Journal: The Journal of Immunology Author Choice

Article Title: Citrullination Controls Dendritic Cell Transdifferentiation into Osteoclasts

doi: 10.4049/jimmunol.1800534

Figure Lengend Snippet: Protein citrullination and high PAD activity levels are associated with differentiation plasticity toward OCs in iDCs. (A) PCA indicates clearly distinct protein profiles in DCs obtained from sparse or dense cultures (principal components 1 and 2 are shown). Triplicate samples were analyzed from three independent experiments. (B) Images showing resorption activity on calcium phosphate–coated surface representing OCs that were generated from sparse or dense culture–derived iDCs (original magnification ×40). (C) Higher PAD activity was observed in the lysates of DCs that were obtained from dense cultures. Activity levels are normalized to sparse cultures; the graph represents four individual experiments, each with three replicates. *p < 0.05. (D) Normalized PAD activity levels are shown in cell lysates of iDCs and mature OCs. (E) Immunohistochemistry images indicate 3,3-diaminobenzidene staining of PAD4 and PAD2 enzymes with their respective irrelevant controls during OC development from DC precursors. Slides were counterstained with Mayer’s hemotoxylin and analyzed in light microscopy (original magnification ×100). (F) Citrullinated actin was identified using mass spectrometry in lysates of DCs developing in sparse or dense cultures. The graph shows Mascot score values obtained for the identified peptide; symbols represent three different DC cultures. Mean ± SD values are shown. (G) Reduced DC–OC transdifferentiation in the presence of the PAD inhibitor Cl-amidine.

Article Snippet: Slides were then incubated with rabbit polyclonal anti-PAD2 (Cosmobio, Tokyo, Japan) and anti-PAD4 (Abcam, Cambridge, U.K.) primary Abs, diluted in PBS containing 0.1% saponin for 90 min at room temperature at a final concentration of 0.02 μg/ml for the anti-PAD2 Ab and 1 μg/ml for the anti-PAD4 Ab.

Techniques: Activity Assay, Generated, Derivative Assay, Immunohistochemistry, Staining, Light Microscopy, Mass Spectrometry

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: 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: mRNA expression of protein arginine deiminase 2 and 4 in patients with different types of ulcerative colitis and Crohn’s disease. mRNA was isolated from the ileal and colonic mucosa of patients with ulcerative colitis (UC; active disease, n = 20; remitted disease, n = 20, A) and Crohn’s disease (CD; active disease, n = 10; remitted disease, n = 10, B). qRT-PCR analysis of the mRNA expression levels of protein arginine deiminase (PAD)2 and PAD4. Each dot represents the value for each patient. Data are presented as the mean ± SE. ** p <0.01.

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

Journal: Nature Communications

Article Title: A citrullinated histone H3 monoclonal antibody for immune modulation in sepsis

doi: 10.1038/s41467-025-62788-6

Figure Lengend Snippet: a Immunofluorescence analysis showing efficient uptake of Alexa Fluor 488-labeled CitH3 (5 µg/mL) by wild-type (WT) BMDMs but not by Tlr2⁻/⁻ BMDMs. Cellular intensity of Alexa-488 was quantified ( n = 5). Statistical analysis was performed using two-sided, Unpaired t test with Welch’s correction. b Cytokine profiling of pro- and anti-inflammatory mediators (IL-6, IL-1β, IL-10, TNFα, IFN-α, IFN-β) in supernatants of WT and Tlr2⁻/⁻ BMDMs treated with H3 or CitH3 peptides (15 µg/mL) for 16 h ( n = per group). LPS (200 ng/mL) served as positive control. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. c Immunocytochemistry demonstrating nuclear translocation of PAD2 in BMDMs after 1 h exposure to CitH3 peptide (15 µg/mL). LPS (200 ng/mL) served as a positive control. PAD2 was stained with Alexa Fluor 488 (green), and nuclei were counterstained with DAPI (blue). Quantification of PAD2 nuclear localization was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 3 per group). d Subcellular fractionation analysis confirming significant nuclear localization and citrullination of PAD2 in WT BMDMs following treatment with H3, CitH3, or LPS. Quantification was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test ( n = per group). e In vitro citrullination assay showing MPB-tagged PAD2-mediated citrullination of H3 in a Ca²⁺-dependent manner. EGTA-mediated Ca²⁺ chelation eliminated CitH3 production. Similar results from three independent replicates. f In vitro citrullination assay showing PAD2 citrullination. PAD2 auto-citrullination was confirmed as His-tagged recombinant PAD2 co-incubated with MBP-tagged PAD2 resulted in PAD2 citrullination. PAD4 also promotes PAD2 citrullination. Quantification was performed by Image J, citrullination signals were normalized to corresponding input signals and further normalized to the matching EGTA-treated group in each replicate experiments. Statistical analysis was performed using two-sided t-test.Data are presented as mean ± SD. Statistical significance in all panels was determined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used in immunocytochemistry assays: PAD2 (12110-1-AP), PAD4 (17373-1-AP), Rab5 (CST, 3547), EEA1 (CST, 3288), MPO (Abcam ab208670)

Techniques: Immunofluorescence, Labeling, Positive Control, Immunocytochemistry, Translocation Assay, Staining, Fractionation, In Vitro, Recombinant, Incubation

Journal: Nature Communications

Article Title: A citrullinated histone H3 monoclonal antibody for immune modulation in sepsis

doi: 10.1038/s41467-025-62788-6

Figure Lengend Snippet: a LDH release from supernatants of BMDMs treated with increasing doses of CitH3 or unmodified H3 peptides for 24 h at 37 °C, showing CitH3-induced cytotoxicity ( n = per group). b Cell viability, assessed using the CCK8 assay, demonstrated significant reduction following CitH3 peptide treatment compared to the H3 peptide ( n = 5 per group). c BMDMs treated with 50 µg/mL CitH3 peptide and increasing doses of hCitH3-mAb or human IgG. LDH release was measured to evaluate cytotoxicity ( n = per group). d , Viability of BMDMs treated as in ( c ) was measured using the CCK8 assay. hCitH3-mAb significantly preserved cell viability compared to control IgG ( n = per group). e Immunoblot analysis of CitH3 in BMDMs treated with 15 μg/mL CitH3 or H3 peptides. Cells were washed three times prior to collection. And given that the CitH3 peptide is only ~30 amino acids in length, the bands observed at ~17 kDa are interpreted as endogenous cellular CitH3 protein detected at various time points. Similar results from three independent replicates. f Western blot analysis of citrullinated proteins in BMDMs from WT, Pad2⁻/⁻, and Pad2/4⁻/⁻ mice following 1 h treatment with 15 μg/mL CitH3 peptide. CitH3-induced citrullination was PAD2-dependent. Similar results from four independent replicates. g Quantification of IL-6, IL-1β, TNFα, IFN-α, and IFN-β levels in the supernatants of BMDMs treated with 25 μg/mL CitH3 peptide for 24 h ( n = 6 per group). hCitH3-mAb effectively reduced CitH3-induced cytokine elevation in a dose-dependent manner, whereas control IgG had no effect. Data are presented as mean ± SD. Statistical analysis: One-way ANOVA followed by Dunnett’s multiple comparisons test was applied throughout. Comparisons were made to the ‘0’ group in ( a –d ), and the CitH3-treated group in ( g ), as indicated. Significance thresholds: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.Source data are provided as a Source Data file.

Article Snippet: The primary antibodies used in immunocytochemistry assays: PAD2 (12110-1-AP), PAD4 (17373-1-AP), Rab5 (CST, 3547), EEA1 (CST, 3288), MPO (Abcam ab208670)

Techniques: CCK-8 Assay, Control, Western Blot