collagen type i sc-25974 antibody  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: LncRNA GAS5 attenuates fibroblast activation through inhibiting Smad3 signaling

doi: 10.1101/2020.02.01.930412

Figure Lengend Snippet: A-D) , TGF-β (5ng/ml) induced the expression of myofibroblast cell marker Col1A and αSMA in 3T3 fibroblasts (A-B) and primary skin fibroblasts (C-D). B) Quantification of protein expression in A by normalizing to α-Tubulin. D) Quantification of protein expression in C by normalizing to α-Tubulin. E) TGF-β down-regulated GAS5 in 3T3 fibroblasts in a dose-dependent manner. 3T3 fibroblasts were cultured in serum-free DMEM with 0, 1, 2, 5, or 10 ng/ml of TGF-β for 12 hrs. GAS5 was detected by RT-qPCR. F) TGF-β (5ng/ml) down-regulated GAS5 in primary mouse skin fibroblasts. G) GAS5 was down-regulated in skin tissues with bleomycin treatment, as analyzed by RT-qPCR. C57BL/6 mice were injected subcutaneously with bleomycin (Bleo; 0.02 U) every other day for 14 days. GAS5 RNA expression was calculated by normalizing to cyclophilin mRNA. H) GAS5 positive cells in skin tissues were decreased by bleomycin treatment. Skin tissues from (G) was fixed in 4% PFA, and GAS5 positive cells were shown by RNA-FISH staining. Positive and false-positive staining were indicated by white and yellow arrows, respectively. I) Quantification of GAS5-expressing cells by counting the positive staining from 10 different fields, shown as the fold change. Bar: 200μm. * p<0.05; ** p<0.01; n=3-5. All values are presented as means ± SEM. T-tests were performed for B, D, F, G, I, and one-way ANOVA test was performed for E.

Article Snippet: PCNA (sc-56) and Type I Collagen (Col1A) (sc-25974) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) ( , ).

Techniques: Expressing, Marker, Cell Culture, Quantitative RT-PCR, Injection, RNA Expression, Staining

Journal: bioRxiv

Article Title: LncRNA GAS5 attenuates fibroblast activation through inhibiting Smad3 signaling

doi: 10.1101/2020.02.01.930412

Figure Lengend Snippet: A-B) GAS5 suppressed αSMA and Col1A protein expression. 3T3 cells were transduced with AdGFP or AdGAS5 followed by vehicle or 5 ng/ml of TGF-β treatment for 24 hrs. αSMA and Col1A expression was assessed by Western blot (A) and quantified by normalizing to α-Tubulin (B). C-D) Knockdown of GAS5 increased αSMA and Col1A protein expression. 3T3 cells were transfected with siCtrl or siGAS5 followed by vehicle or 5 ng/ml of TGF-β treatment for 24 hrs. αSMA and Col1A expression was assessed by Western blot (C) and quantified by normalizing to α-Tubulin (D). E) GAS5 suppressed TGF-β-induced αSMA and Col1A mRNA expression. 3T3 cells were transduced with AdGFP or AdGAS5 followed by vehicle (Basal) or 5 ng/ml of TGF-β treatment for 12 hrs. mRNA expression was assessed by RT-qPCR and normalized to cyclophilin. F) Knockdown of GAS5 increased αSMA and Col1A mRNA expression. 3T3 cells were transfected with siCtrl or siGAS5 followed by vehicle (Basal) or 5 ng/ml of TGF-β treatment for 12 hrs. mRNA expression was assessed by qPCR and normalized to cyclophilin. G) GAS5 suppressed TGF-β-induced αSMA promoter activity. 3T3 cells were transduced with AdGFP or AdGAS5 and transfected with αSMA promoter luciferase reporter for 24 hours prior to the treatment with vehicle (-) or 5 ng/ml of TGF-β for 8 hours. Luciferase assays were performed. NS: not significant; * p<0.05; ** p<0.01; n=3. All values are presented as means ± SEM. one-way ANOVA tests were performed.

Article Snippet: PCNA (sc-56) and Type I Collagen (Col1A) (sc-25974) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) ( , ).

Techniques: Expressing, Transduction, Western Blot, Knockdown, Transfection, Quantitative RT-PCR, Activity Assay, Luciferase

Journal: bioRxiv

Article Title: LncRNA GAS5 attenuates fibroblast activation through inhibiting Smad3 signaling

doi: 10.1101/2020.02.01.930412

Figure Lengend Snippet: A-B) Knockdown of PPM1A rescued the inhibitory effect of GAS5 on Smad3 phosphorylation and myofibroblast marker gene expression. 3T3 cells were transduced with AdGFP or AdGAS5 along with transfection of siCtrl or siPPM1A followed by vehicle (-) or TGF-β treatment (5 ng/ml) for 24 hours. Protein expression was assessed by Western blot (A) and quantified by normalizing to α-Tubulin (for αSMA and Col1A) or total Smad3 (for pSmad3) (B). C-D) Overexpression of Smad3 rescued GAS5-blocked Col1A expression. 3T3 cells were transduced with AdGFP or AdGAS5 and transfected with control or Smad3 expression plasmid followed by vehicle or TGF-βtreatment (5 ng/ml) for 24 hours. Col1A expression was assessed by Western blot (C) and quantified by normalizing to α-Tubulin (D). E-F) Smad3 inhibitor SIS3 blunted GAS5 knockdown-enhanced Col1A expression. 3T3 cells were transfected with siCtrl or siGAS5 followed by vehicle or SIS3 (10 μM) treatment for 24 hours. Col1A expression was assessed by Western blot (E) and quantified by normalizing to α-Tubulin (F). ** p<0.01; n=3. All values are presented as means ± SEM. One-way ANOVA tests were performed.

Article Snippet: PCNA (sc-56) and Type I Collagen (Col1A) (sc-25974) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) ( , ).

Techniques: Knockdown, Phospho-proteomics, Marker, Gene Expression, Transduction, Transfection, Expressing, Western Blot, Over Expression, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: LncRNA GAS5 attenuates fibroblast activation through inhibiting Smad3 signaling

doi: 10.1101/2020.02.01.930412

Figure Lengend Snippet: A-D) Forced expression of GAS5 via local adenoviral delivery suppressed bleomycin-induced skin fibrosis in mice. Mouse skins were treated with vehicle (PBS) or bleomycin (0.02U/day) every other day for 28 days. The skin tissues were stained with H&E for structural changes (A), Masson’s trichrome (MT) for collagen deposition (C). Bar: 200 μm. Skin thicknesses shown in A were averaged from 10 different fields (B), and collagen deposition in C was quantified by measuring the staining intensity from 10 different fields (D). E-H) Forced expression of GAS5 via local adenoviral delivery suppressed bleomycin-induced expression of Col1A (E-F) and αSMA (G-H). Bar: 100 μm. Skin tissue sections underwent immunohistochemistry (IHC) staining using Col1A (E) and αSMA (G) antibodies, respectively. Col1A (F) and αSMA (H)-positive cells were averaged from 10 different fields. The large rectangle inserts are enlarged images from the small rectangle boxes in C, E and G, respectively. * p<0.05; ** p<0.01; n=5. All values are presented as means ± SEM. One-way ANOVA tests were performed.

Article Snippet: PCNA (sc-56) and Type I Collagen (Col1A) (sc-25974) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) ( , ).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: LncRNA GAS5 attenuates fibroblast activation through inhibiting Smad3 signaling

doi: 10.1101/2020.02.01.930412

Figure Lengend Snippet: A) GAS5 suppressed Col1A and αSMA protein expression in skin tissues with bleomycin-induced fibrosis. B) Col1a and αSMA protein levels in (A) were quantified by normalizing to GAPDH. ** p<0.01; n=5. C-F) GAS5 suppressed Smad3 binding to Col1a (C-D) and α-SMA (E-F) promoters in vivo that were significantly enriched during bleomycin-induced skin fibrosis. Smad3 binding to Col1a (C) and α-SMA (E) promoters in a chromatin setting was measured by in vivo chromatin immunoprecipitation (CHIP) assay. Smad3 binding enrichments were quantified by qPCR relative to the Smad3 binding in vehicle-treated skin tissues (D-F). ** p<0.01 vs AdGFP-treated groups; n=5. All values are presented as means ± SEM. One-way ANOVA tests were performed.

Article Snippet: PCNA (sc-56) and Type I Collagen (Col1A) (sc-25974) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) ( , ).

Techniques: Expressing, Binding Assay, In Vivo, Chromatin Immunoprecipitation