Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Characterization of human PDGFR-β-positive pericytes from IPF and non-IPF lungs

doi: 10.1152/ajplung.00289.2018

Figure Lengend Snippet: Cell morphology and characteristics of isolated human lung platelet-derived growth factor receptor-β-positive (PDGFR-β+) pericytes. Representative PDGFR-β+ cells from nonidiopathic pulmonary fibrosis (IPF) controls and IPF lungs were assessed by phase contrast light microscopy after plating on plastic (A and B) or Matrigel (C and D). Pericytes were also assessed by immunofluorescence for PDGFR-β (E and F) and neural glial antigen-2 (NG2; G and H). Scale bar = 500 µm in A, B, and E–H and 100 µm in C and D.

Article Snippet: After being blocked with 1% BSA for 15 min at room temperature, cells were incubated overnight at 4°C with a rabbit monoclonal PDGFR-β antibody (clone Y92; Abcam), a rabbit polyclonal NG2 antibody (H-300; Santa Cruz Biotechnology) or a mouse monoclonal CD31 antibody (clone JC/70A; Novus).

Techniques: Isolation, Derivative Assay, Light Microscopy, Immunofluorescence

Journal: PLOS Pathogens

Article Title: Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease

doi: 10.1371/journal.ppat.1011272

Figure Lengend Snippet: ( A ) Flow cytometry analysis of HeLa and HeLa R5 cells stained with antibodies against CSPG4. (B) Immunoblot analysis of CSPG4 and GAPDH levels using protein lysates from HeLa and HeLa R5 cells. (C) RNA was extracted from HeLa and HeLa R5 cells, and RT-qPCR was used to quantify CSPG4 transcripts. RT-qPCR data are presented as mean (n = 3) ± S.D. ( D ) Immunoblot analysis of CSPG4 in HeLa R5 and HeLa R5 cells transfected with pEF6-CSPG4-myc-his. ( E ) Comparison of TcdB2 CPE in HeLa R5 and HeLa R5 (pEF6-CSPG4-myc-his) after treatment with 4 pM of toxin for 24 h. * p < 0.05 determined by Student’s t-test.

Article Snippet: Primary antibodies used for immunoblots in this study were a mouse monoclonal antibody recognizing nonglucosylated Rac1 (BD Bioscience; catalog no. 610651); a mouse monoclonal antibody recognizing total Rac1 (EMD Millipore; catalog no. 05–389); a mouse monoclonal antibody against GAPDH (Abcam; product # ab8245); a mouse monoclonal against CSPG4 (R&D Biosystems; product # MAB2585); a mouse monoclonal against YAP1 (Santa Cruz Biotechnology; product # sc-101199); and a rabbit monoclonal against TFPI (Abcam; product # ab260041).

Techniques: Flow Cytometry, Staining, Western Blot, Quantitative RT-PCR, Transfection, Comparison

Journal: PLOS Pathogens

Article Title: Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease

doi: 10.1371/journal.ppat.1011272

Figure Lengend Snippet: ( A ) HeLa R5 cells were exposed to 5-AZA-CdR for 48 h and then RT-qPCR was used to quantify CSPG4 transcripts. RT-qPCR data are presented as mean (n = 3) ± S.D. ( B ) The top statistically significant canonical pathways correlating (positive z score) with HeLa R5 cells. ( C ) Upstream regulators predicted to be activated in HeLa R5 cells based on integrated pathway analysis. ( D ) Comparison of ESR1 transcripts between HeLa and HeLa R5 cells presented as the mean (n = 3) ± S.D. ( E ) HeLa cells were cultured in 0.5% charcoal stripped FBS and phenol red free media for 2 h prior to a 24 h exposure to non-encapsulated ß-estradiol or encapsulated ß-estradiol. RT-qPCR was then utilized to quantify CSPG4 transcripts exhibited as mean (n = 3) ± S.D. ( F ) HeLa cells were exposed to encapsulated ß-estradiol for 24 h and then an immunoblot was performed on the resulting protein extracts. *p < 0.05 determined by Student’s t-test.

Article Snippet: Primary antibodies used for immunoblots in this study were a mouse monoclonal antibody recognizing nonglucosylated Rac1 (BD Bioscience; catalog no. 610651); a mouse monoclonal antibody recognizing total Rac1 (EMD Millipore; catalog no. 05–389); a mouse monoclonal antibody against GAPDH (Abcam; product # ab8245); a mouse monoclonal against CSPG4 (R&D Biosystems; product # MAB2585); a mouse monoclonal against YAP1 (Santa Cruz Biotechnology; product # sc-101199); and a rabbit monoclonal against TFPI (Abcam; product # ab260041).

Techniques: Quantitative RT-PCR, Comparison, Cell Culture, Western Blot

Journal: PLOS Pathogens

Article Title: Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease

doi: 10.1371/journal.ppat.1011272

Figure Lengend Snippet: ( A ) Depiction of the Hippo signaling pathway and the effect of inhibition by XMU-MP-1 and TRULI. ( B ) CSPG4 transcript levels in HeLa and HT-29 cells treated with 3 μM XMU-MP-1 for 7 h. CSPG4 transcript levels are presented as mean (n = 3) ± S.D. ( C ) Immunoblot detection of CSPG4 from HeLa and HT-29 cells exposed to 6 μM of XMU-MP-1 for 24 h. ( D ) CSPG4 transcript levels in HeLa and HT-29 cells treated with 30 μM TRULI for 24 h. CSPG4 transcript levels are presented as mean (n = 3) ± S.D. (E) Immunoblot detection of CSPG4 from HeLa and HT-29 cells exposed to 30 μM of TRULI for 24 h. ( F ) CSPG4 transcript levels in HeLa and YAP1 knock out cells (HeLa ΔYAP1 ). CSPG4 transcript levels are presented as mean (n = 3) ± S.D. ( G ) Immunoblot analyzing YAP1 and CSPG4 levels in HeLa cells and HeLa ΔYAP1 . *p < 0.05 determined by Student’s t-test.

Article Snippet: Primary antibodies used for immunoblots in this study were a mouse monoclonal antibody recognizing nonglucosylated Rac1 (BD Bioscience; catalog no. 610651); a mouse monoclonal antibody recognizing total Rac1 (EMD Millipore; catalog no. 05–389); a mouse monoclonal antibody against GAPDH (Abcam; product # ab8245); a mouse monoclonal against CSPG4 (R&D Biosystems; product # MAB2585); a mouse monoclonal against YAP1 (Santa Cruz Biotechnology; product # sc-101199); and a rabbit monoclonal against TFPI (Abcam; product # ab260041).

Techniques: Inhibition, Western Blot, Knock-Out