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