Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of AMF30a on in vitro proliferation of human corneal endothelial cells (hCECs). AMF30a (5µM, PAD2 inhibitor, Cayman, Ann Arbor, Michigan) was applied for 48 h. ( A ) hCECs were identified using immunofluorescence staining of ZO1 and CD166. ( B - C ) Cell area of hCECs were evaluated using cell morphological pictures. Scale bar = 100 μm. ( D and E ) Cell proliferation was measured using CCK-8 cell viability assay and BrdU incorporation assay. ( F ) Cytotoxicity was evaluated using LDH cytotoxicity assay (1.002 ± 0.047 vs. 0.925 ± 0.018). ( G - I ) Cell cycle analysis was assessed using PI staining. (J and K) Wound healing assay was performed using the measurement of scratched ares. Scale bar = 500 μm. ( L and N ) Intracellular oxidative stress levels were measured using DCF-DA assay. Scale bar = 50 μm. * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Article Snippet: Primary antibodies were as follows: rabbit anti-human CD166 antibody (ab109215, Abcam, 1∶500 dilution), rabbit anti-human ZO-1 antibody (sc-10804, Santa Cruz, 1∶500 dilution), mouse anti-human ERK1/2 antibody (sc-514032, Santa Cruz, 1∶500 dilution); mouse anti-human pERK1/2 antibody (sc-136521, Santa Cruz, 1∶500 dilution); mouse anti-human YAP antibody (sc-376830, 1∶500 dilution); rabbit anti-pYAP antibody (PA5-17481, Invitrogen, 1∶500 dilution); rabbit anti-human PAD2 antibody (12110-1-AP, Proteintech, 1∶500 dilution); mouse anti-ATF4 antibody (sc-390063, 1∶500 dilution); mouse anti-human ROCK1 antibody (sc-17794, 1∶500 dilution); mouse anti-ROCK2 antibody (sc-398519, 1∶500 dilution); or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1∶5000 dilution).

Techniques: In Vitro, Immunofluorescence, Staining, CCK-8 Assay, Viability Assay, BrdU Incorporation Assay, LDH Cytotoxicity Assay, Cell Cycle Assay, Wound Healing Assay

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of cytokine on human corneal endothelial cells. ( A ) Immunofluorescence staining of PAD2 shows the distribution of PAD2. Scale bar = 100 μm. ( B and C ) Western blot of PAD2 was used to evaluate the PAD2 levels. ( D and E ) ATF4 levels were evaluated using western blot. ( F ) Immunofluorescence staining of peptidyl-citrulline shows the intensity and distribution of peptidyl-citrulline. Scale bar = 100 μm. * p < 0.05.

Article Snippet: Primary antibodies were as follows: rabbit anti-human CD166 antibody (ab109215, Abcam, 1∶500 dilution), rabbit anti-human ZO-1 antibody (sc-10804, Santa Cruz, 1∶500 dilution), mouse anti-human ERK1/2 antibody (sc-514032, Santa Cruz, 1∶500 dilution); mouse anti-human pERK1/2 antibody (sc-136521, Santa Cruz, 1∶500 dilution); mouse anti-human YAP antibody (sc-376830, 1∶500 dilution); rabbit anti-pYAP antibody (PA5-17481, Invitrogen, 1∶500 dilution); rabbit anti-human PAD2 antibody (12110-1-AP, Proteintech, 1∶500 dilution); mouse anti-ATF4 antibody (sc-390063, 1∶500 dilution); mouse anti-human ROCK1 antibody (sc-17794, 1∶500 dilution); mouse anti-ROCK2 antibody (sc-398519, 1∶500 dilution); or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1∶5000 dilution).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Schematic diagram summarizing PAD2-mediated signaling in hCECs.

Article Snippet: Primary antibodies were as follows: rabbit anti-human CD166 antibody (ab109215, Abcam, 1∶500 dilution), rabbit anti-human ZO-1 antibody (sc-10804, Santa Cruz, 1∶500 dilution), mouse anti-human ERK1/2 antibody (sc-514032, Santa Cruz, 1∶500 dilution); mouse anti-human pERK1/2 antibody (sc-136521, Santa Cruz, 1∶500 dilution); mouse anti-human YAP antibody (sc-376830, 1∶500 dilution); rabbit anti-pYAP antibody (PA5-17481, Invitrogen, 1∶500 dilution); rabbit anti-human PAD2 antibody (12110-1-AP, Proteintech, 1∶500 dilution); mouse anti-ATF4 antibody (sc-390063, 1∶500 dilution); mouse anti-human ROCK1 antibody (sc-17794, 1∶500 dilution); mouse anti-ROCK2 antibody (sc-398519, 1∶500 dilution); or rabbit anti-GAPDH antibody (LF-PA0212, Abfrontier, 1∶5000 dilution).

Techniques:

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of AMF30a on in vitro proliferation of human corneal endothelial cells (hCECs). AMF30a (5µM, PAD2 inhibitor, Cayman, Ann Arbor, Michigan) was applied for 48 h. ( A ) hCECs were identified using immunofluorescence staining of ZO1 and CD166. ( B - C ) Cell area of hCECs were evaluated using cell morphological pictures. Scale bar = 100 μm. ( D and E ) Cell proliferation was measured using CCK-8 cell viability assay and BrdU incorporation assay. ( F ) Cytotoxicity was evaluated using LDH cytotoxicity assay (1.002 ± 0.047 vs. 0.925 ± 0.018). ( G - I ) Cell cycle analysis was assessed using PI staining. (J and K) Wound healing assay was performed using the measurement of scratched ares. Scale bar = 500 μm. ( L and N ) Intracellular oxidative stress levels were measured using DCF-DA assay. Scale bar = 50 μm. * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Article Snippet: The cells were treated overnight with either mouse anti-E-cadherin antibody (sc-8426; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-YAP antibody (sc-376830; Santa Cruz Biotechnology), rabbit anti-PAD2 antibody (sc-293271; Santa Cruz Biotechnology), mouse anti-peptidyl-citrulline antibody (MABN328, Merck Millipore, Feltham, UK), and rabbit anti-NF-κB antibody (ab7970, Abcam, 1:100) at 4 °C and then rinsed with PBS.

Techniques: In Vitro, Immunofluorescence, Staining, CCK-8 Assay, Viability Assay, BrdU Incorporation Assay, LDH Cytotoxicity Assay, Cell Cycle Assay, Wound Healing Assay

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Effect of cytokine on human corneal endothelial cells. ( A ) Immunofluorescence staining of PAD2 shows the distribution of PAD2. Scale bar = 100 μm. ( B and C ) Western blot of PAD2 was used to evaluate the PAD2 levels. ( D and E ) ATF4 levels were evaluated using western blot. ( F ) Immunofluorescence staining of peptidyl-citrulline shows the intensity and distribution of peptidyl-citrulline. Scale bar = 100 μm. * p < 0.05.

Article Snippet: The cells were treated overnight with either mouse anti-E-cadherin antibody (sc-8426; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-YAP antibody (sc-376830; Santa Cruz Biotechnology), rabbit anti-PAD2 antibody (sc-293271; Santa Cruz Biotechnology), mouse anti-peptidyl-citrulline antibody (MABN328, Merck Millipore, Feltham, UK), and rabbit anti-NF-κB antibody (ab7970, Abcam, 1:100) at 4 °C and then rinsed with PBS.

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Scientific Reports

Article Title: AMF30a promotes survival and function of human corneal endothelial cells by regulating TGF-β/ROCK/HIPPO pathway

doi: 10.1038/s41598-025-13656-2

Figure Lengend Snippet: Schematic diagram summarizing PAD2-mediated signaling in hCECs.

Article Snippet: The cells were treated overnight with either mouse anti-E-cadherin antibody (sc-8426; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-YAP antibody (sc-376830; Santa Cruz Biotechnology), rabbit anti-PAD2 antibody (sc-293271; Santa Cruz Biotechnology), mouse anti-peptidyl-citrulline antibody (MABN328, Merck Millipore, Feltham, UK), and rabbit anti-NF-κB antibody (ab7970, Abcam, 1:100) at 4 °C and then rinsed with PBS.

Techniques:

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Reciprocal regulation of protein arginine deiminase 2 and 4 expression in the colonic mucosa of ulcerative colitis

doi: 10.3164/jcbn.23-77

Figure Lengend Snippet: mRNA expression of protein arginine deiminase 2 and 4 in patients with inflammatory bowel diseases. mRNA was isolated from the ileal and colonic mucosa of patients with ulcerative colitis (UC; active disease, n = 20; remitted disease, n = 20) and Crohn’s disease (CD; active disease, n = 10; remitted disease, n = 10). Non-tumorous portions of the colonic mucosa in patients with colon adenoma served as healthy colonic mucosa (HC, n = 4). qRT-PCR analysis of the mRNA expression levels of protein arginine deiminase (PAD)2 and PAD4. Each dot represents the value for each patient. (A) Data are presented as the mean ± SE. ** p <0.01, N.S.; not significant. (B) Correlation between PAD2 and PAD4 mRNA expression in patients with UC and CD. P values and correlation coefficient ( r ) values, as determined by Pearson’s correlation coefficient, are shown.

Article Snippet: PAD2 and PAD4 expression was visualized using the DAKO EnVision+ System (DAKO JAPAN, Tokyo, Japan), as previously described, with rabbit human anti-PAD2 antibody (Proteintech, Rosemont, IL) and anti-PAD4 antibody (GeneTex, Irvine, CA). ( )

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Reciprocal regulation of protein arginine deiminase 2 and 4 expression in the colonic mucosa of ulcerative colitis

doi: 10.3164/jcbn.23-77

Figure Lengend Snippet: Correlation between the mRNA expression of protein arginine deiminase 4 ( PAD4 ) and cytokines in patients with ulcerative colitis (UC). mRNA was isolated from the colonic mucosa of patients with UC. (A) Correlation between the mRNA expression of PAD4 and IFN-α4 , IFN-β , IL12/23p40 , C-X-C motif chemokine ligand 8 ( CXCL8 ), CXCL10 , TNF receptor-associated factor 3 ( TRAF3 ), interferon regulatory factor 3 ( IRF3 ), IRF7 , IL-6 , and TNF -α. (B) Correlation between the mRNA expression of PAD2 and IFN-α4 , IFN-β , CXCL8 , CXCL10 , IL-6 , and TNF -α. Each dot represents the value for each patient. P values and correlation coefficient ( r ) values, as determined by Pearson’s correlation coefficient, are shown.

Article Snippet: PAD2 and PAD4 expression was visualized using the DAKO EnVision+ System (DAKO JAPAN, Tokyo, Japan), as previously described, with rabbit human anti-PAD2 antibody (Proteintech, Rosemont, IL) and anti-PAD4 antibody (GeneTex, Irvine, CA). ( )

Techniques: Expressing, Isolation

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Reciprocal regulation of protein arginine deiminase 2 and 4 expression in the colonic mucosa of ulcerative colitis

doi: 10.3164/jcbn.23-77

Figure Lengend Snippet: Correlation between the mRNA expression of protein arginine deiminase 4 ( PAD4 ) and cytokines in patients with Crohn’s disease (CD). mRNA was isolated from the ileal and colonic mucosa of patients with CD. (A) Correlation between the mRNA expression of PAD4 and IFN-α4 , IFN-β , IL12/23p40 , C-X-C motif chemokine ligand 8 ( CXCL8 ), CXCL10 , TNF receptor-associated factor 3 ( TRAF3 ), interferon regulatory factor 3 ( IRF3 ), IRF7 , IL-6 , and TNF-α . (B) Correlation between the mRNA expression of PAD2 and IFN-α4 , IFN-β , CXCL8 , CXCL10 , IL-6 , and TNF-α . Each dot represents the value for each patient. P values and correlation coefficient ( r ) values, as determined by Pearson’s correlation coefficient, are shown.

Article Snippet: PAD2 and PAD4 expression was visualized using the DAKO EnVision+ System (DAKO JAPAN, Tokyo, Japan), as previously described, with rabbit human anti-PAD2 antibody (Proteintech, Rosemont, IL) and anti-PAD4 antibody (GeneTex, Irvine, CA). ( )

Techniques: Expressing, Isolation