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: Antiviral effects of PAD inhibitors against SARS-CoV-2 in vitro (A and B) Quantification of SARS-CoV-2 2020B.1 (MOI 0.1) viral particle production in Calu-3 and Huh7.5 cells (at 24 hpi and 48 hpi, respectively) treated with the increasing concentrations of BB-Cl (A) or GSK199 (B), determined by plaque assay. Results are expressed as a percentage relative to vehicle-treated cells (DMSO, marked as 0 on the graph). Data are presented as means ± SEM from four independent experiments and are analyzed by one-way ANOVA followed by Bonferroni’s post-test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (C and D) Cell viability of uninfected Calu3 and Huh7.5 cells treated with the indicated concentrations of BB-Cl (C) and GSK199 (D) for 72 h, determined for each concentration by MTT assay. Values are expressed as means ± SEM of three independent experiments. (E) Viral titers of different SARS-CoV-2 variants are measured in Calu-3 cells at 24 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (F) Viral titers of different SARS-CoV-2 variants (as indicated in the figure) are measured in Huh7.5 cells at 48 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

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: In Vitro, Plaque Assay, Concentration Assay, MTT Assay

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: 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: In vivo antiviral activity of PAD inhibitors against SARS-CoV-2 (A) Schematic representation of the treatment and infection protocols in K18-hACE2 transgenic mice. (B) Quantification of viral genome copies in mouse lungs—treated and infected as described in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (C) Plaque assay to determine the number of infectious viral particles in mouse nasal swabs. (D) Graphical representation of cumulative scores from for immunohistochemical staining of mouse lungs using an anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (E) Graphical representation of cumulative scores from for H&E staining of mouse lungs ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. (F) Quantification of viral genome copies in mouse brains—treated and infected, as represented in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (G) Distribution of brains from vehicle- or PAD-inhibitor-treated infected mice based on viral load (low: < below 10 3 ; high: > below 10 3 ). (H) Graphical representation of cumulative scores from for immunohistochemical staining of mouse brains using anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (I) Graphical representation of cumulative scores from for H&E staining of mouse brains ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. Data from all experiments, which represent mean ± SEM, are analyzed using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

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: In Vivo, Activity Assay, Infection, Transgenic Assay, Plaque Assay, Immunohistochemical staining, Staining, Two Tailed Test

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 inhibition modulates SARS-CoV-2-induced inflammation (A–D) Relative mRNA levels of the indicated genes are quantified by comparative RT-qPCR from total RNA of Calu-3 cells infected with the SARS-CoV-2 Delta strain (gray bars; MOI 0.1, 24 hpi) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO (vehicle). Data from three independent experiments were normalized to the housekeeping gene PGK1 and plotted as mean fold change ±SEM over mock-infected cells (white bars). (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test). Red asterisks indicate comparisons with mock control, while black asterisks refer to comparisons among treatments. (E–J) Relative mRNA levels of the indicated cytokines are quantified by RT-qPCR from lung tissues of mice infected with SARS-CoV-2 Delta variant (10 5 PFU, i.n.) for 4 days and treated with BB-Cl (1 mg/kg), GSK199 (30 mg/kg), or DMSO (vehicle). Each dot represents one individual mouse. Data are normalized to the housekeeping gene β-actin and are presented relative to the mean ΔCt value of vehicle-treated mice. Statistical significance is assessed using an unpaired two-tailed t test (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

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, Quantitative RT-PCR, Infection, Control, Variant Assay, Two Tailed Test

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 inhibition protects against SARS-CoV-2-induced pathological changes (A–C) Volcano plots show citrullinated proteins in pooled protein lysates from SARS-CoV-2- vs. mock-infected mouse lungs at 4 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 (A) and between infected mice treated with vehicle (DMSO) or with PAD inhibitors BB-Cl-amidine (B) or GSK199 (C), while the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (D) Heatmap shows the fold changes of differentially citrullinated proteins identified in lung tissues across the indicated pairwise comparisons: uninfected vs. infected, and vehicle-treated infected vs. BB-Cl–treated or GSK199–treated infected mice. The color scale represents relative citrullination levels expressed as log 2 fold change.

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, Incubation, Liquid Chromatography with Mass Spectroscopy