Journal: PLoS ONE

Article Title: CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling

doi: 10.1371/journal.pone.0159490

Figure Lengend Snippet: ( A ) N1 fibroblasts were treated with vehicle (DMSO) or EGFR inhibitor (AG1478, 500 nM) prior to CXCL12 treatment. Phosphorylation of EGFR, Akt, Smad and ERK1/2 were assessed via western blot. ( B ) qRT-PCR analysis of myofibroblast marker expression after CXCL12 (100pM) treatment in the presence or absence of AG1478 (500 nM). Expression levels of α-smooth muscle actin (ACTA2) and collagen 1α1 (COL1α1) were analyzed over the course of 24 hours of treatment. Treatment with AG1478 reduced or ablated the CXCL12/CXCR4 -mediated stimulation of both the ACTA2 and COL1α1 genes. ( C ) Western blot analysis of fibroblast markers, α-smooth muscle actin and collagen 1, at 24 and 48 hours after CXCL12 treatment. Secretion and incorporation of collagen 1 was inhibited in the presence of AG1478, α-smooth muscle actin production was inhibited as well. ( D ) Signal intensity quantification for 7 and collagen 1 western blots. * = p-value < 0.05. Error bars, SE.

Article Snippet: Primary antibodies were diluted in blocking solution and included 1:200 dilution FITC-conjugated mouse monoclonal anti-α-smooth muscle actin (α SMA), and 1:100 dilution biotin-conjugated rabbit polyclonal anti-collagen type 1 (COL1) (Rockland Immunochemicals, Gilbertsville, PA).

Techniques: Phospho-proteomics, Western Blot, Quantitative RT-PCR, Marker, Expressing

Journal: PLoS ONE

Article Title: CXCL12/CXCR4 Axis Activation Mediates Prostate Myofibroblast Phenoconversion through Non-Canonical EGFR/MEK/ERK Signaling

doi: 10.1371/journal.pone.0159490

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of N1 fibroblasts left untreated (-CXCL12) or treated with 100pM CXCL12 (CXCL12) for 48 hours in the absence or presence of CXCR4 (250 μM AMD3100), EGFR (250 uM AG1478), ALK-5 (TGFβRII) (20μM A-83-01) small molecular inhibitors or an antibody against TGFβRII (200 ng/ml TGFβ MAb). Figure depicts photomicrographs images of cells stained for α-smooth muscle actin (green) or collagen 1 (red) proteins or DAPI (blue nuclear stain); orange color indicates α-smooth muscle actin and collagen 1 colocalization in the merged image. ( B ) Western blot analysis of fibroblast markers, α-smooth muscle actin and collagen 1 at 24, 48 and 72 hours after CXCL12 (100pM) of scramble, anti-CXCR4, and anti-TGFβRI siRNAs transfected fibroblasts. CXCL12-driven expression of myofibroblasts markers does not require the presence or activation of TGFβRI. ( C ) Quantification of western blot images for myofibroblast markers.

Article Snippet: Primary antibodies were diluted in blocking solution and included 1:200 dilution FITC-conjugated mouse monoclonal anti-α-smooth muscle actin (α SMA), and 1:100 dilution biotin-conjugated rabbit polyclonal anti-collagen type 1 (COL1) (Rockland Immunochemicals, Gilbertsville, PA).

Techniques: Immunofluorescence, Staining, Western Blot, Transfection, Expressing, Activation Assay

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Article Title:

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Figure Lengend Snippet: Development and characterization of an experimental chronic kidney disease (CKD) model in mice (A) The experimental design for the mouse CKD model. (B) Glomerular filtration rate (GFR) in kidney of the CKD models versus shams in mice (C) Blood urea nitrogen (BUN) in kidney of the CKD models versus shams in mice. (D) Urine protein to creatinine ratio (uPCR) in kidney of the CKD models versus shams in mice. (E and F) Morphology studies in kidney of the CKD models versus shams in mice. (G) Western blot showing collagen 1 and α-SMA protein expressions in the kidney of CKD model and vehicle at the timeline indicated in the figure. (H and I) Representative images of Picrosirius Red stain for fibrillar collagen 45 days after nephrectomy (20x magnification and scale bar, 50 μm) and quantification of interstitial fibrosis as compared with sham control, bilateral ischemia, and nephrectomy controls. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n = 6/group unless otherwise stated. Data are represented as mean ± SEM.

Article Snippet: Rabbit polyclonal anti-Collagen 1 , Abcam , Cat# ab600408; RRID: AB_10556808.

Techniques: Filtration, Western Blot, Staining

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Article Title:

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Figure Lengend Snippet: SMOC2 knockout mice are protected from kidney function loss and fibrogenesis in the experimental CKD model (A) Time course of glomerular filtration rate (GFR) showing the effect of SMOC2 knockout (KO) versus WT in CKD models. (B and C) Time course of blood urea nitrogen (BUN) and urine protein creatinine ratio (uPCR) in CKD models of SMOC2 KO and WT. (D and E) Picrosirius Red staining showing the interstitial fibrosis and quantification in the CKD models of SMOC2 KO and WT. (F) Western blots showing the fibrotic protein levels in the mouse CKD kidney in response to SMOC2 depletion. (G and H) Representative images and quantification of interstitial fibrosis by immunofluorescent staining of collagen 1, α-SMA and fibronectin. (I and J) Expression of collagen 1 and α-SMA and inflammatory genes of IL-1β and TNFα mRNAs in SMOC2 KO and WT mice. (K–M) Representative images and quantification of LTL+, PDGFRβ+, F4/80 + cells in WT versus SMOC2 KO CKD mouse model. Immunofluorescence staining with 40x magnification and scale bar, 20 μm. n = 6/group unless otherwise stated. Data are represented as mean ± SEM.

Article Snippet: Rabbit polyclonal anti-Collagen 1 , Abcam , Cat# ab600408; RRID: AB_10556808.

Techniques: Knock-Out, Filtration, Staining, Western Blot, Expressing, Immunofluorescence

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Article Title:

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Figure Lengend Snippet: Silencing SMOC2 with siRNA prevents development of tubule-interstitial inflammation, fibrosis, and kidney function loss in the experimental mouse CKD model (A) Glomerular filtration rate (GFR) measurement in the CKD mice treated with PBS or SMOC2 siRNA (30 μg/200 μL) twice/week starting day 21 after disease onset till day 43 for a total of seven treatments. (B and C) Time course of blood urea nitrogen (BUN) and total urine protein to creatinine ratio (uPCR) in CKD mice treated with PBS and SMOC2 siRNA. (D and E) Picosirius Red staining showing the interstitial fibrosis and quantification. (F) Western blots showing the effect of silencing SMOC2 on fibrotic protein of collagen 1, α-SMA, and fibronectin in whole kidney lysates. (G and H) Representative immunofluorescent images and quantification of protein expressions of collagen 1, α-SMA, and fibronectin in the CKD kidneys after SMOC2 silencing. (I and J) mRNA levels of genes encoding collagen 1, α-SMA, and inflammatory genes of IL-1β and TNFα in CKD mice treated with PBS or SMOC2 siRNA. (K–M) Representative images and quantification of LTL+, PDGFRβ+, F4/80 + cells in PBS versus SMOC2 siRNA-treated CKD mice. Immunofluorescence staining with 40x magnification and scale bar, 20 μm. n = 6/group unless otherwise stated. Data are represented as mean ± SEM.

Article Snippet: Rabbit polyclonal anti-Collagen 1 , Abcam , Cat# ab600408; RRID: AB_10556808.

Techniques: Filtration, Staining, Western Blot, Immunofluorescence

Journal:

Article Title:

doi:

Figure Lengend Snippet: Lysosome-autophagosome signaling pathways are involved in SMOC2-induced kidney fibrosis (A) In NIH/3T3 cells, the time course of SMOC2 (100 ng/mL)-induced Smad2/3 and mTOR phosphorylation (5 min, 15 min, 30 min, 1 h, 4 h, and 24 h; TGFβ1 stimulation for 30 min was used as a positive control for Smad activation). (B and C) The dose-dependent effect of lysosome and autophagy inhibitor chloroquine (0.01, 0.1, 10, and 100 μM) on SMOC2 (100 ng/mL)-induced Smad 2/3 phosphorylation and collagen 1 expression. (D and E) The dose-dependent effect of lysosome inhibitor, Bafilomycin (0.01, 0.1, 10, and 100 μM) on SMOC2 (100 ng/mL)-induced Smad 2/3 phosphorylation and collagen 1 expression. (F) The effect of mTOR activator, MHY1485 (10 μM), and autophagy inhibitor, 3-MA (10 μM), on SMOC2 (100 ng/mL)-induced collagen 1 expression in NIH/3T3 cells. (G and H) The effect of inhibiting autophagosomes by siRNA transduction of BECN1 on SMOC2 (100 ng/mL)-induced collagen 1 expression. Blots are representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-Collagen 1 , Abcam , Cat# ab600408; RRID: AB_10556808.

Techniques: Positive Control, Activation Assay, Expressing, Transduction

Journal:

Article Title:

doi:

Figure Lengend Snippet: SMOC2 induces kidney fibrosis by cross-activating signaling cascades of TGFβ, Integrin, MAPKs, and Akt (A) Western blots showing collagen 1 production in response to several stimuli at 24 h. Note that SMOC2 stimulation markedly increases collagen 1 expression. ALK5/TGFβR II inhibitor of GW788388 (10 μM), ALK5 inhibitor of SB431542 (10 μM), and TGFβR I/II inhibitor of LY2109761 (10 μM) block the SMOC2 (100 ng/mL, 24 h)-induced collagen 1 expression. (B) Western blots show that SMOC2 (100 ng/mL, 24 h)-induced collagen 1 responses of the different inhibitors, integrin α5 antagonist of ATN-121 (100 μM) and integrin antagonist of SB273005 (1 μM). (C) Western blots show the effects of inhibition of different signaling pathways on the SMOC2-induced collagen 1 expression with p42/44-MAPK inhibition (U0126 [10 μM]), p38-MAPK inhibition (SB203580 [10 μM]), JNK/SAPK inhibition (SP600125 [10 μM]), and PI3K/Akt inhibition (LY294002 [20 μM]). (D and E) Immunostaining for F-Actin shows effects on NIH/3T3 of co-stimulation of SMOC2 (100 ng/mL, 24 h) with inhibitors or antagonists (U0126, SP600125, SB203580, LY294002, LY2109761, GW788388, SB431542, SB273005). Positive controls consisted of predicted modulation of TGFβ1 (10 ng/mL, 24 h) stimulation and cascades inhibitors (SB431542, LY2109761, GW788388). Immunofluorescence staining with 40x magnification and scale bar, 20 μm. Images and blots are representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-Collagen 1 , Abcam , Cat# ab600408; RRID: AB_10556808.

Techniques: Western Blot, Expressing, Blocking Assay, Inhibition, Immunostaining, Immunofluorescence, Staining