Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: Fibroblast-specific ROCK2-deficient (ROCK2Postn–/–) and littermate control (ROCK2flox/flox) mice (A–H), and fibroblast-specific constitutively active ROCK knock-in (caROCKPostn–/–) and littermate control (caROCKflox/flox) mice (I–N) were treated with saline or Ang II for 4 wk. (A and I) Representative immunoblots of ROCK1, ROCK2, and ROCK activity, as assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), in heart tissues from each experimental genotype. (B and C) Quantification of ROCK1 and ROCK2 protein expression, and (D and J) ROCK activity levels by densitometry (n = 4–6 each). (E and K) Representative immunoblots of FGF2, CTGF, and α-SMA in heart tissues from each experimental genotype. (F–H and L–N) Quantification of FGF2, CTGF, and α-SMA protein expression by densitometry (n = 4–6 each). *P < 0.05 vs. saline-treated each genotype. #P < 0.05 vs. Ang II–treated respective controls. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Control, Knock-In, Saline, Western Blot, Activity Assay, Binding Assay, Expressing

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A and B) Quantitative PCR analysis mRNA levels of Rock1 and Rock2; (C) a prohypertrophic mediator of Fgf2 (encoding fibroblast growth factor 2); (D) a profibrotic mediator of Ctgf (connective tissue growth factor); (E) a fibroblast-myoblast differentiation marker, Acta2 (α-smooth muscle actin); (F and G) hypertrophic markers of Nppa (atrial natriuretic factor) and Acta1 (skeletal muscle α-actin); and (H and I) fibrotic markers of Col1a (collagen type I) and Fn1 (fibronectin 1) in heart tissues from ROCK2Postn–/– and littermate control (ROCK2flox/flox) mice at 4 wk after saline or Ang II infusion (n = 4–7 each). *P < 0.05, **P < 0.01 vs. saline-treated ROCK2flox/flox mice. #P < 0.05 vs. Ang II-treated ROCK2flox/flox mice. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Marker, Control, Saline

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A and B) Representative immunoblots and densitometric quantification of ROCK activity, as assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), stimulated by 1 μM Ang II or 10 ng/ml TGF-β1 for 24 hours, with or without 10 μM Y27632, a specific ROCK inhibitor, in MEFs isolated from WT mice (n = 3–4 each). (C–F) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by Ang II or TGF-β1, with or without Y27632, in WT MEFs (n = 3–4 each). *P < 0.05, **P < 0.01 vs. vehicle-stimulated WT MEFs. ##P < 0.01 vs. the same stimulated WT MEFs without Y27632. ††P < 0.01 vs. Ang II–stimulated WT MEFs without Y27632. (G–J) Representative immunoblots and densitometric quantification of ROCK1 and ROCK2 protein expression and ROCK activity, as assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), stimulated by Ang II or TGF-β1, in MEFs isolated from WT, global Rock1-KO (ROCK1–/–), and global Rock2-KO (ROCK2–/–) mice (n = 3–4 each). (K–N) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by Ang II or TGF-β1, in WT, global ROCK1–/–, and global ROCK2–/– MEFs (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated WT MEFs. #P < 0.05, ##P < 0.01 vs. the same stimulated WT MEFs. ††P < 0.01 vs. Ang II–stimulated WT MEFs. ‡P < 0.05, ‡‡P < 0.01 vs. TGF-β1-stimulated global ROCK1–/– MEFs. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Western Blot, Activity Assay, Binding Assay, Isolation, Expressing

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A–C) Quantitative PCR analysis of Fgf2 (encoding fibroblast growth factor 2), Ctgf (connective tissue growth factor), and Acta2 (α-smooth muscle actin) mRNA expression stimulated by 1 μM Ang II or 10 ng/ml TGF-β1 for 24 hours, with or without 10 μM Y27632, a specific ROCK inhibitor, in MEFs isolated from WT (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated WT MEFs. #P < 0.05, ##P < 0.01 vs. the same stimulated WT MEFs without Y27632. †P < 0.05, ††P < 0.01 vs. Ang II–stimulated WT MEFs without Y27632. (D–F) Quantitative PCR analysis of Fgf2, Ctgf, and Acta2 mRNA expression stimulated by 1 μM Ang II or 10 ng/ml TGF-β1 for 24 hours in MEFs isolated from WT, global Rock1-KO (ROCK1–/–), and global Rock2-KO (ROCK2–/–) mice (n = 3–4 each). *P < 0.01, **P < 0.01 vs. vehicle-stimulated WT MEFs. #P < 0.05, ##P < 0.01 vs. the same stimulated WT MEFs. †P < 0.05, ††P < 0.01 vs. Ang II–stimulated WT MEFs. ‡P < 0.05 vs. TGF-β1–stimulated global ROCK1–/– MEFs. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A and B) Representative immunoblots and densitometric quantification of ROCK activityas assessed by the ratio of phosphorylated form of the myosin-binding subunit (MBS) to total MBS (p-MBS/t-MBS), stimulated by 10 ng/ml TGF-β1 for 24 hours, with or without 10 μM Y27632, a specific ROCK inhibitor, in RNCFs (n = 3–4 each). (C–F) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by TGF-β1, with or without Y27632, in RNCFs (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated RNCFs. ##P < 0.01 vs. TGF-β1–stimulated RNCFs without Y27632. (G–J) Representative immunoblots and densitometric quantification of ROCK1 and ROCK2 protein expression and ROCK activity, stimulated by TGF-β1, in RNCFs transfected with control, ROCK1, or ROCK2 siRNA (n = 3–4 each). (K–N) Representative immunoblots and densitometric quantification of FGF2, CTGF, and α-SMA protein expression, stimulated by TGF-β1, in RNCFs transfected with control, ROCK1, or ROCK2 siRNA (n = 3–4 each). **P < 0.01 vs. vehicle-stimulated RNCFs transfected with control siRNA. ##P < 0.01 vs. TGF-β1–stimulated RNCFs transfected with control siRNA. †P < 0.05, ††P < 0.01 vs. TGF-β1–stimulated RNCFs transfected with ROCK1 siRNA. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Western Blot, Binding Assay, Expressing, Activity Assay, Transfection, Control

Journal: JCI Insight

Article Title: Fibroblast deletion of ROCK2 attenuates cardiac hypertrophy, fibrosis, and diastolic dysfunction

doi: 10.1172/jci.insight.93187

Figure Lengend Snippet: (A) Detection of CTGF and FGF2 protein expression in the mixture of supernatant and eluted extracellular matrix of RNCFs, stimulated by 10 ng/ml TGF-β1 for 24 hours, with or without 2.5 μM Y27632. Ponceau S staining of the membrane shows equal loading. (B) ELISA quantification of FGF2 concentration in eluted extracellular matrix of RNCFs, stimulated by TGF-β1 with or without Y27632 (n = 6–9, each in triplicate). **P < 0.01 vs. vehicle-stimulated RNCFs. ##P < 0.01 vs. TGF-β1-stimulated RNCFs without Y27632. (C) Detection of CTGF and FGF2 protein expression in the mixture of supernatant and eluted extracellular matrix of TGF-β1–stimulated RNCFs, transfected with control, ROCK1, or ROCK2 siRNA. (D) ELISA quantification FGF2 concentration in eluted extracellular matrix of TGF-β1–stimulated RNCFs, transfected with control, ROCK1, or ROCK2 siRNA (n = 5–8, each in triplicate). **P < 0.01 vs. vehicle-stimulated RNCFs transfected with each siRNA. ##P < 0.01 vs. TGF-β1–stimulated RNCFs transfected with control siRNA. †P < 0.05 vs. TGF-β1–stimulated RNCFs transfected with ROCK1 siRNA. Data are expressed as mean ± SEM. (E and F) Representative fluorescent images and quantification of cellular hypertrophy of rat H9C2 cardiomyocytes stained with sarcomeric α-actinin (green), in response to 0–100 ng/ml FGF2 for 24 hours. Nuclei are stained with DAPI (blue). (n = 30–50 each). Scale bars: 25 μm. (G–I) Quantification of RT-PCR analysis of hypertrophic markers of Acta1 (encoding skeletal muscle α-actin), Myh7 (β-myosin heavy chain), and Nppa (atrial natriuretic factor) in H9C2 cells stimulated by FGF2 (n = 3–4 each). *P < 0.05, **P < 0.01 vs. vehicle-stimulated H9C2 cells. Data are expressed as mean ± SEM. P values were calculated using one-way ANOVA with Tukey’s HSD test.

Article Snippet: FGF2 protein in this NaCl wash buffer was quantitated using rat FGF2 ELISA kit according to manufacturer’s instructions (MFB00; R&D Systems).

Techniques: Expressing, Staining, Membrane, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Control, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: ColXV Aggravates Adipocyte Apoptosis by Facilitating Abnormal Extracellular Matrix Remodeling in Mice

doi: 10.3390/ijms21030959

Figure Lengend Snippet: ColXV promoted ECM deposition in adipose tissue via fibroblast growth factor 2 (FGF2)/ fibroblast growth factor receptor 1 (FGFR1) axis. ( A ) Representative images of adipocytes in different groups stained by FGFR1 (green) and FGF2 (red). Arrowheads indicate FGF2 enriched to the cell membrane. Scale bar, 10 µm. ( B ) Relative mRNA levels of FGFR1, FGFR2, FGFR3, and FGFR4 of adipocytes in different groups ( n = 4). ( C - D ) Representative images of adipocytes in different groups were stained by FGFR1 (green) and p-FGFR1 Tyr653/Tyr654 (red). Arrowheads indicate FGFR1 transferred into the nucleus. Scale bar, 100 µm ( n = 4). ( E – H ) Relative protein phosphorylation levels of FGFR1 Tyr653/Tyr654 , AKT, and ERK1/2 of two groups in adipocytes with gradients concentration of FGF2 ( n = 4). ( I – J ) Relative protein levels of FN, Col I, MMP-2, MMP-9, and TIMP-1 of adipocytes in different groups ( n = 4). Values are presented as mean ± SEM. * p < 0.05, ** p < 0.01, ns, not significant.

Article Snippet: In experiments involving the administration of Ad-Col15a1 or sh-Col15a1 (24 h or 48 h at the titer of 1 × 10 9 IFU/mL), AMPK inhibitor (Compound C, Selleck, Houston, Texas, USA), mTORC1 inhibitor (rapamycin, Selleck), MMP agonist (4-Aminophenylmercuric acetate, Sigma–Aldrich, St. Louis, Missouri, USA), FGFR1 inhibitor (PD173074, Selleck), LOX inhibitor (β-APN, Sigma–Aldrich), or FGF2 recombinant protein (50037-M07E, Sino Biological, Beijing, China), cells were allowed to adhere for 48 h before treatment.

Techniques: Staining, Concentration Assay

Journal:

Article Title: Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

doi: 10.1096/fj.09-134056

Figure Lengend Snippet: VEGF-A, FGF-2 and VEGF-C-induced corneal angiogenesis and lymphangiogenesis in C57BL/6 and BALB/c mice. A) Pellets containing VEGF-A (200 ng), FGF-2 (100 ng), VEGF-C (400 ng), or vehicle control (PBS) were implanted into corneas of C57BL/6 and BALB/c mice. Corneal angiogenesis and lymphangiogenesis were examined 6 d after implantation. B) Double staining of corneal flat mounts for angiogenic (CD31, green) and lymphangiogenic (LYVE-1, red) endothelium. Scale bar = 400 μm. C, D) Quantitative analysis of angiogenesis (C) and lymphangiogenesis (D) in PBS-, VEGF-A-, FGF-2-, or VEGF-C-implanted corneas on d 6 (n=5–11). E) Quantitation of the number of lymphatic tips in corneas of VEGF-A-, FGF-2-, VEGF-C-, or vehicle control-implanted eyes (n=3–13). *P < 0.05; **P < 0.01.

Article Snippet: Matrigel plug assay Mice were injected subcutaneously with 0.2 ml Matrigel (356230; BD Biosciences) containing PBS, 1 μg FGF-2 (3139-FB; R&D Systems), or 2 μg VEGF-C (2179-VC; R&D Systems).

Techniques: Double Staining, Quantitation Assay

Journal:

Article Title: Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

doi: 10.1096/fj.09-134056

Figure Lengend Snippet: Corneal lymphangiogenesis and conjunctival lymphatic vessels. Corneal flatmounts with lymphangiogenesis were examined on d 6 after implantation and divided into two groups, with or without preexisting lymphatic vessels in the conjunctiva. A) Double staining of corneal flatmounts for angiogenesis (CD31, green) and lymphangiogenesis (LYVE-1, red) with or without lymphatic tube structures in VEGF-A-implanted BALB/c mice. Blood vessels (arrowheads) and lymphatic vessels (arrows) in the conjunctiva are indicated. B) Frequency of the corneas with LYVE-1+ lymphatic tube structures in the pellet-implanted site of C57BL/6 and BALB/c mice. C) Quantitative analysis of lymphangiogenesis (white) and angiogenesis (black) in VEGF-A-implanted BALB/c corneas on d 6 (n=4 and 7). D) Photographs and immunohistochemistry of CD31 and LYVE-1 with or without conjunctival removal. E, F) Corneal angiogenesis and lymphangiogenesis were examined 6 d after PBS or VEGF-A implantation with or without conjunctival removal. Arrows indicate lymphatic hyperplasia around the excised area. G, H) Quantitative analysis of lymphangiogenesis (G) and angiogenesis (H) in PBS- or VEGF-A-implanted corneas on d 6 with or without conjunctival removal (n=4–7). I, J) Correlation between lymphangiogenic area (d 6) and limbus lymphatic ratio in VEGF-A (I) or FGF-2 implantation (J) (n=6–11). *P < 0.05; **P < 0.01. Scale bars = 400 μm.

Article Snippet: Matrigel plug assay Mice were injected subcutaneously with 0.2 ml Matrigel (356230; BD Biosciences) containing PBS, 1 μg FGF-2 (3139-FB; R&D Systems), or 2 μg VEGF-C (2179-VC; R&D Systems).

Techniques: Double Staining, Immunohistochemistry

Journal:

Article Title: Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

doi: 10.1096/fj.09-134056

Figure Lengend Snippet: GF-induced angiogenesis and lymphangiogenesis in Matrigel plug assay. A) Double staining for angiogenic (CD31, green) and lymphatic endothelium (LYVE-1, red) of PBS-, FGF-2-, or VEGF-C-containing Matrigel in C57BL/6 and BALB/c mice, 10 d after subcutaneous injection of Matrigel. B, C) Quantitative analysis of LYVE-1+ area (red, B) and CD31+ area (green, C) in Matrigel (n=4). D) Double staining for lymphatic endothelium (podoplanin, red) and macrophages (CD11b, green) of Matrigel in C57BL/6 and BALB/c mice (d 10). E, F) Quantitative analysis of podoplanin+ area (red, E) and CD11b+ area (green, F) in Matrigel (n=4). G) Double staining for lymphatic endothelium (LYVE-1, red) and proliferating marker (Ki67, green) of FGF-2-containing Matrigel in BALB/c mice (d 10). H) Quantitative analysis of number of LYVE1+ Ki67+ cells in Matrigel (n=4). *P < 0.05; **P < 0.01. Scale bars = 100 μm (A, D); 50 μm (G).

Article Snippet: Matrigel plug assay Mice were injected subcutaneously with 0.2 ml Matrigel (356230; BD Biosciences) containing PBS, 1 μg FGF-2 (3139-FB; R&D Systems), or 2 μg VEGF-C (2179-VC; R&D Systems).

Techniques: Matrigel Assay, Double Staining, Injection, Marker

Journal: eLife

Article Title: Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex

doi: 10.7554/eLife.61432

Figure Lengend Snippet:

Article Snippet: The following plasmids were used in this study: pCDNA (control vector) (Clontech), pCDNA3-EGFP (Addgene Plasmid #13031), pCMV-mCherry-Mem (Addgene Plasmid #55779), pcDNA-Wnt3A (Addgene Plasmid #35908), pIRES-Jag1-HA (Addgene Plasmid #17336), pCMV-R-GECO1 (Addgene Plasmid #32444), pCMV-mCherry-CD9 (Addgene Plasmid #55013), pCMV-mCherry-CD81 (Addgene Plasmid #55012), pEGFP-CD63-C2 (Addgene Plasmid #62964), pCMV-EGFP-Rab18 (Addgene Plasmid #49550), pEGFP-C1-hMyoX (Addgene Plasmid #47608), pCMV6-hGAS1-Myc-DDK (Origene Cat: RC224804), pCMV3-mFGF2-N-GFPSpark (SinoBiological Cat: MG50037-ANG), pCMV3-mBMP2-C-GFPSpark (SinoBiological Cat: MG51115-ACG), pCMV-mCherry2, pCDNA3-mSHH-FL, pCDNA3-mSHH-N, pCDNA3-mSHH-FL-EGFP, pCDNA3-mSHH-FL-mCherry2, pCDNA3-V5-Disp-HA, pCS2-hBOC-EGFP and pCS2-hCDON-EGFP (a gift from A. Salic), pEGFP-C2-bMyo10-HMM ( ) and pEGFP-C2-bMyo10-Δ3PH, a modified version of pEGFP-bMyo10 where the 3 PH domains were removed via deletion of aa 1168–1491.

Techniques: Derivative Assay, Transfection, Construct, Recombinant, In Situ, Software, Imaging, Luciferase, Reporter Assay, Western Blot