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

Journal: iScience

Article Title: Targeting endometrial inflammation in intrauterine adhesion ameliorates endometrial fibrosis by priming MSCs to secrete C1INH

doi: 10.1016/j.isci.2023.107201

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Col1 , Proteintech , Cat# 14695-1-AP; RRID: AB_2082037.

Techniques: Staining, Recombinant, Cell Counting, Enzyme-linked Immunosorbent Assay, Western Blot, Software

Journal: Journal of Clinical Medicine

Article Title: The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype

doi: 10.3390/jcm9040961

Figure Lengend Snippet: Expression of GR, ACTA2 and extracellular proteins in peritubular cells of the monkey testis before and after puberty. Immunohistochemistry of GR of adult rhesus monkeys (right panels) shows strong GR expression in the peritubular compartment (arrows) and moderate staining of Sertoli cells (asterisks). Smooth muscle cells of blood vessels (arrowheads) served as a positive intrinsic control. GR staining of the peritubular wall was not found in all samples from immature rhesus monkeys (186–282 days, left panels and results not shown). Only interstitial cells (asterisks) and blood vessels (arrowheads) show a robust staining. ACTA2 is constantly expressed by vascular smooth muscle cells (arrowheads) in both immature and adult monkeys but only localized in the peritubular wall of mature monkeys (arrows). In immature rhesus, staining of ELN is restricted to the interstitial matrix (asterisks) but is not detected in the peritubular compartment, which in turn is the prevailing expression site in adult monkeys (arrows). Independent of age, Col1 is localized in the peritubular compartment (arrows), in the interstitial area (asterisks), and around blood vessels (arrowheads). Negative control experiments were performed with IgG (not shown). Hematoxylin was used to counterstain nuclei. Scale bars = 50 µm.

Article Snippet: In further IHC experiments, the following primary antibodies were used: anti-ACTA2 (1:200, mouse monoclonal anti-actin, Sigma A5228, St. Louis, MO, USA), Col1 (1:200, affinity-purified, polyclonal rabbit anti-Col1, R1038, Origene, Rockville, MD, USA), and affinity-purified, polyclonal rabbit anti-human ELN (1:200, A5228, Sigma Prestige Antibodies, St. Louis, MO, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Negative Control

Journal: Journal of Clinical Medicine

Article Title: The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype

doi: 10.3390/jcm9040961

Figure Lengend Snippet: Dex treatment of HTPCs rapidly increases FKBP5 mRNA, a known target of GR signaling, and affects mRNA levels of characteristic peritubular ECM markers, as well as cytoskeleton-associated genes. ( A ) Cultured HTPCs stimulated with Dex (1 µM) in the absence or presence of RU486 (1 µM), respond with a highly significantly increase ( p < 0.01) in FKBP5 mRNA levels after 6 h compared to control. RU486 completely blocks this effect ( p < 0.01) and does not alter FKBP5 mRNA expression within 6 h. ( B ) After Dex treatment of HTPCs for 24 h, the mRNAs of the elastic fiber components ELN and FBLN5 are highly significantly ( p < 0.01) increased. A slightly smaller ( p < 0.05) increase is observed for FBN1 mRNA. ( C ) The transcript concentration of the collagen fibers Col1 and Col3 do not change ( p > 0.05) after application of Dex, while ( D ) the mRNAs of cytoskeleton markers ACTA2 and PDLIM1 ( n = 8) are enriched ( p < 0.01) after stimulation of HTPCs with Dex. Data are means ± SEM after 6 ( A ), and 24 h ( B – D ), normalized to control conditions. Asterisks denote statistical significance, * p < 0.05, ** p < 0.01.

Article Snippet: In further IHC experiments, the following primary antibodies were used: anti-ACTA2 (1:200, mouse monoclonal anti-actin, Sigma A5228, St. Louis, MO, USA), Col1 (1:200, affinity-purified, polyclonal rabbit anti-Col1, R1038, Origene, Rockville, MD, USA), and affinity-purified, polyclonal rabbit anti-human ELN (1:200, A5228, Sigma Prestige Antibodies, St. Louis, MO, USA).

Techniques: Cell Culture, Expressing, Concentration Assay