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: Molecular Vision

Article Title: Peptidylarginine deiminase type 2 is over expressed in the glaucomatous optic nerve

doi:

Figure Lengend Snippet: Elevated PAD2 and protein-bound citrulline immunoreactivity in glaucomatous optic nerve. A : Representative western analyses with monoclonal anti-PAD2 of protein extracts from human optic nerve tissue (~25 µg per lane) demonstrating the presence of elevated levels of PAD2 was detected in glaucomatous tissues. Lower panel shows the GAPDH immunoreactivity indicating equal protein loading. B : Western analyses with rabbit polyclonal antibody to citrulline (25 µg protein per lane). Prior to applying antibody, membrane with transferred proteins was treated with 2,3-butanedione monooxime and antipyrine in a strong acid environment to modify the protein-bound citrullines to enable the detection with the antibody. Proteins were extracted from the optic nerve of Caucasian cadaver donor eyes: age and gender are indicated.

Article Snippet: Western analyses were performed with 5 μg of protein, 4%–20% gradient gels (Invitrogen Inc., Carlsbad, CA), electroblotted to PVDF membrane and probed with antibodies: rabbit polyclonal antibody against PAD2 (Abcam Inc., Cambridge, MA), mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore Corporation, Billerica, MA) and against protein-bound monoxime modified citrulline (Millipore Corporation).

Techniques: Western Blot, Membrane

Journal: Molecular Vision

Article Title: Peptidylarginine deiminase type 2 is over expressed in the glaucomatous optic nerve

doi:

Figure Lengend Snippet: The PAD2 and level of deimination using ELISA analyses as described in methods. A and B : PAD2 level represented by hollow bars and dotted bars for control and glaucomatous donors. C and D : Level of deimination are represented by hollow bars and dotted bars for control and glaucomatous donors respectively. The results are standard deviation of three independent experiments.

Article Snippet: Western analyses were performed with 5 μg of protein, 4%–20% gradient gels (Invitrogen Inc., Carlsbad, CA), electroblotted to PVDF membrane and probed with antibodies: rabbit polyclonal antibody against PAD2 (Abcam Inc., Cambridge, MA), mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore Corporation, Billerica, MA) and against protein-bound monoxime modified citrulline (Millipore Corporation).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Standard Deviation

Journal: Molecular Vision

Article Title: Peptidylarginine deiminase type 2 is over expressed in the glaucomatous optic nerve

doi:

Figure Lengend Snippet: Elevated PAD2 activity in glaucomatous optic nerve. PAD activity was measured as described in the methods and absorbance at 435 nm has been shown.

Article Snippet: Western analyses were performed with 5 μg of protein, 4%–20% gradient gels (Invitrogen Inc., Carlsbad, CA), electroblotted to PVDF membrane and probed with antibodies: rabbit polyclonal antibody against PAD2 (Abcam Inc., Cambridge, MA), mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore Corporation, Billerica, MA) and against protein-bound monoxime modified citrulline (Millipore Corporation).

Techniques: Activity Assay

Journal: Molecular Vision

Article Title: Peptidylarginine deiminase type 2 is over expressed in the glaucomatous optic nerve

doi:

Figure Lengend Snippet: Representative immunohistochemical analyses of NTG (70 F) and control (72F) donor optic nerve sections. A : Immunoreactivity for protein-bound citrulline detected by a rabbit polyclonal antibody to citrulline after monoxime modification as described in methods. B : Merged image of anti-citrulline with DAPI. C : Control donor section probed for immunoreacitivty with anti-citrulline. D : Merged image of anti-citrulline and DAPI for control donor section. E : Densitometric analysis of immunohistochemical detection of protein-bound citrulline in NTG eyes. The data was analyzed using Image J software.

Article Snippet: Western analyses were performed with 5 μg of protein, 4%–20% gradient gels (Invitrogen Inc., Carlsbad, CA), electroblotted to PVDF membrane and probed with antibodies: rabbit polyclonal antibody against PAD2 (Abcam Inc., Cambridge, MA), mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore Corporation, Billerica, MA) and against protein-bound monoxime modified citrulline (Millipore Corporation).

Techniques: Immunohistochemical staining, Control, Modification, Software

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