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: A Principal component analysis showed separation of 50 kPa-MDFs ( n = 6) vs 2 kPa-MDFs ( n = 8). B Volcano map between 50 kPa-MDFs and 2 kPa-MDFs. C Metabolites enrichments for significantly upregulated pathways from 50 kPa-MDFs vs 2 kPa-MDFs. D Illustration schematic of arginine and proline metabolism pathway. E Measurement of L-Arginine, L-Ornithine and L-Proline in 2 kPa-MDFs and 50 kPa-MDFs ( n = 5). F Western blot assay for key metabolic enzymes Arg1, OAT, PYCR1 and PRODH in 2 kPa-MDFs/50 kPa-MDFs and quantitative analysis ( n = 3). G Measurement of key metabolic enzymes activity Arg1, OAT, PYCR1 and PRODH in 2 kPa-MDFs/50 kPa-MDFs and quantitative analysis ( n = 5). The results are expressed as the means with SD. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: For immunohistochemistry and immunofluorescence, 5 mm sections were incubated with primary antibodies against Piezo1 (ab128245, 1:1000, Abcam), α-SMA (ab7817, 1:200, Abcam), Collagen I (ab6308, 1:200, Abcam), GFP (ab290, 1:500, Abcam), Arg1 (16001-1-AP, 1:400, Proteintech), OAT (A6235, 1:100, ABclonal Technology), PYCR1 (13108-1-AP, 1:200, Proteintech), PRODH (22980-1-AP, 1:400, Proteintech) overnight at 4 °C.

Techniques: 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: 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: For immunohistochemistry and immunofluorescence, 5 mm sections were incubated with primary antibodies against Piezo1 (ab128245, 1:1000, Abcam), α-SMA (ab7817, 1:200, Abcam), Collagen I (ab6308, 1:200, Abcam), GFP (ab290, 1:500, Abcam), Arg1 (16001-1-AP, 1:400, Proteintech), OAT (A6235, 1:100, ABclonal Technology), PYCR1 (13108-1-AP, 1:200, Proteintech), PRODH (22980-1-AP, 1:400, Proteintech) overnight at 4 °C.

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: 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: For immunohistochemistry and immunofluorescence, 5 mm sections were incubated with primary antibodies against Piezo1 (ab128245, 1:1000, Abcam), α-SMA (ab7817, 1:200, Abcam), Collagen I (ab6308, 1:200, Abcam), GFP (ab290, 1:500, Abcam), Arg1 (16001-1-AP, 1:400, Proteintech), OAT (A6235, 1:100, ABclonal Technology), PYCR1 (13108-1-AP, 1:200, Proteintech), PRODH (22980-1-AP, 1:400, Proteintech) overnight at 4 °C.

Techniques: Expressing, Immunofluorescence, 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: 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: For immunohistochemistry and immunofluorescence, 5 mm sections were incubated with primary antibodies against Piezo1 (ab128245, 1:1000, Abcam), α-SMA (ab7817, 1:200, Abcam), Collagen I (ab6308, 1:200, Abcam), GFP (ab290, 1:500, Abcam), Arg1 (16001-1-AP, 1:400, Proteintech), OAT (A6235, 1:100, ABclonal Technology), PYCR1 (13108-1-AP, 1:200, Proteintech), PRODH (22980-1-AP, 1:400, Proteintech) overnight at 4 °C.

Techniques: Biomarker Discovery, Knockdown, Staining