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

Journal: bioRxiv

Article Title: ACE2 associates with insulin-responsive GLUT4 dynamics in adipocytes

doi: 10.64898/2026.03.10.710920

Figure Lengend Snippet: (A) 3T3L1 fibroblasts expressing ACE2-Halo were incubated with biotinylated recombinant S-protein (30 min, 37 °C). Internalized S-protein was detected using Alexa Fluor 488-conjugated streptavidin following permeabilization. Endosomal structures containing ACE2 and S-protein are shown. Representative images are shown from one of three independent experiments. Scale bars, 20 μm. Right graph : Quantification of fluorescence heterogeneity measured as the coefficient of variation (CV = standard deviation / mean fluorescence intensity) within cellular regions of interest. Each dot represents one cell from a representative experiment. Similar results were obtained in three independent experiments. Data represent mean ± SD of analyzed cells, and statistical significance is indicated as follows: **** P < 0.001. (B) 3T3L1 adipocytes expressing ACE2-Halo and GLUT4-mCherry were treated with S-protein in the absence or presence of insulin (100 nM, 30 min). Internalized S-protein was detected using Alexa Fluor 488-conjugated streptavidin following permeabilization.Endosomal structures containing ACE2 and S-protein are shown. Colocalization of internalized S-protein with GLUT4-positive vesicles was assessed by confocal microscopy. Arrowheads indicate peripheral endosomes containing GLUT4 that also contain internalized S-protein. Asterisks indicate S-protein puncta located at or just beneath the plasma membrane that do not contain detectable GLUT4. Representative images are shown from one of three independent experiments. Scale bars, 20 μm.

Article Snippet: Biotinylated recombinant SARS-CoV-2 Spike protein (Cat. #BT10549) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Incubation, Recombinant, Fluorescence, Standard Deviation, Confocal Microscopy, Clinical Proteomics, Membrane