Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Expressing, In Vivo, In Vitro, Infection, Quantitative RT-PCR, Variant Assay, Nucleic Acid Electrophoresis, Control

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Infection, Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Variant Assay, Multiplex Assay, Immunofluorescence, Staining, Control, Software, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Inhibition, Infection, Quantitative RT-PCR, Western Blot, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: Reciprocal regulation between the protein arginine deiminases and mSWI/SNF chromatin remodelers controls skeletal muscle differentiation and regeneration

doi: 10.64898/2026.01.20.700682

Figure Lengend Snippet: (A) Confocal micrographs of primary myoblasts differentiated for 24 h and immunostained with anti-PAD2 (red) and anti-PAD3 or anti-PAD4 (green) antibodies. Nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. (B) Subcellular fractionation of primary myoblasts differentiated for 24 h showing whole-cell (WC), cytosolic (Cyt), and nuclear (Nuc.) fractions. Representative immunoblots (left) and corresponding quantification (right) of PAD2, PAD3, and PAD4 protein levels are shown. Lamin A/C and α-tubulin were used as purity controls for the nuclear and cytoplasmic fractions, respectively. 20 ug of each extract was loaded onto the gels. (C) Representative immunoblots (left) and quantification (right) of histone H3 citrullination marks (H3CitR2+R8+R17, H3CitR17, and H3CitR26) in proliferating (P or Prol), time 0 (0 h), or differentiating (24 h) primary myoblasts. (D-G) ChIP-qPCR analysis showing enrichment of H3CitR2+R8+R17 (D) , H3CitR26 (E) , H3CitR17 (G) and total H3 (H) at the Pax7 promoter and at regulatory sequences controlling the indicated myogenic genes in proliferating (Prol), time 0 (0 h), and differentiating (24 h) primary myoblasts. The IgH enhancer was included as a negative sequence control. Bar graphs represent mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. n.s., not significant.

Article Snippet: Mouse anti-BRG1(G-7; sc-17796), anti-PAD3 (F6; sc-393622), anti-PAD4 (A11; sc-365369), anti-lamin A/C (4A58), and anti-α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnologies.

Techniques: Fractionation, Western Blot, ChIP-qPCR, Sequencing, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4)

doi: 10.1167/iovs.18-24118

Figure Lengend Snippet: Western blot analysis of PAD4 expression in mouse retinal lysate and testing for antibody cross-reactivity. (A) Retinal lysates from WT and PAD4KO mice were electrophoresed and probed for PAD4 (green). GAPDH (red) served as a loading control. Control, 20 ng PAD4-GST fusion protein (positive control). At 3 months, WT mice exhibit a strong band at 65 kDa, indicative of PAD4, while PAD4KO animals exhibit no band, indicative of loss of PAD4. (B) 20 ng and 40 ng of rPAD4-GST (lanes 2 and 3) and rPAD2-GST (lanes 4 and 5) were electrophoresed and probed for PAD2 and PAD4 (both green), respectively. The anti-PAD2/4 antibody bound to PAD4 with a high affinity while the anti-PAD4 antibody bound negligibly to PAD2 protein.

Article Snippet: To circumvent these challenges, a rabbit anti-PAD2/4 cross-reactive antibody was used to label for both PAD2 and PAD4 (12110-1-AP; Proteintech) in both WT and PAD4KO animals.

Techniques: Western Blot, Expressing, Control, Positive Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4)

doi: 10.1167/iovs.18-24118

Figure Lengend Snippet: IHC analysis of PAD4 localization and expression levels in the mouse retina. (A) PAD4 expression levels were quantified using densitometric analysis of fluorescent intensities across the neural retina using ImageJ and plotted as mean intensity divided by area as a function of age. Data are expressed as the mean ± SEM (error bars, n = 3). A steep decline in immunoreactivity was seen in WT mice between 0.5 and 0.75 months (14 vs. 21 days) of age, with subsequent peaks at 1 month and between 3 and 6 months. (B) Immunolabeling of PAD4 (green) in WT and PAD4KO mouse retinas at serial time points. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS/IS, photoreceptor outer segments/inner segments. Scale bar: 50 μm.

Article Snippet: To circumvent these challenges, a rabbit anti-PAD2/4 cross-reactive antibody was used to label for both PAD2 and PAD4 (12110-1-AP; Proteintech) in both WT and PAD4KO animals.

Techniques: Expressing, Immunolabeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4)

doi: 10.1167/iovs.18-24118

Figure Lengend Snippet: IHC localization and expression levels of total PAD (WT) and PAD2 (PAD4KO) in the mouse retina. (A) Expression levels of total PAD (WT) and residual PAD2 (PAD4KO) were quantified using densitometric analysis of fluorescent intensities across the neural retina using ImageJ and plotted as mean intensity divided by area as a function of age. Data are expressed as the mean ± SEM (error bars, n = 3). A decline in both total PAD immunoreactivity and residual PAD2 reactivity over time was seen in both WT mice and PAD4KO mice, respectively. (B) Immunolabeling (in green) attributable to both PAD2 and PAD4 (total PAD reactivity) in WT mice and residual PAD2 reactivity seen in PAD4KO mouse retinas at serial time points. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS/IS, photoreceptor outer segments/inner segments. Scale bar: 50 μm.

Article Snippet: To circumvent these challenges, a rabbit anti-PAD2/4 cross-reactive antibody was used to label for both PAD2 and PAD4 (12110-1-AP; Proteintech) in both WT and PAD4KO animals.

Techniques: Expressing, Immunolabeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4)

doi: 10.1167/iovs.18-24118

Figure Lengend Snippet: Immunohistochemical analysis of citrullinated peptide localization and levels in the mouse retina. (A) Citrullinated peptide expression levels were quantified using densitometric analysis of fluorescent intensities across the neural retina using ImageJ and plotted as mean intensity divided by area as a function of age. Data are expressed as the mean ± SEM (error bars, n = 6). Reactivity declined over time, reaching negligible levels by 9 months under physiological conditions. (B) Immunolabeling of citrullinated peptides (red) in WT and PAD4KO mouse retinas at increasing ages. The pattern of expression of citrullinated peptides over time mirrored closest the dynamics of PAD4 expression. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS/IS, photoreceptor outer segments/inner segments. Scale bar: 50 μm.

Article Snippet: To circumvent these challenges, a rabbit anti-PAD2/4 cross-reactive antibody was used to label for both PAD2 and PAD4 (12110-1-AP; Proteintech) in both WT and PAD4KO animals.

Techniques: Immunohistochemical staining, Expressing, Immunolabeling