Journal: Nature Communications

Article Title: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

doi: 10.1038/ncomms10893

Figure Lengend Snippet: ( a ) Partial correlation network of DGKA-associated proteins in fibroblasts. Data are based on correlations of relative protein signals (unirradiated versus irradiated) in seven patient fibroblasts. Line thickness indicates the strength of correlation, dotted lines depict inverse correlation ( b ) Effect of inhibitors of DGKA (R59949) and PRKCA (Gö6976) on cell growth of fibroblasts from fibrosis or non-fibrosis patients exposed to ionizing radiation at various dose combinations. Data show cell growth determined in quadruplicates after 96h drug treatment with radiation applied after 48 h. Cell growth was measured by calcein fluorescence assay. ( c ) Additive or synergistic drug effects from b were calculated with the Bliss independence model comparing the expected (additive) effects with the observed effects. Scale bar depicts the observed viability differences in comparison to the expected additive effects as blue (synergistic growth suppression) to red (synergistic growth increase). ( d , e ) Impact of R59949 (5.0 μM) and Gö6976 (0.5 μM) on collagen synthesis in NHDF measured by COL1A1 mRNA expression ( d ) or hydroxyproline release ( e ). Cells were pre-treated with drugs for 48 h and analysed an additional 48 h after exposure to radiation or TGFB1 (4 ng ml −1 ) with concurrent drug treatment. Dots show mean values from duplicate ( d , e ) or quadruplicate ( c ) experiments for each fibroblast, bars depict mean±s.e.m. in each group. * P <0.05, ** P <0.01, Student's t -test.

Article Snippet: Western blot analysis was run with NuPAGE SDS-PAGE gel system (Life Technologies) and primary antibodies against DGKA (11547-1-AP, Proteintech), beta actin (ACTB; sc-47778 HRP, Santa Cruz Biotechnology), PRKCA (2056P, Cell Signaling Technology), collagen 1 (EPR7785, Abcam) and appropriate secondary antibodies (Santa Cruz Biotechnology).

Techniques: Irradiation, Fluorescence, Expressing

Journal: Frontiers in Immunology

Article Title: O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

doi: 10.3389/fimmu.2024.1387197

Figure Lengend Snippet: Pharmacological inhibition of OGT attenuates TGF-β1 induction of collagen expression and α-SMA (markers of FMT). (A) Representative western blot images of O-GlcNAc and β-actin from IPF and non-IPF primary HLFs. (B) Densitometric graphs of the ratio of O-GlcNAc to β-actin, n = 5. (C) Representative western blot images of O-GlcNAc, pan collagen, Col1α1, α-SMA, and β-actin expression from HLFs with or without OSMI-4 inhibition (10 μM) followed by TGF-β1 (5 ng/mL) stimulation for 48 hours. (D–H) Densitometric graphs of the ratio of O-GlcNAc, pan collagen, Col1α1, Col3α1, α-SMA, to β-actin, n = 5-7. (I) Representative immunofluorescence staining of Col1α1 (green) and α-SMA (red) expression; DAPI (blue). Data from multiple replicates are presented as mean ± SEM, each dot represents independent biological replicate. Outliers determined by the 1.5xIQR rule were excluded, and statistical analyses were done using the Student’s t-test and one-way ANOVA with Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Fixed cells were blocked in 1% BSA in 1X PBS at room temperature followed by incubation with primary antibodies: α-SMA-Cy3 (1:100; cat no: C6198; Millapore Sigma) and Collagen I [EPR7785] (1:200; cat no: ab138492; abcam) diluted in 0.1% BSA at 4°C overnight.

Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

doi: 10.3389/fimmu.2024.1387197

Figure Lengend Snippet: OGA inhibition drives the expression of key FMT markers. (A) Representative western blot images of O-GlcNAc, Col1α1, Col3α1, α-SMA, and β-actin expression from HLFs with TMG (1 μM) or TGF-β1 (5 ng/mL) stimulation for 48 hours. (B–E) Densitometric graphs of the ratio of O-GlcNAc, Col1α1, Col3α1, α-SMA, to β-actin, n = 4-5. (F) Representative immunofluorescence images of Col1α1 (green) and α-SMA (red) expression; DAPI (blue). Data from multiple replicates are presented as mean ± SEM, each dot represents independent biological replicate. Outliers determined by the 1.5xIQR rule were excluded, and statistical analyses were done using the Student’s t-test. *p<0.05, **p<0.01.

Article Snippet: Fixed cells were blocked in 1% BSA in 1X PBS at room temperature followed by incubation with primary antibodies: α-SMA-Cy3 (1:100; cat no: C6198; Millapore Sigma) and Collagen I [EPR7785] (1:200; cat no: ab138492; abcam) diluted in 0.1% BSA at 4°C overnight.

Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

doi: 10.3389/fimmu.2024.1387197

Figure Lengend Snippet: O-GlcNAc modified proteins identified by mass spectrometry involved in TGF-β signaling and fibrosis.

Article Snippet: Fixed cells were blocked in 1% BSA in 1X PBS at room temperature followed by incubation with primary antibodies: α-SMA-Cy3 (1:100; cat no: C6198; Millapore Sigma) and Collagen I [EPR7785] (1:200; cat no: ab138492; abcam) diluted in 0.1% BSA at 4°C overnight.

Techniques: Modification, Mass Spectrometry

Journal: Journal of Dietary Supplements

Article Title: Beta-Sitosterol Alters Collagen Distribution in Prostate Fibroblasts

doi: 10.1080/19390211.2023.2276943

Figure Lengend Snippet: Figure 4. Beta-sitosterol inhibits the export of collagen 1 and collagen 3 proteins in N1, but not pHPF, cells. N1 or pHPF cells were grown for 24 h in 5% HIE media, starved in SF HIE media for 48 h, then treated with increasing doses of beta-sitosterol or 4 ng/ml TGFβ for 48 h in SF HIE media. Protein recovered from the cells and media was subjected to immunoblotting against antibodies for collagen 1 (COL1), collagen 3 (COL3), or GAPDH (as a loading control). (A,C) N1 cells produce a low basal level of collagen 1 protein, which is decreasingly secreted into the media at higher beta-sitosterol concen tration (A) resulting in a significantly (p < .001) higher intracellular:Extracellular ratio of collagen 1 with increasing beta-sitosterol dosage compared to vehicle (C). (B,D) pHPF cells produce high levels of both collagen 1 and collagen 3 (B), and secretion of both proteins into the media is unaffected by beta-sitosterol treatment (D).

Article Snippet: Membranes were blocked for one hour using a 5% milk in TBS-T solution followed by primary antibody incubation a 5% milk TBS-T solution with anti-Collagen 1 (#EPR7785) from Abcam, anti-Collagen 3 (#30565), and GAPDH (#2118) from Cell Signaling Technologies.

Techniques: Western Blot, Control