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