Journal: Diabetes

Article Title: Fibroblast Growth Factor 6 Promotes Adipocyte Progenitor Cell Proliferation for Adipose Tissue Homeostasis.

doi: 10.2337/db22-0585

Figure Lengend Snippet: Figure 3—AAV-Adipoq–mediated FGF6 overexpression in iWAT prevents HFD-induced obesity by inducing beige fat biogenesis. A: Schematic representation of animal experiments. Eight-week-old C57BL/6 mice were injected with AAV-Adipoq-FGF6 (AAV-FGF6) or AAV-Adipoq-GFP (AAV-GFP) into the iWAT depot and fed an HFD for 10 weeks (n = 6 per group). B and C: Body weight and fat mass curve of HFD-fed AAV-GFP and AAV-FGF6 mice (n = 6 per group). D and E: GTT and ITT results in 10-week-old HFD-fed AAV-GFP and AAV-FGF6 mice with quantification of area under the curve (AUC) (n = 6 per group). F: The O2 consumption rates of HFD-fed mice were measured using a comprehensive laboratory animal monitoring system (n = 3 per group). ANCOVA with body weight as covariant (P = 0.018). G: Tissue weight as a percentage of body weight in the iWAT, gWAT, BAT, liver, and gastrocnemius (gas) of 10-week-old HFD-fed AAV-GFP and AAV-FGF6 mice (n = 6 per group). H: Representative IHC staining of PDGFRa and UCP1 in iWAT sections (n = 6 per group). Scale bar, 50 mm. I and J: Western blot analysis of PGC1a and UCP1 proteins in the iWAT of 10-week-old HFD-fed AAV-GFP and AAV- FGF6 mice under normal conditions or 7 days of cold exposure (n = 3 per group). K: mRNA levels of fibrosis-, apoptosis-, and hypoxia- related genes in the iWAT (n = 6 per group). L: Representative Masson trichrome staining results confirm iWAT fibrosis. Scale bar, 50 mm. M: Representative TUNEL staining confirms iWAT apoptosis. Scale bar, 12.5 mm. N: Representative IHC staining of a-SMA and hypoxia- inducible factor 1a (HIF1a) in the iWAT sections. Scale bar, 50 mm. O: Western blot analysis of indicated proteins in the iWAT (n = 6 per group). Data are presented as mean ± SEM. Significance was determined using a two-tailed Student t test (B–E and G, J, and K). *P < 0.05, **P < 0.01. a.u., arbitrary unit.

Article Snippet: For immunohistochemistry (IHC), sections of iWAT were pretreated with 3% H2O2 for 30 min to quench endogenous peroxidase activity, blocked in PBS containing 2.5% horse serum for 1 h, and treated overnight with the corresponding primary antibodies against PDGFRa (cat. no. 3174S; Cell Signaling), UCP1 (cat. no. ab209483; Abcam), COX IV (cat. no. GB11250; Servicebio Technology), F4/80 (cat. no. sc-52664; Santa Cruz Biotechnology), hypoxia-inducible factor 1a (cat. nos.

Techniques: Over Expression, Injection, Immunohistochemistry, Western Blot, Staining, TUNEL Assay, Two Tailed Test

Journal: Diabetes

Article Title: Fibroblast Growth Factor 6 Promotes Adipocyte Progenitor Cell Proliferation for Adipose Tissue Homeostasis.

doi: 10.2337/db22-0585

Figure Lengend Snippet: Figure 5—FGF6 overexpression in the iWAT improves aging-induced central fat redistribution and insulin resistance. A and B: AAV-FGF6 or AAV-GFP was injected into the iWAT of 16-month-old mice. Body weight (A) and fat mass (B) curves of AAV-GFP and AAV-FGF6 aging mice (n = 5 per group). C and D: Glucose and insulin tolerance tests of AAV-GFP and AAV-FGF6 aging mice with quantification of the area under the curve (AUC) (n = 5 per group). E: Tissue weight as a percentage of body weight in the iWAT, gWAT, BAT, liver, and gastrocnemius (gas) of aging AAV-GFP and AAV-FGF6 mice (n = 5 per group). F: Representative hematoxylin-eosin (H-E) and Masson trichrome staining in the iWAT of aging AAV-GFP and AAV-FGF6 mice. Scale bar, 50 mm. G: Representative TUNEL staining in the iWAT of aging AAV-GFP and AAV- FGF6 mice. Scale bar, 12.5 mm. H: Representative IHC staining of a-SMA, F4/80, and hypoxia-inducible factor 1a (HIF1a) in the iWAT of aging AAV-GFP and AAV-FGF6 mice. Scale bar, 50 mm. I: mRNA levels of fibrosis- and apoptosis-related genes in the iWAT of aging AAV-GFP and AAV-FGF6 mice (n = 5 per group). J: Representative IHC staining of PDGFRa and COX IV in the iWAT of aging AAV-GFP and AAV-FGF6 mice. Scale bar, 50 mm. K: mRNA levels of indicated APC markers and mitochondrial genes in the iWAT of aging AAV-GFP and AAV-FGF6 mice (n = 5 per group). L: Western blot analysis of indicated proteins in the iWAT of AAV-GFP and AAV-FGF6 mice (n = 5 per group). Data are presented as mean ± SEM. Significance was determined using a two-tailed Student t test (A–E, I, and K). *P < 0.05, **P < 0.01.

Article Snippet: For immunohistochemistry (IHC), sections of iWAT were pretreated with 3% H2O2 for 30 min to quench endogenous peroxidase activity, blocked in PBS containing 2.5% horse serum for 1 h, and treated overnight with the corresponding primary antibodies against PDGFRa (cat. no. 3174S; Cell Signaling), UCP1 (cat. no. ab209483; Abcam), COX IV (cat. no. GB11250; Servicebio Technology), F4/80 (cat. no. sc-52664; Santa Cruz Biotechnology), hypoxia-inducible factor 1a (cat. nos.

Techniques: Over Expression, Injection, Staining, TUNEL Assay, Immunohistochemistry, Western Blot, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: COX2 Enhances Neovascularization of Inflammatory Tenocytes Through the HIF-1α/VEGFA/PDGFB Pathway

doi: 10.3389/fcell.2021.670406

Figure Lengend Snippet: Knockdown of VEGFR2 and PDGFRβ inhibited the tube-forming ability of RAOECs of the conditioned medium from COX2-overexpressing tenocytes. (A,B) Western blot was performed to detect the protein expression level of VEGFR2 and PDGFRβ in RAOECs with VEGFR2-siRNA and PDGFRβ-siRNA transfection. (C,D) A three-dimensional Matrigel assay was conducted to assess the tube formation of RAOECs with VEGFR2-siRNA and PDGFRβ-siRNA transfection. The data were presented as the means ± SD; n = 3; ∗∗ p < 0.01; scale bar: 50 μm; the triangles represent the position of the tubes. Western blot was performed using tenocytes. The three-dimensional Matrigel assay was performed using RAOECs.

Article Snippet: After being blocked, the primary antibodies COX2 (Cell Signaling Technology, United States, #12282), HIF-1α (Cell Signaling Technology, United States, #14179), K-AC (Cell Signaling Technology, United States, #9441), VEGFR2 (Cell Signaling Technology, United States, #9698), PDGFRβ (Cell Signaling Technology, United States, #3161), and β-Actin (Cell Signaling Technology, United States, #8457) were added to membranes overnight at 4°C.

Techniques: Knockdown, Western Blot, Expressing, Transfection, Matrigel Assay

Journal: PLoS ONE

Article Title: Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1371/journal.pone.0209786

Figure Lengend Snippet: (A) A flow cytometric analysis showed that PDGFR-α and -β were both detected on C57B/L6 lung fibroblasts. (B) Mouse alveolar type 2 epithelial cells were isolated as described in the Materials and Methods section. The cells obtained were investigated by a FACS analysis. Neither PDGFR-α nor -β was detected in freshly isolated mouse alveolar type 2 epithelial cells. Similar results were obtained at least two independent experiments.

Article Snippet: As the primary antibodies, a rabbit anti-PDGFR-α antibody (Cell Signaling Technology) and a rabbit anti-PDGFR-β antibody (Cell Signaling Technology) and a FITC anti-mouse CD326(Ep-CAM) antibody (Biolegend) or anti-alpha smooth muscle actin antibody (abcam, Cambridge, UK) were used as the primary antibodies.

Techniques: Isolation

Journal: PLoS ONE

Article Title: Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1371/journal.pone.0209786

Figure Lengend Snippet: Immunoblotting was performed for the detection of PDGFRs, Akt, and their phosphorylation in lung fibroblasts. Murine lung fibroblasts were stimulated with PDGF-AA (10 ng/ml) and -BB (10 ng/ml) in the presence of the anti-PDGF-α antibody APA5, anti-PDGFR-β antibody APB5, or control Ab. APA5 inhibited the phosphorylation of PDGFR-α induced by PDGF-AA. APB5 inhibited the phosphorylation of PDGFR-β induced by PDGF-BB. The phosphorylation of Akt was also inhibited by both antibodies. Representative immunoblot (A) and corresponding densitometric quantification of PDGFRs phosphorylation and Akt phosphorylation (B-D) are shown.

Article Snippet: As the primary antibodies, a rabbit anti-PDGFR-α antibody (Cell Signaling Technology) and a rabbit anti-PDGFR-β antibody (Cell Signaling Technology) and a FITC anti-mouse CD326(Ep-CAM) antibody (Biolegend) or anti-alpha smooth muscle actin antibody (abcam, Cambridge, UK) were used as the primary antibodies.

Techniques: Western Blot, Phospho-proteomics, Control

Journal: PLoS ONE

Article Title: Blockade of platelet-derived growth factor receptor-β, not receptor-α ameliorates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1371/journal.pone.0209786

Figure Lengend Snippet: (A)The expression of PDGFRs in the lungs of BLM-treated mice was examined. Staining for PDGFRs was detected in fibrotic lungs. Several cells on alveolar wall showed weak staining for PDGFR-α. PDGFR-β was not clearly detected on alveolar wall. Mesenchymal cells clustering in fibrotic regions showed strong staining for PDGFR-α and -β; however, PDGFR-β was more strongly stained than PDGFR-α. (B) The double-staining with anti-PDGFR antibody and anti-EpCAM or anti-α SMA antibody. PDGFR-α and -β were expressed on α-SMA-positive fibroblasts, not EpCAM-positive lung epithelial cells.

Article Snippet: As the primary antibodies, a rabbit anti-PDGFR-α antibody (Cell Signaling Technology) and a rabbit anti-PDGFR-β antibody (Cell Signaling Technology) and a FITC anti-mouse CD326(Ep-CAM) antibody (Biolegend) or anti-alpha smooth muscle actin antibody (abcam, Cambridge, UK) were used as the primary antibodies.

Techniques: Expressing, Staining, Double Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice

doi: 10.1016/j.jcmgh.2021.09.001

Figure Lengend Snippet: CEP-1347 does not inhibit phosphorylation of JNK, ERK1, ERK2, or SMAD2 but reduces phosphorylation of p70S6K in human HSCs (LX-2). Human LX-2 cells were stimulated with ( A ) TGF-β or ( B , C ) PDGF-BB and co-stimulated with 1000 nM CEP-1347 (CEP) for ( A ) 24 hours or ( B , C ) 1 hour. ( A ) Densitometry data of phosphorylated protein expression levels were normalized to that of unphosphorylated proteins. The results are illustrated as representative blots (at least 6 per group; ∗ P < .05; ∗∗ P < .01; analysis of variance). ( B ) Phosphorylation of p70S6K (pp70S6K) was assessed by immunomicroscopy (green) and compared with unphosphorylated p70S6K. (C) Protein expression of pp70S6K was quantified by densitometry. Densitometric values of p70S6K were normalized to unphosphorylated p70S6K (n = 13; ∗ P < .05; analysis of variance).

Article Snippet: Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich).

Techniques: Phospho-proteomics, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice

doi: 10.1016/j.jcmgh.2021.09.001

Figure Lengend Snippet: CEP-1347 inhibits TGF-β–dependent activation of human HSCs (LX-2). ( A–E ) Human HSCs (LX-2) were activated by TGF-β in the presence or absence of CEP-1347 at the concentration of 1000 nM (CEP) for 24 hours before being harvested for immunoblotting or fluorescence microscopy. ( A ) Representative Western blot of α-SMA, PDGF receptor-β, and collagen 1α1, and ( B–D ) corresponding quantitative optical densitometry are shown. Densitometry data of protein-expression levels from at least 11 replicates were quantified (at least 11 per group; ∗ P < .05; ∗∗ P < .01; analysis of variance). ( E ) Representative fluorescence microscopy pattern of HSCs for α-SMA staining (green). Cell nuclei, counterstained with Hoechst, are visible in blue.

Article Snippet: Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich).

Techniques: Activation Assay, Concentration Assay, Western Blot, Fluorescence, Microscopy, Expressing, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice

doi: 10.1016/j.jcmgh.2021.09.001

Figure Lengend Snippet: CEP-1347 inhibits TGF-β–dependent migration and contractility of human HSCs (LX-2). ( A , B ) Representative figures of a migration assay upon stimulation with PDGF-BB and CEP-1347 (CEP). The reduction in gap size was quantified at baseline (left) and at 48 h (right) (n = 4; ∗∗ P < .01; t test). ( C , D ) Collagen matrix was populated with human LX-2 cells and contractility induced by addition of TGF-β with or without CEP-1347 for 120 hours. The gel area is expressed as a percentage of the well-area (n = 5; ∗∗ P < .01; analysis of variance).

Article Snippet: Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich).

Techniques: Migration

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice

doi: 10.1016/j.jcmgh.2021.09.001

Figure Lengend Snippet: CEP-1347 induces G2/M cell cycle phase arrest in human HSCs (LX-2). Cell cycle was evaluated using fluorescence-activated cell sorting analysis after being incubated for 24 hours with PDGF-BB, CEP-1347, or their combination (n = 6; ∗∗ P < .01; analysis of variance).

Article Snippet: Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich).

Techniques: Fluorescence, FACS, Incubation