Journal: iScience

Article Title: Integrative analysis reveals marker genes for intestinal mucosa barrier repairing in clinical patients

doi: 10.1016/j.isci.2023.106831

Figure Lengend Snippet: Protein validation of key genes contributing to intestinal mucosal barrier recovery using the in-depth proteins

Article Snippet: Primary antibodies used for western blotting were anti-PADI2 (Bioss, bs-11662R, 1:1000), anti-FAM162A (Abcam, ab122295, 1:1000), anti-AQP8 (Bioss, bs-6786R, 1:500), anti-SULT1A1 (Bioss, bs-6283R, 1:1000) and anti-GAPDH (Abmart, P30008 M, 1:4000).

Techniques:

Journal: iScience

Article Title: Integrative analysis reveals marker genes for intestinal mucosa barrier repairing in clinical patients

doi: 10.1016/j.isci.2023.106831

Figure Lengend Snippet: Validation of identified potential biomarkers in clinical patients and correlation analysis on their expression levels and the degree of intestine functional recovery (A) mRNA expression levels of PADI2 , FAM162A , AQP8 and SULT1A1 at different timepoints of intestinal injury repair after surgery in patients. (B) Protein expression levels of PADI2, FAM162A, AQP8 and SULT1A1 at different timepoints of intestinal injury repair after surgery in patients. (C) AQP8 and SULT1A1 mRNA levels in patients with and without oral dietary intolerance 48 h after surgery and in patients with and without infectious complications 7 days after surgery. (D) Correlation analysis of the mRNA levels of AQP8 and SULT1A1 at 48 h after surgery and the time of passing flatus and stool. ∗p ≤0.05, ∗∗p ≤0.01, ∗∗∗p ≤0.001, ∗∗∗∗p ≤0.0001, one-way ANOVA with Tukey’s multiple comparisons test. Data are represented as mean ± standard deviation.

Article Snippet: Primary antibodies used for western blotting were anti-PADI2 (Bioss, bs-11662R, 1:1000), anti-FAM162A (Abcam, ab122295, 1:1000), anti-AQP8 (Bioss, bs-6786R, 1:500), anti-SULT1A1 (Bioss, bs-6283R, 1:1000) and anti-GAPDH (Abmart, P30008 M, 1:4000).

Techniques: Expressing, Functional Assay, Standard Deviation

Journal: iScience

Article Title: Integrative analysis reveals marker genes for intestinal mucosa barrier repairing in clinical patients

doi: 10.1016/j.isci.2023.106831

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for western blotting were anti-PADI2 (Bioss, bs-11662R, 1:1000), anti-FAM162A (Abcam, ab122295, 1:1000), anti-AQP8 (Bioss, bs-6786R, 1:500), anti-SULT1A1 (Bioss, bs-6283R, 1:1000) and anti-GAPDH (Abmart, P30008 M, 1:4000).

Techniques: Expressing, Software

Journal: Journal of Translational Medicine

Article Title: Increased citrullination and expression of peptidylarginine deiminases independently of P. gingivalis and A. actinomycetemcomitans in gingival tissue of patients with periodontitis

doi: 10.1186/s12967-018-1588-2

Figure Lengend Snippet: PAD2 and PAD4 protein and mRNA expression in gingival tissues of patients with and without periodontitis. Mean positive cell scores (± SD) for a and c PAD2 and b and d PAD4 in connective and epithelial tissue sections (respectively) from patients with periodontitis (n = 15) and subjects without periodontitis (n = 15). Gene expression of e PADI2 and f PADI4 in gingival tissue biopsies of patients with periodontitis (n = 13) and without periodontitis (n = 11) using RT-qPCR. The mRNA expression is shown as fold change of periodontitis samples relative to healthy controls and calculated according to the ΔΔCt method, where periodontitis samples were normalized to the non-periodontitis samples and to corresponding GAPDH sample. * p < 0.05; ** p < 0.01

Article Snippet: TaqMan probes were used as follows: PADI2 (Hs00247108_m1), PADI4 (Hs00202612_m1) and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ; Hs02758991_g1).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: Proteomic profiling of FBXW7 ‐mutant serous endometrial cancer cells reveals upregulation of PADI2, a potential therapeutic target

doi: 10.1002/cam4.3013

Figure Lengend Snippet: Unsupervised hierarchical clustering of normalized signal:noise for total proteome peptides significantly ( P < .05) differently expressed in CRISPR‐edited FBXW7 ‐mutated cells compared to respective FBXW7 nonmutant parental cells in replicate lysates prepared for each cell line. ARK4 replicate 2 was run twice (to fill extra 10‐plex runs; both values for replicate 2 were used in final calculations). Fold change is not shown on this figure and was calculated within replicates, comparing mutant line expression to parental. Proteins significantly differentially expressed in all three ARK1 or ARK4 FBXW7 ‐mutant lines compared to respective parental lines are boxed. Protein names and fold change values are provided in Figure and Tables . Arrows indicate PADI2, which was significantly differentially expressed in all three ARK1 and ARK4 FBXW7 ‐mutant lines compared to respective parental controls

Article Snippet: Blots were blocked in Tris‐buffered saline containing 1% Tween 20 (TBST) + 5% milk and incubated in the following antibodies from CST ® according to their suggested protocols: PADI2 (#97647), UCHL1 (#13179), P‐MARCKS (#8722), MARCKS (#5607), NDRG1 (#9485), TGM2 (#3557), and EPLIN (#50311).

Techniques: CRISPR, Mutagenesis, Expressing

Journal: Cancer Medicine

Article Title: Proteomic profiling of FBXW7 ‐mutant serous endometrial cancer cells reveals upregulation of PADI2, a potential therapeutic target

doi: 10.1002/cam4.3013

Figure Lengend Snippet: (A) Venn diagram of proteins significantly ( P < .05) differentially expressed in CRISPR‐edited FBXW7 ‐mutated ARK1 and ARK4 cell lines compared to matched parental cells. Proteins validated via Western blot are labeled. (B) Western blot results confirming altered protein expression in ARK1 and ARK4 CRISPR‐edited FBXW7 ‐mutated lines compared to matched parental controls. All results are representative of a minimum of three lysate replicates from each cell line. (C) Intensity plot of average fold change ( FBXW7 ‐mutated line compared to parental line) of total protein expression generated by mass spectrometry. Yellow boxes indicate values that met filtering criteria (≥±2.0 average fold change, P < .05 for difference between FBXW7 ‐mutated cell line compared to respective parental line). *Increased MARCKS total protein expression in ARK4 FBXW7 R505C cells compared to parental cells was only observed in one of three replicate Western blot experiments; blot shown is representative of the other two replicates. (D) CRISPR editing of JHUEM‐1 endometrial endometrioid adenocarcinoma cells (which endogenously harbor FBXW7 p.R505C) to a FBXW7 nonmutant genotype resulted in decreased PADI2 expression. Results are representative of three lysate replicates. (E) Western blot detection of PADI2 protein expression in six serous endometrial tumors

Article Snippet: Blots were blocked in Tris‐buffered saline containing 1% Tween 20 (TBST) + 5% milk and incubated in the following antibodies from CST ® according to their suggested protocols: PADI2 (#97647), UCHL1 (#13179), P‐MARCKS (#8722), MARCKS (#5607), NDRG1 (#9485), TGM2 (#3557), and EPLIN (#50311).

Techniques: CRISPR, Western Blot, Labeling, Expressing, Generated, Mass Spectrometry

Journal: Arthritis Research & Therapy

Article Title: Increased peptidylarginine deiminases expression during the macrophage differentiation and participated inflammatory responses

doi: 10.1186/s13075-019-1896-9

Figure Lengend Snippet: Protein expression of PADI2, PADI4, and citrullinated histone 3 in U937 cells, differentiated macrophages, and macrophages stimulated with lipopolysaccharides (LPS). a The protein expression of PADI2 increased in differentiated macrophages compared with U937 cells as detected by Western blot analysis. The addition of LPS slightly increased the protein expression of PADI2 in macrophages. b The protein expression of PADI4 increased in differentiated macrophages compared with U937 cells. The addition of LPS did not affect the protein expression of PADI4 in macrophages. c The protein level of citrullinated histone 3 (cit-H3) slightly increased in differentiated macrophages compared with U937 cells. The addition of LPS significantly increased the protein level of cit-H3 in macrophages

Article Snippet: U937 cells were electroporated with PADI2 short hairpin RNA (shRNA) plasmid (Santa Cruz Biotechnology, Dallas, TA, USA), which contained three 19–25 nt shRNA designed to knock down PADI2 expression.

Techniques: Expressing, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Increased peptidylarginine deiminases expression during the macrophage differentiation and participated inflammatory responses

doi: 10.1186/s13075-019-1896-9

Figure Lengend Snippet: Effects of PADI inhibitors on PADI2 and PADI4 expression in macrophages stimulated with lipopolysaccharides (LPS). a The protein expression of PADI2 was not different in LPS-stimulated macrophages cocultured with sanguinarine (SANG), ruthenium red (RUR), or Cl-amidine compared with the control. b The protein expression of PADI4 was not different in LPS-stimulated macrophages cocultured with SANG, RUR, or Cl-amidine compared with the control. c A representative example

Article Snippet: U937 cells were electroporated with PADI2 short hairpin RNA (shRNA) plasmid (Santa Cruz Biotechnology, Dallas, TA, USA), which contained three 19–25 nt shRNA designed to knock down PADI2 expression.

Techniques: Expressing, Control

Journal: Arthritis Research & Therapy

Article Title: Increased peptidylarginine deiminases expression during the macrophage differentiation and participated inflammatory responses

doi: 10.1186/s13075-019-1896-9

Figure Lengend Snippet: Using short hairpin (sh) RNA to inhibit the expression of PADI2 and its effect on PAI-2 protein expression. a Protein expression of PADI2 decreased in U937 cells transfected PADI2 shRNA plasmid compared with those transfected with control plasmid. b Protein expression of PAI-2 decreased in U937 cells transfected with PADI2 shRNA plasmid compared with the controls (empty plasmid). c Protein expression of PAI-2 decreased in differentiated macrophages transfected with PADI2 shRNA plasmid compared with the controls. d Protein expression of PAI-2 decreased in macrophages stimulated with LPS transfected with PADI2 shRNA plasmid compared with the controls

Article Snippet: U937 cells were electroporated with PADI2 short hairpin RNA (shRNA) plasmid (Santa Cruz Biotechnology, Dallas, TA, USA), which contained three 19–25 nt shRNA designed to knock down PADI2 expression.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Control

Journal: Arthritis Research & Therapy

Article Title: Increased peptidylarginine deiminases expression during the macrophage differentiation and participated inflammatory responses

doi: 10.1186/s13075-019-1896-9

Figure Lengend Snippet: Using short hairpin (sh) RNA to inhibit the expression of PADI2 and its effect on citrullinated PAI-2 (citPAI-2) protein levels. a Protein levels citPAI-2 did not change in U937 cells transfected with PADI2 shRNA plasmid compared with the controls (empty plasmid). b Protein levels of citPAI-2 decreased in differentiated macrophages transfected with PADI2 shRNA plasmid compared with the controls. c Protein levels of citPAI-2 decreased in macrophages stimulated with LPS transfected with PADI2 shRNA plasmid compared with the controls

Article Snippet: U937 cells were electroporated with PADI2 short hairpin RNA (shRNA) plasmid (Santa Cruz Biotechnology, Dallas, TA, USA), which contained three 19–25 nt shRNA designed to knock down PADI2 expression.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation

Journal: Arthritis Research & Therapy

Article Title: Increased peptidylarginine deiminases expression during the macrophage differentiation and participated inflammatory responses

doi: 10.1186/s13075-019-1896-9

Figure Lengend Snippet: A schematic diagram of this study showing that increased PADI2 and PADI4 expression during the differentiation of macrophages leads to IL-1β and TNF-α production and PAI-2 expression and citrullination. The citrullination of PAI-2 impaired its binding ability with PSMB1

Article Snippet: U937 cells were electroporated with PADI2 short hairpin RNA (shRNA) plasmid (Santa Cruz Biotechnology, Dallas, TA, USA), which contained three 19–25 nt shRNA designed to knock down PADI2 expression.

Techniques: Expressing, Binding Assay

Journal: JCI Insight

Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

doi: 10.1172/jci.insight.124729

Figure Lengend Snippet: (A) Quantification of splenomegaly assessed by spleen weight (% of body weight) in untreated (white bars) and imiquimod-treated (black bars) WT FVB, Padi2–/–, and Padi4–/– mice; n = 10–15 mice/group. Compiled data from 3 independent experiments. (B and C) Levels of circulating anti-dsDNA autoantibodies (B) and total IgG (C) in untreated (white whisker plots) and imiquimod-treated (black whisker plots) FVB, Padi2–/–, and Padi4–/– mice. n = 9–15 mice/group. Compiled data from 3 independent experiments. (D) Gene expression data of splenocytes from treated FVB, Padi2–/–, and Padi4–/– mice normalized to gene expression in the corresponding control mice. n = 7 mice/group. Compiled data from 2 independent experiments. Box-and-whisker plots show median, lower and upper quartiles, and minimum and maximum values. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001. Statistical analysis was performed by 2-tailed Mann-Whitney U test. IMQ, imiquimod.

Article Snippet: Splenic CD4 + CD62L + CD44 – naive T cells and CD4 + CD62L – CD44 + memory T cells were sorted using FACSAria IIIu, and expression of the 2 genes analyzed with Padi2 - or Padi4 -specific TaqMan Gene Expression Assays ( Padi2 [Mm01341648_m1] and Padi4 [Mm01341658_m1]; Applied Biosystems) and normalized to 18s ribosomal RNA (Mm03928990_g1).

Techniques: Whisker Assay, Gene Expression, Control, MANN-WHITNEY

Journal: JCI Insight

Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

doi: 10.1172/jci.insight.124729

Figure Lengend Snippet: (A–C) Aortic rings were isolated from untreated and imiquimod-treated FVB (A), Padi2–/– (B), and Padi4–/– (C) mice. Acetylcholine-dependent relaxation following phenylephrine (PE) precontraction was determined. n = 6–10 mice/group. Results represent mean ± SEM % vasorelaxation; ****P < 0.0001. A 2-way ANOVA with post-hoc Tukey’s test was used to compare differences between the groups.

Article Snippet: Splenic CD4 + CD62L + CD44 – naive T cells and CD4 + CD62L – CD44 + memory T cells were sorted using FACSAria IIIu, and expression of the 2 genes analyzed with Padi2 - or Padi4 -specific TaqMan Gene Expression Assays ( Padi2 [Mm01341648_m1] and Padi4 [Mm01341658_m1]; Applied Biosystems) and normalized to 18s ribosomal RNA (Mm03928990_g1).

Techniques: Isolation

Journal: JCI Insight

Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

doi: 10.1172/jci.insight.124729

Figure Lengend Snippet: (A and B) BM neutrophils of naive FVB, Padi2–/–, and Padi4–/– mice were stimulated with the calcium ionophore A23817 for 2 hours to induce NET formation and stained for Cit-H3 (A) and MPO (B). Total magnification is 40×. (C) Lack of Cit-H3 in Padi4–/– NETs was confirmed by Western blot of imiquimod-induced NETs. Results are representative of 3 experiments (D). Aorta rings were cultured in the presence of imiquimod-induced NETs prior to assessment of vasorelaxation capacity. Results represent % mean ± SEM vasorelaxation, n = 3 mice/group. (E) Splenocytes from naive FVB, Padi2–/–, and Padi4–/– mice (n = 3/group) were cultured for 24 hours in the presence of imiquimod-induced NETs, and the expression of type 1 IFN–inducible genes was analyzed by RT-PCR. *P < 0.05, **P < 0.005. Statistical analysis was performed with 2-tailed unpaired t test. For D, analysis was by 2-way ANOVA with post-hoc Tukey’s test to compare differences between the groups; *P < 0.05 for PAD4 KO vs. FVB; #P < 0.05 for PAD4 KO vs. PAD2 KO.

Article Snippet: Splenic CD4 + CD62L + CD44 – naive T cells and CD4 + CD62L – CD44 + memory T cells were sorted using FACSAria IIIu, and expression of the 2 genes analyzed with Padi2 - or Padi4 -specific TaqMan Gene Expression Assays ( Padi2 [Mm01341648_m1] and Padi4 [Mm01341658_m1]; Applied Biosystems) and normalized to 18s ribosomal RNA (Mm03928990_g1).

Techniques: Staining, Western Blot, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: JCI Insight

Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

doi: 10.1172/jci.insight.124729

Figure Lengend Snippet: (A) MA plots of gene expression data in lymph nodes following in vivo imiquimod exposure between Padi2–/–, Padi4–/–, and WT FVB (WT) (n = 4 mice/group). Differential expression was calculated by ANOVA test on normalized gene counts. Genes with P < 0.05 are colored in red. (B) Venn diagrams depicting the number of significantly downregulated and upregulated genes between Padi2–/– or Padi4–/– and WT lymph nodes. (C) Gene Ontology (GO) biological pathway analysis of significant genes between Padi4–/– and Padi2–/– lymph nodes. Statistical analysis of functional profiles for genes and related pathways was performed using the clusterProfiler package in RStudio.

Article Snippet: Splenic CD4 + CD62L + CD44 – naive T cells and CD4 + CD62L – CD44 + memory T cells were sorted using FACSAria IIIu, and expression of the 2 genes analyzed with Padi2 - or Padi4 -specific TaqMan Gene Expression Assays ( Padi2 [Mm01341648_m1] and Padi4 [Mm01341658_m1]; Applied Biosystems) and normalized to 18s ribosomal RNA (Mm03928990_g1).

Techniques: Gene Expression, In Vivo, Quantitative Proteomics, Functional Assay

Journal: JCI Insight

Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

doi: 10.1172/jci.insight.124729

Figure Lengend Snippet: (A and B) Splenocytes isolated from naive FVB, Padi2–/–, and Padi4–/– mice were sorted, and PAD2 expression was quantified by Western blot and densitometry. Data are shown as mean ± SD; 2 mice per group (1 and 2).

Article Snippet: Splenic CD4 + CD62L + CD44 – naive T cells and CD4 + CD62L – CD44 + memory T cells were sorted using FACSAria IIIu, and expression of the 2 genes analyzed with Padi2 - or Padi4 -specific TaqMan Gene Expression Assays ( Padi2 [Mm01341648_m1] and Padi4 [Mm01341658_m1]; Applied Biosystems) and normalized to 18s ribosomal RNA (Mm03928990_g1).

Techniques: Isolation, Expressing, Western Blot

Journal: JCI Insight

Article Title: Peptidylarginine deiminases 2 and 4 modulate innate and adaptive immune responses in TLR-7–dependent lupus

doi: 10.1172/jci.insight.124729

Figure Lengend Snippet: (A and B) Splenocytes from untreated and imiquimod-treated (IMQ-treated) FVB, Padi2–/–, and Padi4–/– mice were stimulated in vitro with PMA and ionomycin, and CD4+ T cell cytokine production was measured by flow cytometry to determine Th1 (A) and Th17 (B) responses. Box-and-whisker plots show median, lower and upper quartiles, and minimum and maximum % values and are representative of 2 independent experiments, each performed in 4-6 mice/group. *P < 0.05, **P < 0.005, ****P < 0.0001. (C) Splenic naive T cells from FVB, Padi2–/–, and Padi4–/– mice were activated by α-CD3 and α-CD28 in the presence/absence of polarizing cytokines and antibodies. At day 5, the cells were analyzed for gene expression levels of Ifng in Th1 conditions and Il17a in Th17 conditions; n = 4. Box-and-whisker plots show median, lower and upper quartiles, minimum and maximum fold induction relative to polarization response in FVB mice. *P < 0.05, **P < 0.005. Statistical analysis was performed by 2-tailed Mann-Whitney U test.

Article Snippet: Splenic CD4 + CD62L + CD44 – naive T cells and CD4 + CD62L – CD44 + memory T cells were sorted using FACSAria IIIu, and expression of the 2 genes analyzed with Padi2 - or Padi4 -specific TaqMan Gene Expression Assays ( Padi2 [Mm01341648_m1] and Padi4 [Mm01341658_m1]; Applied Biosystems) and normalized to 18s ribosomal RNA (Mm03928990_g1).

Techniques: In Vitro, Flow Cytometry, Whisker Assay, Gene Expression, MANN-WHITNEY