicated represent relative fold increase (decrease) as compared to unstimulated keratinocytes.

Article Snippet: Reagents Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, Rnase Protection Assay, Western Blot

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: TGF-β1 modulates PPARβ expression via Smad3. (A) The activity of the full-length (PPARβ(−1880)) PPARβ promoter carrying various deletions (PPARβ(−587), (−445) and (−277)) and site-directed mutations was quantified in transactivation assays in primary keratinocytes. The PPARβ(−445) promoter construct contains three putative AP-1 sites (solid bar). The mutations of the AP-1 sites and their position relative to the transcription start site are indicated by the hatched bars and associated italic numbers. The PPARβ(Δ308/Δ197) promoter construct is similar to PPARβ(−445), but has two putative AP-1-binding sites (at positions −308 and −197) mutated, while the −414 was left intact. The PPARβ(Δ414) promoter construct contains a mutated AP-1 at −414. The AP-1(−414) site is the only site critical for both TNF-α (5 ng/ml) responsiveness and TGF-β1-mediated (5 ng/ml) repression. The means of at least six independent experiments are shown. (B) A dominant-negative c-JUN (TAM-67) or wild-type c-JUN was cotransfected in increasing amounts with either PPARβ(−445), PPARβ(Δ308/Δ197) or PPARβ(Δ414) promoter constructs. TNF-α-induced PPARβ promoter activity was repressed upon overexpression of TAM-67 (left panel). Overexpression of c-JUN overcomes TGF-β1-mediated repression (right panel). The means of at least six independent experiments are shown. (C) Cotransfections of PPARβ(−445) reporter construct with Smad1, 2, 3 or 4 (left panel) or with dominant-negative Smad 3(Δ3) or Smad 4(Δ4) (middle panel). As positive control, cotransfections were performed using Smad3, 4, SmadΔ3 and Δ4 with a luciferase reporter promoter construct containing three copies of the Smad-binding elements (3SBE-luc) (right panel). The means of at least three independent experiments are shown. Smad3 and Smad4 as well as dominant-negative Smad 3(Δ3) or Smad 4(Δ4) are equally expressed and protein integrity equally well preserved when transfected in primary keratinocytes. All Smad expression constructs contain an N-terminal FLAG tag. Integrity and expression levels of Smad3, 4, SmadΔ3 and Δ4 were verified by Western blot analysis using anti-FLAG antibody (see the right of the third panel). As a control of the FLAG antibody, the unrelated Flag-PPARγ2 protein was in vitro transcribed and translated. (D) Smad3+/+ and Smad3−/− keratinocytes were transfected with the PPARβ(−445) construct and stimulated with TNF-α in the presence or absence of TGF-β1. Smad3 deficiency blocked TGF-β1 repression of TNF-α-induced PPARβ promoter activity. Cotransfection with increasing Smad3, in Smad3−/− keratinocytes, restored responsiveness to TGF-β1 inhibition. The means of at least six independent experiments are shown. V: empty expression vector.

Article Snippet: Reagents Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, Activity Assay, Construct, Binding Assay, Dominant Negative Mutation, Over Expression, Positive Control, Luciferase, Transfection, FLAG-tag, Western Blot, In Vitro, Cotransfection, Inhibition, Plasmid Preparation

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Differential regulation of PPARβ expression of in vitro model mimicking different stages of wound repair. (A, B) RPA analyses of PPARβ, keratin 5 (K5) and 6 (K6) after exposure of the keratinocytes to various treatments, as indicated. NM: necrosis-derived conditioned medium mimicking early inflammation, initiated by using either minced skin (MS) or freezed/thawed fibroblasts (FT); AM: apoptotic-derived conditioned medium mimicking the context of late remodeling stages of wound healing, initiated by using either UV (UV)- or dexamethasone (Dex)-treated fibroblasts. The importance of TGF-β1 in AM was assessed by preincubation of AM with anti-TGF-β1 antibody (50 μg/ml). TGF-β1 (5 ng/ml), TNF-α (5 ng/ml) and LPS (1 ng/ml) were used. In all RPA analyses, data were normalized using the ribosomal protein L27 mRNA levels. Values represent the mean of at least three independent experiments. (C) ChIP performed on primary keratinocytes treated as in (B) with AM or NM in the presence of TNF-α or TGF-β1, as indicated. The results show a PCR amplification of the AP-1(−414) site (right panel) or control sequence (left panel) after chIP with anti-c-JUN antibody. Preimmune serum serves as a control for chIP. (D) JunB is not binding to the AP-1 site of the PPARβ promoter. ChIP assays were performed with the anti-JunB antibody. Keratinocytes exposed to NM, in the absence or presence of TGF-β1, were used for chIP. The AP-1(−414) site of the PPARβ promoter could not be amplified, indicating the lack of JunB binding to this site (top panel). As a positive control, the AP-1 site at position −783 of the mouse p16INK4a gene was amplified using senescent (passages 6 and 12) primary fibroblasts (bottom panel). For (C) and (D), the figure shows a representative result out of four independent experiments.

Article Snippet: Reagents Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, In Vitro, Derivative Assay, Amplification, Sequencing, Binding Assay, Positive Control

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Effects of Smad3–c-JUN interaction are promoter context dependent. (A) Cotransfection of the PPARβ(−445) promoter construct with increasing amounts of Smad3, Smad3(4A) (left panel), SmadΔ3-NLS (middle panel) and Smad3ΔC (right panel). Similar expression levels of Smad3 and Smad3(4A) were verified by immunoblotting with anti-FLAG antibody, whereas SmadΔ3-NLS and Smad3ΔC were verified by anti-myc antibody (see top of each panel); V: empty expression vector. The means of at least six independent experiments are shown. (B) Smad3–c-JUN interaction inhibits PPARβ expression. Protein–protein crosslink and chIP with anti-c-JUN (top panel) and anti-p300 (middle panel) antibodies, followed by Western blot (WB) with anti-Smad3, anti-Smad4 antibody or PCR amplification of the AP-1(−414) site on the PPARβ promoter are shown. In re-chIP experiment (bottom panel), first chIP was performed with anti-c-JUN antibody. After dissociation from the anti-c-JUN antibody, a second chIP (or re-chIP) was performed with anti-p300 antibody. Prior chIP, equal input was verified by immunoblotting with anti-tubulin antibody. A representative result out of three independent experiments is shown. (C) p300–c-Jun and p300–Smad3 complexes stimulate human PAI expression. ChIPs were performed on keratinocytes transfected with a 800 bp hPAI luciferase promoter construct and coincubated with TNF-α, AM, NM or TGF-β1 as indicated. ChIP assays with anti-c-JUN (top panel), chIP/re-chIP with anti-c-JUN/anti-p300 (middle panel) and anti-FLAG/anti-p300 (bottom panel) antibodies are shown. Our results also suggest that both transcription factors can recruit p300 coactivator to the hPAI promoter in response to either TNF-α/NM or TGF-β1/AM. One representative result out of three independent experiments is shown.

Article Snippet: Reagents Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Cotransfection, Construct, Expressing, Western Blot, Plasmid Preparation, Amplification, Transfection, Luciferase

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Smad3 inhibits AP-1-mediated transactivation of PPARβ expression. (A) Primary keratinocytes were transfected with the PPARβ(−445) reporter construct and promoter activity was measured upon induction by TNF-α (5 ng/ml). Inhibition of TNF-α-induced PPARβ promoter activities by TGF-β1 was obtained either by additional treatment with TGF-β1 (5 ng/ml) (left panel) or cotransfection with a Smad3 expression plasmid (right panel). This repression was partially overcome by overexpression of increasing amounts of p300. V: empty expression vector. Values represent the mean of four independent experiments. Top panel: Control by Western blot of the amounts of p300 expression in the corresponding keratinocyte whole-cell extracts, using baculovirus-produced His-tagged p300 as positive control. (B) ChIP and re-chIP of the AP-1(−414) site with indicated antibodies on keratinocytes treated with TNF-α and/or TGF-β1 as indicated, and transfected with increasing amounts of the c-JUN expression plasmid (top panel) or p300 expression plasmid (middle panel). TGF-β1 signaling was mimicked by overexpressing Smad3 (FLAG-Smad3) and chIP performed with an anti-FLAG antibody, since the commercially available anti-Smad3 antibodies were not suitable for chIP of endogenous Smad3 (bottom panel). A representative result out of three independent experiments is shown.

Article Snippet: Reagents Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Construct, Activity Assay, Inhibition, Cotransfection, Plasmid Preparation, Over Expression, Western Blot, Produced, Positive Control

Journal:

Article Title: Essential role of Smad3 in the inhibition of inflammation-induced PPAR?/? expression

doi: 10.1038/sj.emboj.7600437

Figure Lengend Snippet: Smad3 is essential for inhibition of inflammation-induced PPARβ expression and downregulation of its target genes. (A) RPA analysis of PPARβ mRNA expression after treatment of Smad3+/+ and Smad3−/− primary keratinocytes with TNF-α (5 ng/ml) in the presence or absence of TGF-β1 (5 ng/ml). Ribosomal protein (L27) mRNA was used as internal control. Vehicle (PBS)-treated keratinocytes served as control. (B) RPA analyses of PPARβ, ILK and PDK1 mRNA expression in keratinocytes exposed to day 1 wound fluid (WFD1; 0.1 and 0.5% v/v) in the presence or absence of day 7 wound fluid (WFD7; 0.1 and 0.5% v/v). The importance of TGF-β1 in WFD7 was assessed by preincubation of WFD7 with anti-TGF-β1 antibody (10 and 50 μg/ml). ChIP analysis with anti-c-JUN antibody confirmed reduced c-JUN binding to AP-1(−414) of the PPARβ promoter in the presence of untreated or control Ig-treated (10 and 50 μg/ml) WFD7. The means of at least four independent experiments are shown. (C) RPA analyses of expression levels of PPARβ, ILK and PDK1 in day 1 (inflammation phase) and 7 (re-epithelialization phase) wound biopsies from Smad3+/+ and Smad3−/− mice, normalized against ribosomal protein (L27) mRNA levels. The experimental value, from unwounded wild-type skin, used as normalization unit was arbitrarily assigned a value of 1. The other values indicate relative fold increase (decrease) as compared to unwounded wild-type skin. Values represent the mean of three independent experiments. (D) Model for the role of Smad3 in PPARβ regulation. Upon injury, early inflammation signals, like TNF-α, predominate at the wound site. TNF-α stimulates PPARβ expression and activity via the SAPK signaling cascade resulting in the activation of AP-1 complex that binds at the AP-1(−414) site and the production of PPARβ ligands, respectively (Tan et al, 2001). Activated PPARβ upregulates the expression of ILK and PDK1, which activate Akt1 by phosphorylation (Di Poi et al, 2002). Phosphorylated Akt1 interacts with Smad3, resulting in a decrease in Smad3-mediated transcription (Conery et al, 2004; Remy et al, 2004). Hence, during early phases of wound repair, the growth–survival Akt1 pathway is dominant. As wound repair proceeds into the re-epithelialization/remodeling phase, TGF-β1 produced by both the infiltrating immune cells and the wound fibroblasts leads to strong activation of the TGF-β1/Smad3 pathway. Phosphorylation of Smad3 prevents the formation of Akt1–Smad3 complex, forms a complex with Smad4, which translocates to the nucleus and regulates gene expression. Smad3 either interacts with c-JUN or sequesters the p300 coactivator, which inhibits inflammation-induced PPARβ expression. Reduced Akt1 phosphorylation amplifies the effect of Smad3. Importantly, this mechanism allows for a shift in the control of PPARβ and downstream effects, from the prevalence of growth–survival pathway to the prevalence of TGF-β1-mediated growth arrest.

Article Snippet: Reagents Antibody providers are as follows: anti-JunB and anti-PPARβ/δ (Affinity Bioreagents); anti-PAI (BD Biosciences); anti-ILK, anti-PDK1 and agarose-conjugated anti-c-JUN (Santa Cruz Biotechnology); anti-FLAG (Sigma); anti-p300 (Upstate Biotechnology), anti-tubulin (Pharmingen); neutralizing TGF-β1 antibody ( Shah et al , 1995 ) (R&D System, AB-101-NA) and corresponding control antibody (Upstate Biotechnology, 12-332); anti-c-JUN and Akt Kinase Assay Kit (Cell Signaling); anti-Smad3 (Zymed; Western blot analyses) and anti-Smad3 (Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Binding Assay, Activity Assay, Activation Assay, Produced

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Immunoblot analysis of mouse CMT-93 cells infected with M1 wt, and recombinant M1-ΔE1A-G and M1-IX-G viruses using an MOI of 3. Cell lysate samples were harvested at six time points and analyzed with the indicated rabbit antibodies raised against early E1A-M1, E1B-19K-M1, intermediate protein IX-M1, and the late hexon protein-M1, plus mouse antibodies against GFP and control actin. Staining with protein IX-specific antibodies revealed a weak band corresponding to processed IX-2A (Mr 14.1 kDa), and a major form corresponding to unprocessed IX-2A-GFP (Mr 41 kDa). Staining with GFP-specific antibodies revealed two major processing forms, corresponding to processed GFP (Mr 27 kDa), and the unprocessed IX-2A-GFP, respectively. Both of these stainings gave rise to additional individual protein forms (denoted by *). (B) Mouse CMT-93 cells and (C) human M000216 cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber-chimeric H5-ΔE3B-CG-FK-M1 and–FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (upper panel) and percent infected cells (lower panel) were determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. Data represent triplicates, shown as mean ± SEM.

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Western Blot, Infection, Recombinant, Control, Staining, Flow Cytometry

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A-D) CMT-93 cells were incubated for 1 h on ice using 5-fold dilution series of the indicated FK proteins starting with 0.8 μg/ml as highest concentration, followed by addition of the different GFP-expressing viruses and transfer to 37°C for 48 h. The virus input amounted to an MOI of 1 and included M1-IX-G (A), M3-IX-G (B), H5-ΔE3B-CG-FK-M1 (C), M2-ΔE1A-G (D). GFP analysis was performed 48 h pi, and expression index was normalized to FK-H3 control protein. (E) M000216 cells were preincubated and processed as described for CMT-93 cells, except that M1-IX-G was used at an MOI of 3. (F) The M3-IX-G and H5-ΔE3B-CG-FK-M3 viruses were pre-incubated for 1 h at RT with serial 5-fold dilutions of the different antisera ranging from 1:1,250 to 1:781,250, followed by addition of the mixes to CMT-93 cells for 48 h at 37°C. The rabbit antisera tested were raised against recombinant FK-M3, FK-M2 and FK-H3, respectively. The virus input amounted to an MOI of 1, and samples were processed for analysis as described above. (G) To check for cross-neutralization of the rabbit anti-FK-M3 for M1, M1-IX-G was preincubated with serial dilutions of rabbit anti-FK-M3, -FK-M2 and -FK-H3 sera and further processed as described above. For all experiments, data represent triplicates, shown as mean ± SEM. For highest concentrations of FKs and anti-FK sera, asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005); ns: not significant ( P >0.05).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Incubation, Concentration Assay, Expressing, Virus, Control, Recombinant, Neutralization, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Flow cytometry profiles of B16, B16-mβ6, B16-mβ8, A549 and A549-hβ6 cells. Green and red histograms show β6 and β8 specific staining, respectively, and grey histograms show background staining using an isotype control. Numbers indicate MFI values of specific or control antibodies. (B, C) Virus binding and dependency on divalent ions. Detached mouse (B) and human cells (C) were incubated with control medium (not containing virus) or medium containing the indicated viruses for 1 h on ice, followed by washing and staining with primary rabbit anti-FK-M3 antibodies and secondary fluorescently labeled antibodies for flow cytometry analysis. Incubation/washing buffers were adjusted to contain either Mg 2+ /Ca 2+ , 1 mM each, 1/0.2 mM Mn 2+ /Ca 2+ , or EDTA 2.5 mM. (D) Virus-receptor antibody competition experiment. Detached cells were first incubated on ice with control medium or the indicated viruses, followed by washing and incubation with either the anti-αvβ6 antibody (CMT-93 and B16-mβ6 cells), or the αvβ8 antibody (M000216 cells). Subsequently the cells were stained with secondary fluorescently labeled antibodies for flow cytometry analysis. (E) The indicated mouse and human cells were infected with recombinant M1-/M3-IX-G, M2-ΔE1A-G and fiber chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 viruses at an MOI of 3. Cells were harvested at the indicated six time points and GFP intensity (MFI) was determined by flow cytometry. Cellular autofluorescence of uninfected cells was included as 0 h infection time point. (F) For analysis of virus progeny, CMT-93, B16, B16-mβ6 and B16-mβ8 cells were infected with M1-/M3-IX-G using an MOI of 1.5. After 14 h, the cells were thoroughly washed, trypsinized and re-seeded. Virus-containing supernatant samples were collected 48 (d2) and 72 h (d3) pi and used for titration analyses. Based on the virus input, fold increases of progeny virus were calculated. For B16 cells no measurable levels of viruses were detected, which translated to a virus progeny production of less than a factor of 0.01, based on the sensitivity level of this assay. Data in (B) to (F) represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Flow Cytometry, Staining, Control, Virus, Binding Assay, Incubation, Labeling, Infection, Recombinant, Titration, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A) Virus binding interference in CMT-93 and M000216 cells by β6/-β8 function blocking antibodies. Detached cells were sequentially incubated for 1 h on ice with control antibody, or the anti-β6/-β8 antibodies, followed by incubation with control medium or medium containing the indicated viruses at an MOI of 4, the rabbit anti-FK-M3 antibodies, and finally the secondary PE-conjugate antibodies. Incubation and washing buffer contained either Mg 2+ /Ca 2+ , 1 mM each, or 1/0.2 mM Mn 2+ /Ca 2+ . (B-C) Virus infection interference in CMT-93 and M000216 cells by β6- and β8-specific antibodies. CMT-93 (B) and M000216 cells (C) were pre-incubated for 1 h on ice using 5-fold dilution series of the specific β6- or β8-integrin antibodies, respectively, starting with 800 ng/ml as highest concentration, followed by addition of the different indicated GFP-expressing viruses and transfer to 37°C for 48 h. An MOI of 1 was used for CMT-93 cells and MOI of 3 for M000216 cells in all experiments shown in this figure. GFP analysis was performed 48 h pi, and expression index was normalized to a control antibody. IC 50 values determined in this experiment are summarized in . (D, E) Infection blocking assays by sITGs. M1-IX-G virus was incubated for 1 h at RT with 5-fold serial dilutions of the indicated sITGs starting from 800 ng/ml to 6.4 ng/ml, followed by addition to CMT-93 cells (D) and M00216 cells (E) and cultivated and further processed as above. (F-H) Infection blocking assays by peptides. (F) The 20-mer peptides tested for virus infection inhibition included peptides A20FMDV2 derived from the VP1 coat protein of FMDV2, A20FMDV2-E containing a D to E mutation in the critical RGD motif, A20M1 and A20M3 derived from M1-/M3-FK, respectively, as compared to LAP-hTGFβ1, all containing the critical αvβ6/αvβ8-binding RGDLXX(L/I) motif. (G, H) Cells were pre-incubated on ice with 5-fold serial dilutions of peptides resulting in final concentrations from 5,000 to 0.32 nM. Subsequently, M1-IX-G virus was added to CMT-93 cells (G) or M000216 cells (H), followed by processing as described above. (I) Comparative flow cytometry profiles of αvβ8 expression in 3T6 cells. Blue and red show β8-specific staining in 3T6-sgNT and 3T6-sgItgβ8 cells, respectively, and grey histogram shows background staining of 3T6-sgItgβ8 cells using a matched isotype control. Numbers indicate MFI values of specific or control antibodies. (J, K) Transduction of 3T6-sgNT and 3T6-sgItgβ8 cells using M1-/M3-IX-G, the fiber-chimeric H5-ΔE3B-CG-FK-M1/-FK-M3 and control H5-ΔE3B-CG at an MOI of 9. Cells were processed as described in . (L) Comparative flow cytometry MFI αvβ6 expression values in control CMT-93-sgNT versus β6 integrin shRNA knock down CMT-93-sgItgβ6 cells. (M-O) Infection of control CMT-93-sgNT and CMT-93-sgItgβ6 cells using M1-IX-G (M), M3-IX-G (N) and H5-ΔE3B-CG (O) at an MOI of 1. Cells were processed as described above. Except for the representative flow cytometry histogram in (I), data represent triplicates, shown as mean ± SEM. Asterisks indicate level of significance for comparison of indicated values (*, P <0.05; **, P <0.005; ***, P <0.0005).

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Virus, Binding Assay, Blocking Assay, Incubation, Control, Infection, Concentration Assay, Expressing, Inhibition, Derivative Assay, Mutagenesis, Flow Cytometry, Staining, Transduction, shRNA, Knockdown, Comparison

Journal: PLoS Pathogens

Article Title: The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

doi: 10.1371/journal.ppat.1010083

Figure Lengend Snippet: (A, B) Sensor chips containing immobilized biotinylated FK-M1 and FK-M3 were probed with mouse sITG αvβ6. Following consecutive analyte injections over 120 s, dissociation was monitored for 600 s (black). Sensorgrams were fitted with a 1:1 kinetic model (red). (C-E) FK saturation cell binding assays using cells with defined αvβ6/αvβ8 expression included FK-M1 binding to B16-mβ6 (C), FK-M3 binding to B16-mβ6 (D) and FK-M3 binding to B16-mβ8 (E). Parental B16 cells were included in order to subtract background levels when calculating equilibrium dissociation constant K D values by Scatchard plot analyses.

Article Snippet: Integrin antibodies included human αv mAb Sc-9969 (Santa Cruz), human β1 mAb sc-59829 (Santa Cruz), hu β3 mAb AP3 (ATCC), human β5 mAb B5-IVF2 (provided by M. Hemler, Harvard Medical School, Boston, USA), human αvβ3 mAb 23C6 (sc-7312, Santa Cruz), human and mouse αvβ5 mAb ALULA (provided by D. Sheppard, UCSF [ ]), human and mouse αvβ6 mAb 10D5 (function blocking [ ], ab77906, Abcam), human and mouse αvβ8 mAb ADWA-11 (function blocking, provided by D. Sheppard, UCSF [ ]), mouse αv rAb RMV7 (14–00512, Affymetrix eBioscience), mouse β1 mAb (MAB2405, R&D systems, Bio-techne brand, USA) mouse β3 (rAb MAB41182, R&D systems, Bio-techne brand, USA).

Techniques: Binding Assay, Expressing