Journal: Redox Biology

Article Title: BMX-001, a clinically relevant radioprotector, can reverse radiation-induced fibrosis when given three weeks after radiation, in part, by restoring methylation

doi: 10.1016/j.redox.2026.104020

Figure Lengend Snippet: BMX-001 reverses the expression of fibrotic markers in irradiated fibroblasts when administered after radiation. A. Representative images of fibroblasts immunostained for alpha smooth muscle actin (α-SMA, in green). Scale bar represents 100 μm. B. Quantification of alpha smooth muscle actin. ∗ = p < 0.05, n = 3 independent experiments and 6 images per group. C. Collagen contraction assay quantification as a function of area and representative images in fibroblasts collected four days after radiation. ∗ = p < 0.05, n = 3 independent experiments. D. Collagen contraction assay quantification as a function of area and representative images in fibroblasts collected six days after radiation. ∗ = p < 0.05, n = 3 independent experiments. B + R denotes BMX-001 given before radiation, R + B 2 denotes BMX-001 given two days after radiation, and R + B 4 denotes BMX-001 given four days after radiation. E. Representative images of beta-galactosidase staining on prostate fibroblasts six days after radiation. Scale bar represents 100 μm. F. Quantification of cells positive for beta-galactosidase. ∗ = p < 0.05 compared to PBS control, n = 3 independent experiments.

Article Snippet: Primary antibodies used were α-SMA (1:100, Novus Bio, NB300-978), CaMKIIβ (1:100, ThermoFisher, 13–9800), and 8-OHdG (1:100, Abcam, Waltham, MA, ab62623).

Techniques: Expressing, Irradiation, Contraction Assay, Staining, Control

Journal: Redox Biology

Article Title: BMX-001, a clinically relevant radioprotector, can reverse radiation-induced fibrosis when given three weeks after radiation, in part, by restoring methylation

doi: 10.1016/j.redox.2026.104020

Figure Lengend Snippet: BMX-001 decreases hallmarks of fibrosis in irradiated tissues and ex vivo cells when administered to mice three weeks after radiation. A. Representative images of α-SMA staining on skin isolated from mouse pelvis. Scale bar represents 100 μm. B. Quantification of number of α-SMA positive foci per field. ∗ = p < 0.05, n = 5 biological replicates per group, 6 images quantified per replicate. C. Representative images of α-SMA staining on bladder tissue. Scale bar represents 100 μm. D Quantification of α-SMA mean fluorescent intensity. ∗ = p < 0.05, n = 5 biological replicates per group, 6 images quantified per replicate. E. Representative images of col1A1 staining on bladder tissue. Scale bar represents 100 μm. F. Quantification of col1A1 mean fluorescent intensity. ∗ = p < 0.05, n = 5 biological replicates per group, 6 images quantified per replicate. G. Representative images of prostate fibroblasts stained for β-galactosidase with corresponding brightfield images. Scale bar represents 100 μm. H. Quantification of total cells positive for β-galactosidase in mouse prostate fibroblasts. ∗ = p < 0.05 as compared to PBS controls, n = 3 biological replicates per group, 6 images quantified per replicate. I. Representative images of bladder fibroblasts stained for β-galactosidase with corresponding brightfield images. Scale bar represents 100 μm. J. Quantification of total cells positive for β-galactosidase in mouse bladder fibroblasts. ∗ = p < 0.05 as compared to PBS controls, n = 3 biological replicates per group, 6 images quantified per replicate.

Article Snippet: Primary antibodies used were α-SMA (1:100, Novus Bio, NB300-978), CaMKIIβ (1:100, ThermoFisher, 13–9800), and 8-OHdG (1:100, Abcam, Waltham, MA, ab62623).

Techniques: Irradiation, Ex Vivo, Staining, Isolation

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Transgelin expression pattern in colorectal cancer. (A) Transgelin expression in normal colonic tissues. (B) Transgelin expression in colorectal cancer tissues. (C) Transgelin expression in cancer tissues. (D) Fluorescent immunostaining showed that transgelin was expressed in desmin‐positive smooth muscles as well as in other cancer stromal regions; transgelin, desmin are shown in dark green, green, and purple, respectively. (E) Immunostaining results for transgelin (dark green), smooth muscle Actin (SMA; red), and 4′,6‐diamidino‐2‐phenylindole (DAPI; purple) in the cancer cell stroma. (F) Immunostaining for AE1/3 (green), transgelin (dark green), and DAPI (purple).

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Expressing, Immunostaining, Muscles

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Knockdown of transgelin in cSPFs does not alter the expression of α‐SMA, COL1A1, and TNC. (A) Comparison of transgelin and α‐SMA expression levels in cSPFs under unstimulated (blue) and stimulated (orange) conditions. Data are shown as mean ± standard deviation ( n = 3). ** p < 0.01. B Unstimulated and stimulated conditions, with sh‐Luc, sh‐transgelin #1 (#1), or sh‐transgelin #2 (#2), and transgelin expression was quantified using reverse transcription polymerase chain reaction. Data are shown as mean ± standard deviation ( n = 3). ** p < 0.01. (C) Protein levels of transgelin were compared using western blotting following transfection with sh‐Luc, sh‐transgelin #1, or sh‐transgelin #2 in cSPF‐stimulated and unstimulated conditions. (D) Changes in α‐SMA, COL1A1, and TNC expression in cSPFs transfected with sh‐Luc, sh‐transgelin #1, or sh‐transgelin #2 under unstimulated conditions with the cancer cell‐culture supernatant (blue) and after stimulation with cancer cell‐culture supernatants (orange). Different treatment groups were compared using a two‐tailed t ‐test. ** p < 0.01. cSPF: Colonic subperitoneal fibroblast; α‐SMA: Alpha‐smooth muscle Actin; COL1A1: Collagen type I alpha 1; TNC: Tenascin C; sh‐Luc: Short hairpin RNA targeting luciferase.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Knockdown, Expressing, Comparison, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Transfection, Cell Culture, Two Tailed Test, shRNA, Luciferase

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Transgelin knockdown suppresses cSPF‐induced contractility in vitro and tumor growth by co‐transplantation of cSPFs and DLD‐1 cells in vivo. (A) To establish the conditions for the contraction assay, five cell counts were performed: 5 × 10 4 , 1 × 10 5 , 2 × 10 5 , 4 × 10 5 , and 8 × 10 5 . No stimulation by the cancer cell‐culture supernatant (blue) and stimulation by the cancer cell‐culture supernatant (orange). Data are shown as mean ± standard deviation ( n = 3) of at least three independent experiments. (B) The results of the contraction assay under unstimulated conditions with cancer cell‐culture supernatant with sh‐Luc (Luc), sh‐transgelin #1 (#1), or sh‐transgelin #2 (#2). Data are shown as mean ± standard deviation ( n = 5) of at least two independent experiments. * p < 0.05. (C) The results of the contraction assay under stimulation with the cancer cell‐culture supernatant are shown. Data are shown as mean ± standard deviation ( n = 5) of at least two independent experiments. * p < 0.05. (D) Schematic illustration of the experiment. The left, middle, and right panels show the groups implanted with DLD‐1 cells only, co‐implanted with DLD‐1 cells and cSPFs, and co‐implanted with DLD‐1 cells and cSPFs with transgelin knockdown, respectively. (E) The growth of tumors was compared with the co‐transplantation of DLD‐1 cells, cSPFs (orange), compared with the transplantation of DLD‐1 cells only (blue), and co‐transplantation (gray) of DLD‐1 cells with cSPFs with knockdown. Data are presented as mean (tumor volume) ± standard deviation, n = 9 per group. (F) In sh‐transgelin #2, co‐transplantation (gray) of transgelin‐knockdown cSPFs and DLD‐1 cells suppressed the tumor growth‐promoting effect. Data are presented as mean (tumor volume) ± standard deviation, n = 12 per group. (G) Photograph of mice 4 weeks following transplantation. cSPF, colonic subperitoneal fibroblast; sh‐Luc, short hairpin RNA targeting luciferase.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Knockdown, In Vitro, Transplantation Assay, In Vivo, Contraction Assay, Cell Culture, Standard Deviation, shRNA, Luciferase

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: RNA sequencing results in cSPFs with knockdown of transgelin before and after CM stimulation. (A) Experimental flow diagram. The stimulated and unstimulated cSPFs of the control and transgelin‐knockdown groups were assessed ( n = 3 each; total n = 12). (B) Principal component analysis of the correlation between expression levels showed a correlation with inter‐individual effects rather than knockdown. (C) Heatmap analysis showed a correlation with inter‐individual effects rather than with transgelin‐knockdown effects. cSPF, colonic subperitoneal fibroblast; CM, conditioned medium.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: RNA Sequencing, Knockdown, Control, Expressing

Journal: Cancer Medicine

Article Title: Transgelin Expression in Activated Cancer‐Associated Fibroblasts Regulates Stromal Contractility and Promotes Colon Cancer Progression

doi: 10.1002/cam4.71789

Figure Lengend Snippet: Assessment of transgelin expression and progression‐free survival in patients with colorectal cancer. (A) A total of 359 of 388 colorectal cancer surgery cases were analyzed after excluding those that met the exclusion criteria. (B) The assessment of the transgelin‐positive area ratio and the α‐SMA‐positive area ratio in the tissue core showed a positive correlation. (C) A high transgelin‐positive area ratio is significantly associated with poor prognosis. (D) A high α‐SMA‐positive area ratio is significantly associated with poor prognosis. α‐SMA, alpha‐smooth muscle Actin; PFS, progression‐free survival.

Article Snippet: The primary antibodies used were mouse monoclonal anti‐human transgelin antibody (OriGene Technologies, Rockville, MD, USA; used at 1:200 dilution), rabbit monoclonal anti–human α‐SMA (SPRING BIOSCIENCE, Pleasanton, CA, USA; used at 1:200 dilution), Desmin (Nichirei, Tokyo, Japan; used without dilution), 4′,6‐diamidino‐2‐phenylindole (DAPI; Akoya Biosciences, Menlo Park, CA, USA; used without dilution), and AE1/3 antibody (Dako, Agilent Technologies, Santa Clara, CA, USA; used without dilution).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Fibronectin synthesis, but not α-smooth muscle expression, is regulated by periostin in gingival healing through FAK/JNK signaling

doi: 10.1038/s41598-018-35805-6

Figure Lengend Snippet: α-SMA is not expressed at the protein level following gingivectomy in vivo . ( A ) Representative images of immunoreactivity for α-SMA of gingiva at 1, 3, 7, and 14 days post-wounding and unwounded gingiva (day 0). Histological sections from rats after 1, 3, 7, and 14 days post-wounding and unwound gingiva were incubated with a primary antibody against α-SMA. The primary antibody was detected by using peroxidase-conjugate secondary antibody and DAB. All sections were counterstained with haematoxylin. Panels (a–e) are higher magnifications of the corresponding regions indicated by rectangular boxes. Black arrowheads denote α-SMA-positive blood vessels. Few myofibroblasts were evident at day 7 indicated by hollow arrowhead. ( B ) Representative images of immunoreactivity for FSP-1 of gingiva following gingivectomy. Anti-FSP-1 was detected by using peroxidase-conjugate secondary antibody and DAB, and the sections were counterstained with haematoxylin. FSP-1-positive cells in the regenerated connective tissue are denoted by black arrows. Scale bar , 50 μm.

Article Snippet: In brief, tissues were deparaffinized and immune-labeled using primary antibodies against periostin (sc49480; Santa Cruz Biotechnology; Dallas, TX; 1:100), α-SMA (ab5694; Abcam; Cambridge, MA; 1:400), PCNA (ab2426-1; Abcam; 1:100), and FSP-1 (07-2274; Millipore; Billerica, MA; 1:100).

Techniques: In Vivo, Incubation

Journal: Scientific Reports

Article Title: Fibronectin synthesis, but not α-smooth muscle expression, is regulated by periostin in gingival healing through FAK/JNK signaling

doi: 10.1038/s41598-018-35805-6

Figure Lengend Snippet: rhPN does not affect HGF proliferation nor modulate myofibroblast differentiation in vitro . ( A ) HGFs cultured on collagen or collagen + rhPN coated plates for 1, 3, 5, 7, and 9 days were assessed for proliferation using CyQUANT assay kit to determine DNA contents. A standard curve was used to extrapolate cell number. Data represents fold cell number increase ± s.d. relative to day 1. Data was analyzed via two-way ANOVA d ( p > 0.05). ( B ) HGFs cultured on collagen or collagen + rhPN coated plates for 1 day and 7 days were assessed for gene expressions of ACTA2 . Target gene expression was normalized to 18 S using the ΔΔCt method. Data represents mean fold gene expressions ± s.d. relative to control day 1 (collagen alone) of 3 independent experiments in triplicates. Data was analyzed via Student’s t-test (unpaired) within each time-point. ( C ) Western blot was used to assess α-SMA protein level of HGFs cultured on collagen or collagen + rhPN coated plates. GAPDH was used as a loading control. ( D ) Immunocytochemistry was performed with HGFs cultured on collagen or collagen + rhPN coated plates for 7 days to visualize myofibroblasts. Representative fluorescent images of HGFs labeled for α-SMA (green), F-actin (red), and nuclei (blue). Myofibroblasts were revealed by α-SMA-positive stress-fibers. Scale bar , 50 μm. ( E ) Fixed collagen gel contraction assay of HGFs in the absence and presence of rhPN (5 μg/ml). Gel contraction was quantified by loss of gel weight, compared with gels lacking cells. Data was analyzed via Student’s t-test (unpaired) within each time-point ( p > 0.05; ns, not significant).

Article Snippet: In brief, tissues were deparaffinized and immune-labeled using primary antibodies against periostin (sc49480; Santa Cruz Biotechnology; Dallas, TX; 1:100), α-SMA (ab5694; Abcam; Cambridge, MA; 1:400), PCNA (ab2426-1; Abcam; 1:100), and FSP-1 (07-2274; Millipore; Billerica, MA; 1:100).

Techniques: In Vitro, Cell Culture, CyQUANT Assay, Targeted Gene Expression, Control, Western Blot, Immunocytochemistry, Labeling, Collagen Gel Contraction Assay

Journal: Cell death discovery

Article Title: Mechanical stiffness promotes skin fibrosis through Piezo1-mediated arginine and proline metabolism.

doi: 10.1038/s41420-023-01656-y

Figure Lengend Snippet: Fig. 1 MDFs Function in different stiffness substrate in vitro and in vivo. A Western blot assay for α-SMA and Fn in MDFs on 2 versus 50 kPa hydrogel and quantitative analysis (n = 3). B Soluble collagen concentration of MDFs on 2 versus 50 kPa hydrogel (n = 3). C Immunofluorescence staining for α-SMA of MDFs on 2 versus 50 kPa hydrogel and quantitative analysis (n = 3). D Collagen contraction assay of MDFs on 2 versus 50 kPa hydrogel (n = 3). E Schematic depicting MDFs labeling, culture and transplantation in dorsal wounding and ECM deposition experiments. F Quantitative analysis of wound area. Percentage means day 0 wound size versus days since wounding (n = 10). G Imaging of transplanted GFP-positive 2 kPa or 50 kPa MDFs in wounding and colocalization between GFP and or Col-I signaling. H Imaging of transplanted GFP-positive 2 kPa or 50 kPa MDFs in skin fibrosis and colocalization between GFP and or Col-I signaling. I Trichrome staining of 2 kPa and 50 kPa MDFs transplanted wounds or mouse skin (n = 3). The results are expressed as the means with SD. ***P < 0.005, ****P < 0.001.

Article Snippet: After blocking with 5% BSA at room temperature (RT) for 1 h, the membranes were incubated with the following primary antibodies: α-SMA (#19245, 1:1000, Cell Signaling Technology, Danvers, MA), fibronectin (ab2413, 1:1000, Abcam, Cambridge, UK), arginase-1 (Arg1) (16001-1-AP, 1: 5000, Proteintech, Wuhan, China), ornithine δ-aminotransferase (OAT) (A6235, 1:1000, ABclonal Technology, Wuhan, China), PYCR1 (13108-1-AP, 1:4000, Proteintech), proline dehydrogenase (oxidase) 1 (PRODH) (22980-1- AP, 1:2000, Proteintech) and β-actin (20536-1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: In Vitro, In Vivo, Western Blot, Concentration Assay, Staining, Contraction Assay, Labeling, Transplantation Assay, Imaging

Journal: Cell death discovery

Article Title: Mechanical stiffness promotes skin fibrosis through Piezo1-mediated arginine and proline metabolism.

doi: 10.1038/s41420-023-01656-y

Figure Lengend Snippet: Fig. 3 Detection of key metabolic enzymes in human and mouse samples. A Immunostaining of Arg1, OAT, PYCR1 and PRODH in human normal skin, hypertrophic scar and keloid tissues and quantitative analysis (n = 8). B Colocalization of Arg1, OAT, PYCR1, PRODH with α-SMA in human normal skin, hypertrophic scar and keloid tissues. C Immunostaining of Arg1, OAT, PYCR1 and PRODH in mouse normal skin and skin fibrosis tissues (n = 8). D Colocalization of Arg1, OAT, PYCR1, PRODH with α-SMA in mouse normal skin and skin fibrosis tissues. The results are expressed as the means with SD. ****P < 0.001.

Article Snippet: After blocking with 5% BSA at room temperature (RT) for 1 h, the membranes were incubated with the following primary antibodies: α-SMA (#19245, 1:1000, Cell Signaling Technology, Danvers, MA), fibronectin (ab2413, 1:1000, Abcam, Cambridge, UK), arginase-1 (Arg1) (16001-1-AP, 1: 5000, Proteintech, Wuhan, China), ornithine δ-aminotransferase (OAT) (A6235, 1:1000, ABclonal Technology, Wuhan, China), PYCR1 (13108-1-AP, 1:4000, Proteintech), proline dehydrogenase (oxidase) 1 (PRODH) (22980-1- AP, 1:2000, Proteintech) and β-actin (20536-1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: Immunostaining

Journal: Cell death discovery

Article Title: Mechanical stiffness promotes skin fibrosis through Piezo1-mediated arginine and proline metabolism.

doi: 10.1038/s41420-023-01656-y

Figure Lengend Snippet: Fig. 4 The role of Piezo1 in increased matrix stiffness-induced MDFs arginine and proline metabolism in vitro. A Piezo1 expression in human normal skin, hypertrophic scar, keloid (Scale bar = 200 μm, Zoom scale bar = 50 μm) and mouse normal/fibrotic skin (Scale bar = 100 μm, Zoom scale bar = 20 μm). B Images of immunofluorescence co-staining of Piezo1 (red) and α-SMA (green) in human normal skin, hypertrophic scar, keloid (Scale bar = 200 μm, Zoom scale bar = 50 μm) and mouse normal/fibrotic skin (Scale bar = 100 μm, Zoom scale bar = 20 μm). C Measurement of L-Arginine, L-Ornithine and L-Proline in 2 kPa-MDFs/50 kPa-MDFs with or without Piezo1 knockdown (n = 5). D Western blot assay for key metabolic enzymes Arg1, OAT, PYCR1 and PRODH in 2 kPa-MDFs/50 kPa-MDFs with or without Piezo1 knockdown and quantitative analysis (n = 3). E Measurement of key metabolic enzymes activity Arg1, OAT, PYCR1 and PRODH in 2 kPa-MDFs/50 kPa-MDFs with or without Piezo1 knockdown and quantitative analysis (n = 5). F Western blot assay for α-SMA and Fn in MDFs with or without Yoda1 (25 μM) and quantitative analysis (n = 3). G Measurement of L-Arginine, L-Ornithine and L-Proline in MDFs with or without Yoda1 (25 μM) (n = 5). H Western blot assay for key metabolic enzymes Arg1, OAT, PYCR1 and PRODH in MDFs with or without Yoda1 (25 μM) and quantitative analysis (n = 3). I Measurement of key metabolic enzymes activity Arg1, OAT, PYCR1 and PRODH in MDFs with or without Yoda1 (25 μM) and quantitative analysis (n = 5). The results are expressed as the means with SD. *P < 0.05, **P < 0.01, ****P < 0.001.

Article Snippet: After blocking with 5% BSA at room temperature (RT) for 1 h, the membranes were incubated with the following primary antibodies: α-SMA (#19245, 1:1000, Cell Signaling Technology, Danvers, MA), fibronectin (ab2413, 1:1000, Abcam, Cambridge, UK), arginase-1 (Arg1) (16001-1-AP, 1: 5000, Proteintech, Wuhan, China), ornithine δ-aminotransferase (OAT) (A6235, 1:1000, ABclonal Technology, Wuhan, China), PYCR1 (13108-1-AP, 1:4000, Proteintech), proline dehydrogenase (oxidase) 1 (PRODH) (22980-1- AP, 1:2000, Proteintech) and β-actin (20536-1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: In Vitro, Expressing, Staining, Knockdown, Western Blot, Activity Assay

Journal: Cell death discovery

Article Title: Mechanical stiffness promotes skin fibrosis through Piezo1-mediated arginine and proline metabolism.

doi: 10.1038/s41420-023-01656-y

Figure Lengend Snippet: Fig. 5 The role of Piezo1 in increased matrix stiffness-induced MDFs arginine and proline metabolism in vivo. A Validation of Piezo1 knockdown in mouse skin and quantitative analysis of Piezo1 staining (n = 6). B Schematic illustration of the experimental procedure. C Representative images and quantitative analysis of H&E and Trichrome staining (n = 10). D Representative images and quantitative analysis of α-SMA staining (n = 10). E Representative images and quantitative analysis of Arg1 and OAT staining (n = 10). F Representative images and quantitative analysis of PYCR1 and PRODH staining (n = 10). The results are expressed as the means with SD. *P < 0.05, **P < 0.01, ****P < 0.001.

Article Snippet: After blocking with 5% BSA at room temperature (RT) for 1 h, the membranes were incubated with the following primary antibodies: α-SMA (#19245, 1:1000, Cell Signaling Technology, Danvers, MA), fibronectin (ab2413, 1:1000, Abcam, Cambridge, UK), arginase-1 (Arg1) (16001-1-AP, 1: 5000, Proteintech, Wuhan, China), ornithine δ-aminotransferase (OAT) (A6235, 1:1000, ABclonal Technology, Wuhan, China), PYCR1 (13108-1-AP, 1:4000, Proteintech), proline dehydrogenase (oxidase) 1 (PRODH) (22980-1- AP, 1:2000, Proteintech) and β-actin (20536-1-AP, 1:5000, Proteintech) at 4 °C overnight.

Techniques: In Vivo, Biomarker Discovery, Knockdown, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer

doi: 10.1186/s13046-024-03077-w

Figure Lengend Snippet: 95D-derived EVs packaged miR-1290 activated NFs to CAFs. A The representative images of EVs derived from 95C and 95D were analyzed by TEM. Scale bar, 100 nm. B EVs were detected by NTA analysis. C Immunoblotting analyses for EVs markers CD63, TSG101 and negative control Calnexin. D NFs were pre-treated with 95C or 95D derived EVs for 24 h. Confocal imaging showed the delivery of PKH67-labeled EVs (green) to Phalloidin labelled NFs (red) and the localization of DAPI-stained nuclei (blue). Yellow arrows represented delivered EVs. Scale bar, 20 μm. E After NFs were incubated with 95C or 95D derived EVs for 48 h, the protein levels of α-SMA and FAP were analyzed by immunoblotting. F Representative images of the contraction ability were analyzed by collagen contraction assay. Isolated primary CAFs were used as the positive control group. G-H After NFs transfected with indicated mimics, the mRNA levels of α-SMA and FAP were analyzed by qPCR. Negative control (NC), Untreated (UN). I The protein levels of α-SMA and FAP were detected by immunoblotting. J Immunoblotting analysis of α-SMA and FAP in NFs treated with blank control (PBS) or EVs derived from 95C/vec and 95C/miR-1290 or 95D/control and 95D/anti-miR-1290. K Representative images and quantification analysis of IF of α-SMA in NFs treated as indicated. Scale bar, 20 μm. L Collagen contraction assay was assessed for the contraction ability of NFs treated as indicated. Data were shown as the mean ± SD of at least three independent experiments. *** P < 0.001, ns: not significant

Article Snippet: The antibodies were used as follows, α-SMA (14395–1-AP), Vimentin (10366–1-AP), PINK1 (23274–1-AP), AKT (60203–2-IG), p-AKT (66444–1-IG), Calnexin (10427–2-AP), E-cadherin (20874–1-AP) and Snail (13099–1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Derivative Assay, Western Blot, Negative Control, Imaging, Labeling, Staining, Incubation, Contraction Assay, Isolation, Positive Control, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer

doi: 10.1186/s13046-024-03077-w

Figure Lengend Snippet: EVs packaged miR-1290 activates NFs to CAFs via MT1G/AKT and promotes lung cancer metastasis. A The binding sites and corresponding mutation sites between miR-1290 and the target gene MT1G. B Luciferase reporter assay in 293 T cells cotransfected with the wild or mutant-type MTIG 3' UTR and miR-1290 mimics. Untreated (UN). Blank Vector (Control). C qPCR analysis of MT1G, α-SMA and FAP expression in NFs treated with miR-1290 mimics. Negative control (NC). D Immunoblotting analysis of MT1G, p-AKT, AKT, α-SMA and FAP in NFs transfected with miR-1290 mimics. E IF analysis and quantification data of α-SMA in NFs treated as indicated. Scale bar, 20 μm. F–H pDONR233-MT1G plasmid was transfected in NFs treated with miR-1290 mimics and 95C/miR-1290-EVs, respectively, F qPCR assays for MT1G, α-SMA and FAP, G Immunoblotting analysis of MT1G, p-AKT, AKT, α-SMA and FAP, H IF analysis for α-SMA. Scale bar, 20 μm. I-J H1299 was cocultured with conditional medium (CM) from different groups indicated, I CCK8 assay for cell proliferation, and J Transwell assays for the cell migration analysis. Scale bar, 100 μm. K Schematic representation of the establishment process of the EVs-educated tumor metastasis model. L The representative tissue images and HE images of lung foci. Scale bar, 200 μm. M Quantitative metastatic area in lung metastatic foci. N ISH images of miR-1290 and IHC images of MT1G, p-AKT and α-SMA in lung foci of different experimental groups. Red arrow: positive signal in the stroma; Black arrow: positive signal in the tumor. Data were shown as the mean ± SD of at least three independent experiments.** P < 0.01, ** *P < 0.001, ns: not significant

Article Snippet: The antibodies were used as follows, α-SMA (14395–1-AP), Vimentin (10366–1-AP), PINK1 (23274–1-AP), AKT (60203–2-IG), p-AKT (66444–1-IG), Calnexin (10427–2-AP), E-cadherin (20874–1-AP) and Snail (13099–1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Plasmid Preparation, Control, Expressing, Negative Control, Western Blot, Transfection, CCK-8 Assay, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer

doi: 10.1186/s13046-024-03077-w

Figure Lengend Snippet: ROS enhances mitophagy and increases release of mtDNA in iCAFs. A-C NFs were treated with EVs derived from 95C or 95D for 48 h. Isolated NFs and CAFs were used as blank control and positive control, respectively. A Immunoblotting analysis of PINK1, BNIP3 and LC3 in the indicated groups. B The fluorescence intensity reflecting the MMP in the different groups. C qPCR analyses for ND1, COX1, D-Loop in the supernatants of indicated cells. D Immunoblotting analysis of PINK1, BNIP3 and LC3 in NFs treated with blank control (PBS) or EVs derived from 95C/vec and 95C/miR-1290 or 95D/control and 95D/anti-miR-1290. E The fluorescence intensity reflecting the MMP in NFs treated as indicated. F qPCR analyses for ND1, COX1, D-Loop in the supernatants of indicated cell. G-I NFs were treated with 95D-derived EVs for 48 h, and then exposed to H 2 O 2 (0.25 mM) or CCCP (10 μM), respectively. G Immunoblotting analysis of PINK1, BNIP3 and LC3 in the indicated groups. H The fluorescence intensity reflecting the MMP in the different groups. I qPCR analyses for ND1, COX1, D-Loop in the supernatants of indicated cells. J ISH images of miR-1290 and IHC images of α-SMA and BNIP3 in lung cancer tissue sections. Stromal and tumor are separated by dashed lines (left). The correlation analysis (right) between miR-1290 and α-SMA (upper), and between α-SMA and BNIP3 (lower). Data were shown as the mean ± SD of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant

Article Snippet: The antibodies were used as follows, α-SMA (14395–1-AP), Vimentin (10366–1-AP), PINK1 (23274–1-AP), AKT (60203–2-IG), p-AKT (66444–1-IG), Calnexin (10427–2-AP), E-cadherin (20874–1-AP) and Snail (13099–1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Derivative Assay, Isolation, Control, Positive Control, Western Blot, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer

doi: 10.1186/s13046-024-03077-w

Figure Lengend Snippet: iCAFs promote the growth and metastasis of mitochondria damaged tumor cells in vivo. A-C After H1299 (2 × 10 6 ), H1299ρ 0 cells (2 × 10 6 ), the mixture of 2 × 10 6 H1299ρ 0 cells with 5 × 10 5 NFs or 5 × 10 5 CAFs cells (4:1) were injected subcutaneously into 5-week-old nude mice, A the tumor growth, B tumor volume and C tumor weight was monitored. D PCR was used to detect the ND2 and CYTB in the indicated groups. E IHC analysis of TOM20 was performed in mouse xenograft tissues. F–H After H1299 (5 × 10 5 ), H1299ρ 0 cells (5 × 10 5 ), the mixture of 5 × 10 5 H1299ρ 0 cells with 5 × 10 5 NFs or 5 × 10 5 iCAFs cells (1:1) were injected into the lateral tail vein of 5-week-old nude mice, F the representative tissue macrographs and HE images of lung foci, and G quantitative analysis of lung metastatic foci. Scale bar, 200 μm. H IHC images of α-SMA and TOM20 in lung foci of different experimental groups. Data were shown as the mean ± SD of at least three independent experiments. ** P < 0.01, *** P < 0.001

Article Snippet: The antibodies were used as follows, α-SMA (14395–1-AP), Vimentin (10366–1-AP), PINK1 (23274–1-AP), AKT (60203–2-IG), p-AKT (66444–1-IG), Calnexin (10427–2-AP), E-cadherin (20874–1-AP) and Snail (13099–1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: In Vivo, Injection

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: FGFR1OP2/wit3.0 and α-SMA in oral wound. A: Gene structures of FGFR1 and FGFR1OP2/wit3.0. In a case report of acute myeloid leukemia,23 the exons 1 to 4 of FGFR1OP2/wit3.0 were trans-inserted to the intron between exons 8 and 9 of FGFR1, and the resulting chimerical peptide formed a ligand-independent dimer through the FGFR1OP2/wit3.0-derived coiled-coil structure. The native FGFR1OP2/wit3.0 gene contains seven highly conserved exons. B: Western blot analysis of monospecific polyclonal antibody (D1042) recognized 41 kDa and 32 kDa bands, corresponding to FGFR1OP2/wit3.0β and FGFR1OP2/wit3.0α, respectively, in NIH fibroblasts. The transfected 3XFLAG-FGFR1OP2/wit3.0β fusion peptide was recognized by M2 monoclonal antibody against 3XFLAG epitope at 41 kDa. C: Oral open wound (asterisk) created by tooth extraction showed progressive wound contraction (arrows) by wound margin approximation (e: proliferation/migration front of oral epithelium; b: alveolar bone; s: tooth extraction socket, H&E staining). D1042 antibody stained a highly restricted zone of oral connective tissue (arrowheads) immediately adjacent to the migration/proliferation front of oral epithelium (e). α-SMA (small arrows) was identified along the ligament-like structure (double arrowhead in H&E stained section) connecting alveolar bones (b). D1042 stained the cytoplasma of fibroblasts with cuboidal shapes (arrowheads), whereas α-SMA was found in vascular smooth muscles (small arrows). D: Real-time RT-PCR revealed that FGFR1OP2/wit3.0α and FGFR1OP2/wit3.0β were up-regulated in oral wound tissues but not in skin wound tissues, whereas α-SMA was up-regulated in both oral and skin wound tissues albeit at different levels (*P < 0.05).

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: Derivative Assay, Western Blot, Transfection, Extraction, Migration, Staining, Muscles, Quantitative RT-PCR

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: SiRNA Target Sequences for FGFR1OP2/wit3.0

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: Sequencing, Control

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: Available Mouse ES Cell Lines Containing Disrupted FGFR1OP2/wit3.0 Allele (146526432-146547719) through the International Gene Trap Consortium

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques:

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: FGFR1OP2/wit3.0 coiled-coil structure and its association with cytoskeletal network in oral wound fibroblasts. A: Hydrophylicity plot of FGFR1OP2/wit3.0β and the coiled-coil domains predicted by the Human Protein Reference Database and COILS. B: The Imperial College Protein Homology/Analogy Recognition Engine predicted that three-dimensional structures of FGFR1OP2/wit3.0β would share similar structures of a-spectrin (top) and apoliprotein A-I (bottom), both molecules were known to form dimers and oligomers. C: In denatured Western blot analysis, D1042 antibody depicted FGFR1OP2/wit3.0α and FGFR1OP2/wit3.0β in NIH3T3 fibroblasts (lane 1), NIH3T3 fibroblasts overexpressing FGFR1OP2/wit3.0β (lane 2), and rat oral wound fibroblasts (lane 3). In oral wound fibroblasts, FGFR1OP2/wit3.0 molecules formed dimers and oligomers that were not affected by the reducing agent, β-melcaptoethanol. D: Nondenature gel Western blot analysis of cytoplasmic peptide of naïve oral fibroblasts (OFs) and oral wound fibroblasts (W-OFs) with D1042 indicated a single band at 50 kDa, which was found modified by SUMO-1 (small arrow). The D1042 positive FGFR1OP2/wit3.0 oligomers at 100 kDa and 160 kDa (small arrowheads) were visible in OFs but not in W-OFs. The involvement of FGFR1OP2/wit3.0 in a large molecular moiety of over 190 kDa (large arrowhead) was also noted in both OFs and W-OFs; however, the size of the large molecular moiety and D1042 signal intensity increased in W-OFs. E: Immunocytology of OFs and W-OFs with D1042 antibody (FGFR1OP2/wit3.0: green) as well as phallotoxins (F-actin: red) and DAPI (nuclei: blue). FGFR1OP2/wit3.0 appeared to aggregate in perinucleic cytoplasma. Whereas stress fibers were formed in thick bundles in OF (white arrows), cuboidal shaped W-OFs formed a thin web of cytoskeleton networks (white arrows). Scale bar = 25 μm.

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: Western Blot, Modification

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: SiRNA knockdown of FGFR1OP2/wit3.0 attenuated collagen gel contraction of oral wound fibroblasts. A: Floating collagen gel contraction assay showed the increased gel contraction by oral wound fibroblasts (W-OFs) as compared with naïve oral fibroblasts (OFs) and skin fibroblasts (SFs) at 12 hours and 24 hours (*P < 0.05). B: Efficacy of five siRNA constructs was evaluated by real time RT-PCR (*P < 0.05 against negative control). The treatment by siRNAs number 4 and number 5 did not affect the expression of contractile and procontractile molecules: α-SMA, radixin, ezrin, and moesin. C: FACS analysis demonstrated the establishment of the optimal transfection protocol for primary oral wound fibroblasts achieving >80% transfection rates (black line) compared with untransfected control (gray line). D: Floating collagen gel contraction assay of oral wound fibroblasts showed that FGFR1OP2/wit3.0 siRNA number 5 significantly attenuated gel contraction (*P < 0.05).

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: Knockdown, Collagen Gel Contraction Assay, Construct, Quantitative RT-PCR, Negative Control, Expressing, Transfection, Control

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: FGFR1OP2/wit3.0 (+/−) mutation reduced the migration ability of mouse ES cell-derived fibroblastic cells. A: Gene Trap mutation was accomplished by inserting a fusion construct of β-galactosidase and neomysin resistant genes (β-geo), which contains splice acceptance sequence (SA) and bovine fibroblast growth factor poly A signal (bpA) into the first intron of mouse fgfrop2/wit3.0 allele. B: Genotype of wild-type (+/+) and FGFROP2/wit3.0 heterogyzous null mutant (+/−) mouse ES cells was determined by PCR. Primers a and d flanked the β-geo construct (Figure 4A) giving rise to the wild-type PCR product (a/d), whereas the combinations of primers a and b (a/b) and c and d (c/d) recognized the β-geo insert. C: Fibroblastic cells derived from wild-type (+/+) and FGFR1OP2/wit3.0 (+/−) mutant ES cells expressed the comparative mRNA level of collagen 1 α 1 chain, whereas the FGFR1OP2/wit3.0 mRNA level was significantly decreased in the mutant cells (*P < 0.5). D:In vitro scratch test revealed that FGFR1OP2/wit3.0 (+/−) mutation significantly decreased fibroblastic cell migration.

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: Mutagenesis, Migration, Derivative Assay, Construct, Sequencing, In Vitro

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: FGFR1OP2/wit3.0β overexpression increased collagen gel contraction by both oral and skin fibroblasts. A: Nonsynonymous SNPs in human FGFR1OP2/wit3.0β sequence. B: A schematic structure of lentiviral vector carrying humanized FGFR1OP2/wit3.0β (wild-type, SNP1, SNP2, or SNP1/2) and internal ribosome entry site-green fluorescent protein. C: FACS analysis of the transduction rate of the lentiviral vector for primary oral fibroblasts assessed by green fluorescent protein. D: Floating collagen gel contraction assay revealed that both oral and skin fibroblasts transduced by FGFR1OP2/wit3.0β lentiviral vectors (dotted lines) increased gel contraction rates. E: SNP1, SNP2, and SNP1/2 of FGFR1OP2/wit3.0β increased collagen gel contraction rates. In both oral and skin fibroblasts, SNP1 FGFR1OP2/wit3.0β showed the largest gel contraction rate. F: The treatment of TGF-β1 did not modulate the expression of FGFR1OP2/wit3.0 in oral and skin fibroblasts, whereas it increased the α-SMA expression. *P < 0.05, **P < 0.01.

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: Over Expression, Sequencing, Plasmid Preparation, Transduction, Collagen Gel Contraction Assay, Expressing

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: FGFR1OP2/wit3.0β induced skin wound closures in vivo. A: Schematic diagram of full-thickness wound created in mouse dorsal skin. B: Unlike in oral wounds, the mRNA levels of FGFR1OP2/wit3.0α and FGFR1OP2/wit3.0β remained unchanged in mouse skin excisional wounds during the healing period of day 0 to day 6 (P > 0.05). C: The single-view three-dimensional optical imaging depicting the luciferase reporter gene derived biofluorescence at the mouse dorsal skin excisional wound with plasmid DNA or lentiviral vectors mixed with CG SM50. The luciferase assay measuring the transfection/transduction efficiency of plasmid DNA or lentiviral vectors mixed with collagen gel carrier (Cell Prime, CP) or CG. Lentiviral vectors mixed with CG showed the highest transfection/transduction efficiency. D: Mouse dorsal skin excisional wound treated with wild-type or SNP1 FGFR1OP2/wit3.0β lentiviral vectors mixed with CG carrier. The wound areas contraction measured at day 7 indicated the significant closure of wounds treated with FGFR1OP2/wit3.0β or SNP1 FGFR1OP2/wit3.0β. *P < 0.05, ***P < 0.001.

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: In Vivo, Optical Imaging, Luciferase, Derivative Assay, Plasmid Preparation, Transfection, Transduction

Journal: The American Journal of Pathology

Article Title: Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

doi: 10.2353/ajpath.2010.090256

Figure Lengend Snippet: Decreased granulation tissue and scarring after FGFR1OP2/wit3.0β treatment. A: Histological specimen of mouse dorsal skin showed a clear demarcation of excisional wounding (arrow) flanking the highly cellular granulation tissue (asterisk, left; 7 days after wounding Goldner's trichrome staining). Immunostaining with D1042 was negative in the Mock-transducation group using green fluorescent protein lentiviral vector. FGFR1OP2/wit3.0β lentiviral vector treatment resulted in the strong D1042 immunostaining in fibroblasts of the granulation tissue juxtaposing the excisinal wound site (arrow). B: Mock-treated mouse skin excisional wound healing (day 7). While the blunted panniculus carnosus layer (arrowheads) remained patent, the granulation tissues asterisk were formed between the dermis wound edges (arrows) and covered by new epithelial keratinocytes. The expenditures of skin such as hair follicles (H) were not observed in the granulation tissue (left; Goldner's trichrome staining). Confocal laser scanning micrograph of skin wound stained by Sirius Red showed that collagen fibers were strongly stained in the dermis containing hair follicles (arrowheads). The dermis wound edge was clearly visible (arrows) after 7 days of wounding. The granulation tissue asterisk contained less organized collagen fibers (right). C: Histological specimen of mouse dorsal skin excisional wound treated with FGFR1OP2/wit3.0β. The blunted panniculus carnosus layer indicates the original wound edge (arrowheads). The dermis was significantly approximated toward the wound center (arrows) leaving a small area of granulation tissue asterisk covered by the keratinocyte layer. The hair follicles (H) were present in the wound area following the dermis closure (left; Goldner's trichrome staining). Confocal laser scanning micrograph of the Sirius Red stained mouse skin wound treated with FGFR1OP2/wit3.0β. The dermis wound edges (arrows) were approximated toward the wound center, surrounding a small granulation tissue (asterisk, right).

Article Snippet: The genotype was determined by PCR, and the mRNA levels of collagen I α 2 chain (Mm00483937_m1) and FGFR1OP2/wit3.0 (Mm00470836_m1) were determined by Taqman-based real time PCR. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Cell line ID Identification status Source Insertion location CA0175 Localized + transcript SIGTR 146526450-146526731 Q017D08 Localized + transcript GGTC 146526450-146526731 {"type":"entrez-protein","attrs":{"text":"PST26232","term_id":"1371709067","term_text":"PST26232"}} PST26232 -NR Localized only ESDB 146527049-146527145 IST10830D12BBF1 Localized only TIGM 146531786-146532310 IST10830D12HMF1 Localized only TIGM 146531799-146532314 IST12138D4BBF1 Localized only TIGM 146544488-146544618 Open in a separate window SIGTR, Sanger Institute Gene Trap Resource (Cambridge, UK).

Techniques: Staining, Immunostaining, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: AMP-activated protein kinase alpha2 activity is not essential for contraction- and hyperosmolarity-induced glucose transport in skeletal muscle.

doi: 10.1074/jbc.M504208200

Figure Lengend Snippet: FIGURE 1. AMPK activity and expression of AMPK subunits in muscle from wild type and 2i TG mice. A, representative immunoblots for hemagglutinin (HA), AMPK 2, 1, 2, and 3 in EDL muscle lysates from wild type and 2i TG mice. The upper arrow shows exogenously expressed 2 inactive form, and the lower arrow shows the endogenous 2 protein. B, isolated EDL muscles were incubated with or without AICAR (2 mM) for 20 min in vitro, and then the muscles were used for isoform-specific AMPK activity assays. The left panel shows 2 activity, and the right panel shows 1 activity. C, isolated EDL muscles were incubated in KRB buffer for 20 min with or without DNP (500 M), and the muscles were used for iso- form-specific AMPK activity assays. The left panel shows 2 activity, and the right panel shows 1 activity. Data are the means S.E. n 4–6/group. *, significantly different from basal of the same genotype (*, p 0.05; **, p 0.01). †, significantly different from wild type with the same interven- tion (†, p 0.05; ††, p 0.01).

Article Snippet: Measurement of Glucose Transport in Vitro—Immediately aftermuscle incubation or contraction, the muscles were transferred to KRB buffer containing 1 mmol/liter 2-deoxy-D-glucose (1.5 Ci/ml) and 7 mmol/liter D-[14C]mannitol (0.3 Ci/ml) (PerkinElmer Life Sciences) at 30 °C and incubated for 10 min.

Techniques: Activity Assay, Expressing, Western Blot, Isolation, Muscles, Incubation, In Vitro

Journal: The Journal of biological chemistry

Article Title: AMP-activated protein kinase alpha2 activity is not essential for contraction- and hyperosmolarity-induced glucose transport in skeletal muscle.

doi: 10.1074/jbc.M504208200

Figure Lengend Snippet: FIGURE 2. Predominant inhibition of AMPK 2 activity in skeletal muscle from both 1 and 2 inactive TG mice. Isolated EDL muscles from wild type, 1i TG, 2i TG, and 1/2i TG mice were incubated in KRB buffer with or without AICAR (2 mM) for 20 min in vitro.A,representativeimmunoblots(IB)ofAMPK1and2protein.AMPK1activity(B) and AMPK 2 activity (C) were measured in immune complexes prepared by immuno- precipitation using each isoform-specific antibody from lysates of the muscles. Ba, basal. Ai, AICAR-stimulated. Data are means S.E., n 4/group. WT, wild type.

Article Snippet: Measurement of Glucose Transport in Vitro—Immediately aftermuscle incubation or contraction, the muscles were transferred to KRB buffer containing 1 mmol/liter 2-deoxy-D-glucose (1.5 Ci/ml) and 7 mmol/liter D-[14C]mannitol (0.3 Ci/ml) (PerkinElmer Life Sciences) at 30 °C and incubated for 10 min.

Techniques: Inhibition, Activity Assay, Isolation, Muscles, Incubation, In Vitro, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: AMP-activated protein kinase alpha2 activity is not essential for contraction- and hyperosmolarity-induced glucose transport in skeletal muscle.

doi: 10.1074/jbc.M504208200

Figure Lengend Snippet: FIGURE 3. 2i TG mice have abolished AICAR-stimulated muscle glucose transport in vitro and AICAR resistance in vivo. A, isolated EDL muscles were incubated in KRB bufferwithorwithoutAICAR(2mM)for20minfollowedbythemeasurementof2-deoxy-

Article Snippet: Measurement of Glucose Transport in Vitro—Immediately aftermuscle incubation or contraction, the muscles were transferred to KRB buffer containing 1 mmol/liter 2-deoxy-D-glucose (1.5 Ci/ml) and 7 mmol/liter D-[14C]mannitol (0.3 Ci/ml) (PerkinElmer Life Sciences) at 30 °C and incubated for 10 min.

Techniques: In Vitro, In Vivo, Isolation, Muscles, Incubation

Journal: The Journal of biological chemistry

Article Title: AMP-activated protein kinase alpha2 activity is not essential for contraction- and hyperosmolarity-induced glucose transport in skeletal muscle.

doi: 10.1074/jbc.M504208200

Figure Lengend Snippet: FIGURE 4. Glucose transport in skeletal muscle of 2i TG mice measured in vitro. Isolated EDL muscles were incubated in KRB buffer with or without 3 M rotenone for 40 min (n 5–6/group) (A) or 120 mM sorbitol for 30 min (n 5–6/group) (B) or subjected to electrical stimulation to induce contractions (n 16/group) (C). Muscle lysates were usedforthemeasurementofglucosetransport.CTXN,contraction.Dataarethemeans S.E. *, significantly different from basal of the same genotype (*, p 0.05; ** p 0.01). †, significantly different from wild type with the same intervention (†, p 0.05; ††, p 0.01).

Article Snippet: Measurement of Glucose Transport in Vitro—Immediately aftermuscle incubation or contraction, the muscles were transferred to KRB buffer containing 1 mmol/liter 2-deoxy-D-glucose (1.5 Ci/ml) and 7 mmol/liter D-[14C]mannitol (0.3 Ci/ml) (PerkinElmer Life Sciences) at 30 °C and incubated for 10 min.

Techniques: In Vitro, Isolation, Muscles, Incubation