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) Representative light micrographs of primary myoblasts differentiated for 24 h and immunostained for MHC under control (untreated), DMSO, AFM32A, or GSK484 treatment. Scale bar, 280 μm. (B,C) Representative immunoblots (left) and corresponding quantification (right) of myogenin, MHC, PAD2, PAD3, PAD4, and the histone H3 citrullination mark, H3CitR2+R8+R17, in primary myoblasts differentiated for 24 h in the presence of AFM32A (640 µM; B ) or GSK484 (40 µM; C ). (D) Schematic representation of the in vivo experimental design to assess the effects of PAD inhibition on skeletal muscle regeneration. Cardiotoxin (CTX) was injected into the tibialis anterior (TA) muscle, followed by intraperitoneal administration of AFM32A or GSK484 at 3-, 4-, and 5-days post-injury (dpi), with analysis at 7 dpi in male and female mice. ( E ) Hematoxylin & Eosin (H&E) staining of representative 10 μm cross-sections of contralateral undamaged TA muscle and injured TA at 7 dpi under vehicle control, AFM32A, or GSK484 treatment. (F) Myofiber regeneration was quantified by measuring fiber diameter. Scale bars, 270 μm. (G) Representative PicroSirius Red and Masson’s trichrome staining of 10 μm TA muscle cross-sections from contralateral undamaged and injured muscles under vehicle, AFM32A, or GSK484 treatment. Bar graphs represent mean ± SEM from three independent experiments (N = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Primary myoblasts were cultured under differentiation conditions with the pan-PAD enzyme inhibitor BB-Cl amidine (HY-111347; MedChemExpress), the PAD2 inhibitor, AFM32A synthesized as previously described , and the PAD4 inhibitor GSK484 , (SML1658; MilliporeSigma).

Techniques: Control, Western Blot, In Vivo, Inhibition, Injection, Staining, Muscles

Journal: Arthritis Research & Therapy

Article Title: Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model

doi: 10.1186/s13075-020-02348-z

Figure Lengend Snippet: Effect of PgPAD on the onset of joint arthritis. The effect of PgPAD on the onset of arthritis was determined. In this experiment, PgPAD deletion mutant 33277 strain (⊿ pad ) and Pg 33277 wild-type (WT) strain were used, unless Pg W83 strain was specifically described in caption of Figures. ABL of the upper jaw in each group was measured ( a ). Ctrl, PBS inoculation; WT, Pg wild-type inoculation; ⊿ pad , Pg pad knockout strain inoculation. To evaluate inflammation of the gingival tissue 6 weeks after Pg inoculation, IL-6 production in gingival tissue of SKG mice was measured by ELISA ( b ). AS was measured 3 and 6 weeks after Pg inoculation with LA i.p. injection ( c ). To assess CP synthesis, ELISA was performed ( d gingival tissue, e joint tissue, f small intestine, g large intestine). The titer of serum ACPA 6 weeks after Pg inoculation with LA i.p. injection in mice from each group was measured by ELISA ( h ). The production of endogenous PADI2 and PADI4 in joint tissue was determined by Western blotting, and then the density of each band was measured by NIH Image-J ( i and j ). Data represent mean ± SD ( b – j ) of 5 mice per group. Statistical analyses were performed using the Tukey-Kramer test and Bonferroni-corrected Mann-Whitney U test for multiple comparisons (* P < 0.05, ** P < 0.01)

Article Snippet: As a control, the amount of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected by anti-GAPDH antibody (10 μg/ml, HRP-60004, PROTEINTECH JAPAN, Japan) The density of target PADI2 and PADI4 bands was measured by NIH Image-J software.

Techniques: Mutagenesis, Knock-Out, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Citrullination alters immunomodulatory function of LL-37 essential for prevention of endotoxin-induced sepsis

doi: 10.4049/jimmunol.1303062

Figure Lengend Snippet: Human MDMs (A) and mouse RAW 264.7 (B) macrophages were stimulated with 10 ng/mL LPS in the presence of native or citrullinated LL-37 at the indicated concentrations (0.1–10 μg/mL). Citrullinated LL-37 was obtained by treatment of the native peptide with human PAD2 or PAD4 at 23.3 U/mg peptide). The level of TNF-α (A) and NO (B) in the culture supernatants was determined using ELISA or the Griess assay at 6 or 20 h post-stimulation, respectively. Since neither the LL-37 nor the PAD enzymes alone induced the release of NO or TNF-α, for the sake of clarity these controls are not shown in the figure. Data represent the mean ± SD of three independent experiments. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Recombinant human PAD2 and PAD4 were obtained from Modiquest (The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: PAD activity in NAWM of MS patients. (A) Quantification of PAD2 protein in white matter from normal and MS brain by immunoslot blot ( n =5, P <0.0001). (B) Citrullinated protein in white matter from normal and MS brains by immunoslot blot as pixel density ( n =4, P <0.01). (C) PAD enzyme activity in normal and MS tissue, with or without pre-incubation with 2CA ( n =5, P <0.05). Each dot represents one patient analysed three times. The means (horizontal bar) for all the MS patients were compared with the normal.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Incubation

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: Interaction of PAD with 2CA. (A) PAD2 and PAD4 inhibition curves in the presence of increasing 2CA concentrations. Insert: PAD1-4 enzymes contain a common C-terminal active-site cysteine residue (Cys656) bound by 2CA, confirmed by ESI mass spectrometry of tryptic digests of PAD2-acetamidine adducts. (B) Schematic of the nucleophilic reaction between 2CA and the Cys656 residue in the active site of PAD2 . (C) Tabular summary of peptide fragment atomic masses for 2CA-modified and native PAD2.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Inhibition, Residue, Mass Spectrometry, Modification

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: mAb4E12 (anti-2CA adduct) immunogold labeled optic nerve cryosections from control and 2CA-treated PAD2 transgenic mice. Minimal labeling in untreated mice (black arrows). Numerous gold particles (white arrows) in nuclei (N) and cytoplasm of oligodendrocytes, myelin and axonoplasm (Ax) of 2CA-treated mice. Scale bars: 500 nm.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Labeling, Control, Transgenic Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates demyelinating disease in ND4 mice. (A) ND4 mice treated with PBS, 2CA (5 mg/kg), or 2CA+B12 (5 mg/kg and 10 mg/kg) starting at 2 months before disease onset ( n =5, P <0.0001). (B) ND4 mice treated at disease onset ( n =4, P <0.0001). (C) Stopping 2CA, but continuing B12 at 3.5 months in ND4 mice ( n =5, P <0.0001) demonstrates that B12 alone does not attenuate disease. (D) PAD activity in brains from animals shown in ( n =5, P <0.05). The first four bars represent results from animals at 6 months of age, whereas the post-treatment animals were 8 months of age. (E) PAD2 RT PCR in white matter extracts from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice ( n =9, P <0.05). (F) LFB and hematoxylin stain of cerebella from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice (40×). WM, white matter; GM, grey matter.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates PAD2 overexpressor. (A) Demyelinating disease in PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12 starting at 6 months of age ( n =5, P <0.0001). (B) PAD activity in brain extracts of PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12, and non-transgenic littermates ( n =4, P <0.05). PAD activity is reduced to normal levels by treatment.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Transgenic Assay, Activity Assay