Journal: The American journal of pathology

Article Title: Epithelial calreticulin up-regulation promotes profibrotic responses and tubulointerstitial fibrosis development.

doi: 10.1016/j.ajpath.2013.07.014

Figure Lengend Snippet: Figure 5 Calreticulin (CRT) overexpression in tubular epithelial cells (TECs) induces selective productionofextracellularmatrix(ECM)components. A: Representative blot shows significantly increased fibronectin expression in CRT-overexpressing cells. Quantification corresponds to means SD of three independent experiments. B: Representative images of collagen IV immunostaining on control and CRT- overexpressing cells show marked up-regulation in overexpressors. C: Representative blots show COL1A1 expression in conditioned medium, ECM, and cell fractions of control, and CRT-overexpressing cells. Ponceau S staining was used as loading control. COL1A1, a fibroblast-specific collagen, is not in- creased by CRT up-regulation in TECs. D: Relative mRNA amount (RT-qPCR) of Col1a1, Col4a1, and Fn1. Transcript levels follow the same pattern as protein expression. E: Relative mRNA amount (RT-qPCR) of ECM molecules in CRT knockdown TECs shows signif- icant suppression of Col1a1 and no change in Col4a1 and Fn1. Data represent means SD of three inde- pendent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. Scale bars: 100 mm (B).

Article Snippet: The primary antibodies used were CRT (catalog No. 06- 661; Upstate Biotechnology, Inc., Lake Placid, NY), Ecadherin (BD 610181; BD Biosciences, San Jose, CA), vinculin (Sigma V9131; Sigma), vimentin (catalog No. MS-129; Thermo Scientific, Inc., Waltham, MA), GRP78 (AP06149PU-N; Acris Antibodies GmbH, Herford, Germany), fibronectin (sc-8422; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), COL1A1 (sc-25974; Santa Cruz Biotechnology), phospho-Smad3 (ab52903; Abcam, Cambridge, England), Smad3 (catalog No. 3102; Cell Signaling Technology, Inc., Danvers, MA), b-actin (catalog No. A5316; Sigma), and b-tubulin (ab6046; Abcam).

Techniques: Over Expression, Expressing, Immunostaining, Control, Staining, Quantitative RT-PCR, Knockdown

Journal: The American journal of pathology

Article Title: Epithelial calreticulin up-regulation promotes profibrotic responses and tubulointerstitial fibrosis development.

doi: 10.1016/j.ajpath.2013.07.014

Figure Lengend Snippet: Figure 8 Decreased calreticulin (CRT) levels in heterozygous mice change the fibrotic profile in unilateral ureteric obstruction (UUO)-induced tubu- lointerstitial fibrosis. A: Relative mRNA amount (RT-qPCR) of the CRT gene in kidney homogenates of control kidneys at 8 and 17 days after UUO in WT and CRTþ/ mice shows that CRT is significantly reduced in CRTþ/ compared with WT mice, with variable degree of reduction in control kidneys and kidneys at 8 and 17 days after UUO. B: Relative mRNA amount of key profibrotic genes (Col1a1, Col3a1, Col4a1, Fn1, TGFb1, Snai1, and Snai2) in WT and CRTþ mice at 8 or 17 days after UUO shows a small repression at 8 days and notably increased repression at 17 days in heterozygous mice, consistent with the expression pattern of CRT at the corresponding UUO time points. C: Representative blot of E-cadherin expression in WT and CRTþ/ mice at 8 or 17 days after UUO shows that E-cadherin is substantially reduced at 17 days in WT but not in CRTþ/ mice, implicating better preservation of the epithelial structure in heterozygous mice. D: Representative images of a-SMAestained kidney sections from WT and CRTþ/ mice at 17 days after UUO and quantification of the staining in non-overlapping images of the entire cortex shows significantly reduced a-SMAepositive area in heterozygous mice. E: Relative mRNA amount of the proinflammatory genes TNF-a and MCP1 in WT and CRTþ/ mice at 8 or 17 days after UUO shows that the proinflammatory mediators are repressed in heterozygous mice after UUO to a degree consistent with the level of CRT expression (E). n Z 3 per group. *P < 0.05, **P < 0.01 versus control WT, L8WT, or L17WT (A, B, D, and E); yyP < 0.01 versus control (C); zzP < 0.01 versus L17þ/ (C). Scale bars: 50 mm (D).

Article Snippet: The primary antibodies used were CRT (catalog No. 06- 661; Upstate Biotechnology, Inc., Lake Placid, NY), Ecadherin (BD 610181; BD Biosciences, San Jose, CA), vinculin (Sigma V9131; Sigma), vimentin (catalog No. MS-129; Thermo Scientific, Inc., Waltham, MA), GRP78 (AP06149PU-N; Acris Antibodies GmbH, Herford, Germany), fibronectin (sc-8422; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), COL1A1 (sc-25974; Santa Cruz Biotechnology), phospho-Smad3 (ab52903; Abcam, Cambridge, England), Smad3 (catalog No. 3102; Cell Signaling Technology, Inc., Danvers, MA), b-actin (catalog No. A5316; Sigma), and b-tubulin (ab6046; Abcam).

Techniques: Quantitative RT-PCR, Control, Expressing, Preserving, Staining

Journal: World journal of gastroenterology

Article Title: Pathophysiology of chronic pancreatitis induced by dibutyltin dichloride joint ethanol in mice.

doi: 10.3748/wjg.v22.i10.2960

Figure Lengend Snippet: Figure 4 Fibrotic parameter changes in pancreas of mice with chronic pancreatitis induced DBTC-induced by dibutyltin dichloride joint ethanol drinking at the indicated time. A: FN expression in pancreas with immunofluorescence double staining of FN and DAPI showing co-expression in the pancreas (original magnification: 200 ×); B: COL1A1 expression in pancreas with western blot.

Article Snippet: The membranes were probed with the following antibodies: anti- COL1A1 (Santa Cruz Biotechnology Inc. sc-25974).

Techniques: Expressing, Immunofluorescence, Double Staining, Western Blot

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

Journal: Molecular cancer research : MCR

Article Title: Increased expression of Beige/Brown adipose markers from host and breast cancer cells influence xenograft formation in mice

doi: 10.1158/1541-7786.MCR-15-0151

Figure Lengend Snippet: Presence of cells with brown adipocyte phenotype and characteristics in HMLEHRASV12xenografts over time. A–B, Xenografts at various stages of growth were fixed with glutaraldehyde, post-fixed with osmium tetroxide and embedded in Technovit. Low (A) and high (B) magnification pictures of hematoxylin/eosin-stained sections are shown. Cells (2×106) from a transplantable HMLEHRASV12 xenograft were mixed with matrigel (1:1) and implanted at a subcutaneous site (posterior dorsolateral) in nude mice to develop xenografts, which were excised at various time points. C, Representative bright field images of xenografts at 1, 5, and 15 week. D, Western blot analysis of pooled lysates (n=3 mice/time point) from xenografts excised at various time points after growth subcutaneously in nude mice. A representative western blot analysis is shown (n=3). E, FACS analysis of PRDM16 (1- and 3 week xenograft), and UCP1, and UCP3 (1- and 5 week xenograft) labeled cells. Data shown is a representation of multiple experiments (n=3). F, Western blot analysis of key markers of angiogenesis (VEGF), proliferation (cyclin D1), and extracellular matrix proteins (ColA1, SMA and FN) in NT and T xenografts. G, Analysis of cellular bioenergetics of cells (4×104) obtained from 7-and 15-week xenografts (n=3,*p<.05, and **p<.001). Bar graphs show mean ± SD.

Article Snippet: The membranes were incubated with the following primary antibodies at 1:1000 dilutions: UCP1 (ab10983, Abcam), UCP2 (AB3226, Millipore, Billerica, MA), UCP3 (ab3477, Abcam, Cambridge, MA), PRDM16 (sc130243, Santa Cruz Biotech, Dallas, TX), PGC-1α (sc-13067, Santa Cruz Biotech, CA), C/EBP-α (sc-16, Santa Cruz Biotech, Dallas, TX), PPARγ (sc-7273, Santa Cruz Biotech, Dallas, TX), COX IV (ab14744, Abcam, Cambridge, MA), COX-2 (160112, Cayman Chemicals, Ann Arbor, MI), ALDH1 (611195, BD Transduction, San Jose, CA), CD44 (3570S, Cell Signaling, Beverly, MA), Myf5 (sc-302, Santa Cruz Biotech, Dallas, TX); CD137 (ab3169, Abcam, Cambridge, MA), p38 MAPK (9212S, Cell Signaling, Beverly, MA), VEGF (ab46154, Abcam, Cambridge, MA), Cyclin D1 (sc 717, Santa Cruz Biotech, Dallas, TX), ColA1 (sc 25974, Santa Cruz Biotech, Dallas, TX), α-SMA (sc 53142, Santa Cruz Biotech, Dallas, TX), FN (HFN 7.1, Hybridoma Bank, Iowa City, IA), pp38 MAPK (9211S, Cell Signaling, Beverly, MA), and β-actin (sc-81178, Santa Cruz Biotech, Dallas, TX).

Techniques: Staining, Western Blot, Labeling