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: Biochimica et Biophysica Acta

Article Title: Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

doi: 10.1016/j.bbamcr.2014.02.018

Figure Lengend Snippet: PAD expression in the developing human central nervous system (brain and spinal cord) and in hNSCs. A) Real time RT-PCR analysis of PAD3 and PAD2 in fetal brains (left panel) and spinal cords (right panel) from human embryos at 42, 63 and 70 days of gestation. PAD2 expression increases while PAD3 expression decreases with development. Human liver was used as a positive control for all PADs. Asterisk indicates statistically significant differences (p < 0.05). B) PAD2 and PAD3 transcript detected by in situ hybridization in human spinal cord at 46 days of gestation (dg). Scale bars are 200 μm. C) PAD2 and PAD3 protein detected by Western blot in developing human brain (Br) and spinal cord (SC); no dramatic change in PAD protein expression is observed.

Article Snippet: The primary antibodies used were rabbit anti-PAD3 (Covalab), rabbit anti-PAD2 (Covalab), rabbit anti-Cleaved Caspase 3 (Cell Signaling), goat anti-AIF (Santa Cruz), mouse anti-ß3-tubulin (Promega), mouse anti-vimentin (Dako), and Alexa 568-conjugated anti-Annexin V (Life Technologies).

Techniques: Expressing, Quantitative RT-PCR, Positive Control, In Situ Hybridization, Western Blot

Journal: Biochimica et Biophysica Acta

Article Title: Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

doi: 10.1016/j.bbamcr.2014.02.018

Figure Lengend Snippet: PADs are expressed in human neural stem cells (hNSCs) and PAD3 inhibition increases hNSC proliferation. A) PAD2 and PAD3 transcript detected by RT-qPCR in hNSC derived from embryonic brain and spinal cord (SC). B) Detection of PAD2 and PAD3 by immunocytochemistry (red) in hNSCs: both proteins are detected in cytoplasm and nucleus (counterstained with Hoechst dye). Scale bars: 25 μm. All pictures are at the same magnification. C) Analysis of cell growth determined by the methylene blue assay after treatment with 100 μM Cl-amidine for 24, 48 or 96 h. Cl-amidine significantly increases hNSC proliferation as compared to controls at 48 and 96 h. * = p < 0.05, ** = p < 0.01 by ANOVA and Student's t -test. D) Analysis of cell growth determined by the methylene blue assay after transfection with siRNA against PAD2 (siPAD2) and PAD3 (siPAD3) or scrambled siRNA. A significant increase in cell growth as compared to controls is observed at 48 h only in cells transfected with siPAD3 (p < 0.05; two-way ANOVA). Error bars indicate SDM; n ≥ 3.

Article Snippet: The primary antibodies used were rabbit anti-PAD3 (Covalab), rabbit anti-PAD2 (Covalab), rabbit anti-Cleaved Caspase 3 (Cell Signaling), goat anti-AIF (Santa Cruz), mouse anti-ß3-tubulin (Promega), mouse anti-vimentin (Dako), and Alexa 568-conjugated anti-Annexin V (Life Technologies).

Techniques: Inhibition, Quantitative RT-PCR, Derivative Assay, Immunocytochemistry, Transfection

Journal: Biochimica et Biophysica Acta

Article Title: Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

doi: 10.1016/j.bbamcr.2014.02.018

Figure Lengend Snippet: Effect of thapsigargin on PAD expression and citrullination in hNSCs. A) RT-qPCR analysis of PAD2 and PAD3 transcripts after thapsigargin treatment: note up-regulation of PAD3, but not PAD2 transcript, in treated cells; * = p < 0.05 by ANOVA and Student's t -test (n ≥ 3; error bars indicate SDM). B) Western blot analysis of citrullinated proteins detected by F95 monoclonal antibody and of citrullinated histone H3 (Cit-H3) following treatment with either Cl-amidine (100 μM) or thapsigargin (5 μM) alone, or both compounds for 24 h. Cl-amidine was added to the culture medium 15 min before thapsigargin treatment. Actin was used as a loading control. Note that PAD activation by thapsigargin results in protein citrullination and this is reduced by the PAD inhibitor, Cl-amidine.

Article Snippet: The primary antibodies used were rabbit anti-PAD3 (Covalab), rabbit anti-PAD2 (Covalab), rabbit anti-Cleaved Caspase 3 (Cell Signaling), goat anti-AIF (Santa Cruz), mouse anti-ß3-tubulin (Promega), mouse anti-vimentin (Dako), and Alexa 568-conjugated anti-Annexin V (Life Technologies).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activation Assay

Journal: Biochimica et Biophysica Acta

Article Title: Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase–AIF pathway

doi: 10.1016/j.bbamcr.2014.02.018

Figure Lengend Snippet: PAD3 is the main PAD involved in hNSC cell death. Death/survival of hNSCs treated for 24 h with thapsigargin was determined by the methylene blue assay and staining for the apoptosis marker annexin V. A) Cell survival is significantly (p < 0.05; two-way ANOVA) reduced in hNSCs carrying PAD3-EGFP as compared to cells transfected with PAD3 lacking the active site (ΔPAD3-EGFP), EGFP alone, or no transfection (WT). B) Example of cells expressing PAD3-EGFP (PAD, green) and Annexin-V (AnV, red) counted for quantification (arrows); note the significantly higher percentage of PAD3-EGFP/Annexin-V-positive cells; * = p < 0.05 by ANOVA and Student's t -test. C) Cell survival is significantly (p < 0.05; two-way ANOVA) increased in hNSC transfected with PAD3 but not PAD2 siRNA. Scale bars are 50 μmin B. Error bars indicate SDM; n ≥ 3.

Article Snippet: The primary antibodies used were rabbit anti-PAD3 (Covalab), rabbit anti-PAD2 (Covalab), rabbit anti-Cleaved Caspase 3 (Cell Signaling), goat anti-AIF (Santa Cruz), mouse anti-ß3-tubulin (Promega), mouse anti-vimentin (Dako), and Alexa 568-conjugated anti-Annexin V (Life Technologies).

Techniques: Staining, Marker, Transfection, Expressing

Journal: Arthritis Research & Therapy

Article Title: Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue

doi: 10.1186/s13075-016-1140-9

Figure Lengend Snippet: OA synovial tissue histology. a H&E, ( b ) citrulline and homocitrulline (F95), ( c ) PAD2, ( d ) PAD3, ( e ) PAD4 and ( f ) MPO staining. The scale bar indicates 50 μm. L lumen of synovial cavity, blue arrow synovial lining layer, double-headed arrow synovial stroma

Article Snippet: PAD2 enzyme was stained with anti-human PAD2 antibody 1:250 (ab16478, Abcam) and PAD3 with 1:50 (ab183209, Abcam) after heat-mediated antigen retrieval in Tris-EDTA, pH 9 and myeloperoxidase without antigen retrieval with anti-human myeloperoxidase antibody 1:5000 (A0398, Dako).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue

doi: 10.1186/s13075-016-1140-9

Figure Lengend Snippet: RA synovial tissue histology. a H&E, ( b ) citrulline and homocitrulline (F95), ( c ) PAD2, ( d ) PAD3, ( e ) PAD4 and ( f ) MPO staining. The scale bar indicates 50 μm. L lumen of synovial cavity, blue arrow synovial lining layer, double-headed arrow synovial stroma, black arrow intact neutrophil, arrowhead disrupted neutrophil

Article Snippet: PAD2 enzyme was stained with anti-human PAD2 antibody 1:250 (ab16478, Abcam) and PAD3 with 1:50 (ab183209, Abcam) after heat-mediated antigen retrieval in Tris-EDTA, pH 9 and myeloperoxidase without antigen retrieval with anti-human myeloperoxidase antibody 1:5000 (A0398, Dako).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue

doi: 10.1186/s13075-016-1140-9

Figure Lengend Snippet: Histology of necrotic RA synovial tissue consisting of fibrinoid necrosis and sporadic intact cells. a H&E, ( b ) citrulline and homocitrulline (F95), ( c ) PAD2, ( d ) and PAD3, ( e ) PAD4 and ( f ) MPO staining. The scale bar indicates 50 μm. Arrowhead intact cells, purple double-headed arrow fibrinoid necrosis

Article Snippet: PAD2 enzyme was stained with anti-human PAD2 antibody 1:250 (ab16478, Abcam) and PAD3 with 1:50 (ab183209, Abcam) after heat-mediated antigen retrieval in Tris-EDTA, pH 9 and myeloperoxidase without antigen retrieval with anti-human myeloperoxidase antibody 1:5000 (A0398, Dako).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue

doi: 10.1186/s13075-016-1140-9

Figure Lengend Snippet: Histology of rheumatoid nodule. a H&E, ( b ) citrulline and homocitrulline (F95), ( c ) PAD2, ( d ) PAD3, ( e ) PAD4 and ( f ) MPO staining. The scale bar indicates 50 μm. Purple double-headed arrow fibrinoid necrosis, blue double-headed arrow palisading histiocytes, black double-headed arrow stromal cells

Article Snippet: PAD2 enzyme was stained with anti-human PAD2 antibody 1:250 (ab16478, Abcam) and PAD3 with 1:50 (ab183209, Abcam) after heat-mediated antigen retrieval in Tris-EDTA, pH 9 and myeloperoxidase without antigen retrieval with anti-human myeloperoxidase antibody 1:5000 (A0398, Dako).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue

doi: 10.1186/s13075-016-1140-9

Figure Lengend Snippet: Cellular localization of ( a ) F95 in RA, ( b ) PAD2 in RA, ( c ) PAD3 in RA, ( d ) MPO in RA, ( e ) PAD4 in RA and ( f ) PAD4 staining in OA. The scale bar indicates 50 μm. L lumen of synovial cavity, black arrow intact neutrophil, arrowhead disrupted neutrophil

Article Snippet: PAD2 enzyme was stained with anti-human PAD2 antibody 1:250 (ab16478, Abcam) and PAD3 with 1:50 (ab183209, Abcam) after heat-mediated antigen retrieval in Tris-EDTA, pH 9 and myeloperoxidase without antigen retrieval with anti-human myeloperoxidase antibody 1:5000 (A0398, Dako).

Techniques: Staining

Journal: Arthritis Research & Therapy

Article Title: Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue

doi: 10.1186/s13075-016-1140-9

Figure Lengend Snippet: PAD3 ( c , g ) co-localization with citrulline- and homocitrulline-binding F95 antibody ( b ) and macrophage marker KP1 (CD68) ( g ) in the intact tissue adjacent to necrosis in rheumatoid nodule. PAD3 HIER was performed in pH 9 Tris-EDTA. PAD3 and F95 bound abundantly to the cytoplasm of cells adjacent to necrosis ( d ). PAD3 co-localized with KP1 in the cells adjacent to necrosis ( h ). Asterisk necrosis, double-headed arrow palisading histiocytes

Article Snippet: PAD2 enzyme was stained with anti-human PAD2 antibody 1:250 (ab16478, Abcam) and PAD3 with 1:50 (ab183209, Abcam) after heat-mediated antigen retrieval in Tris-EDTA, pH 9 and myeloperoxidase without antigen retrieval with anti-human myeloperoxidase antibody 1:5000 (A0398, Dako).

Techniques: Binding Assay, Marker