Journal: bioRxiv

Article Title: Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

doi: 10.1101/2025.02.28.639952

Figure Lengend Snippet: a, The enzymatic reaction mediated by PADs converts arginine to citrulline. b, c, Western blot images of PAD2 and PAD4-mediated citrullination of H3 (b) and TDP-43 (c). In (c) arrows indicate the observed shift in TDP-43 molecular weight. d, Coomassie staining of PAD2 (bands 1, 4), PAD4 (bands 2, 6), unmodified TDP-43 (band 3), PAD2-mediated citR TDP-43 (band 5), and PAD4-mediated citR TDP-43 (band 7). e, Bar diagrams showing the MW shift (kDa) of TDP-43 protein following citrullination. f, Schematic representation of arginine epitopes positioned in the human TDP-43 protein sequence. Position of the 11 citrullinated arginine epitopes are indicated in red. g, Six out of eleven arginine epitopes susceptible to citrullination laid within common RX(X)R (red box) or RXG/RGGG (green box) motifs in TDP-43 sequence. h, MS/MS spectrum showing b- and y-ion coverage of modified citR83 peptide and extracted ion chromatograms (XICs) showed abundance and retention time of unmodified R83 vs. citR83 with PAD2 and PAD4 treatment (intact peptide monoisotopic m/z (+2) 719.8457, (+3) 480.2329). i, Retention time peaks for TDP-43 peptides surrounding the unmodified or modified R83 epitope and base peak m/z undergoing methionine (Met85) oxidation, citrullination or both showed reliable time separation.

Article Snippet: PAD2 and PAD4 primary antibodies were purchased from Proteintech, US (#12110-1-AP and 17373-1-AP) and diluted at 1:10000 vs. 1:1000, respectively.

Techniques: Western Blot, Molecular Weight, Staining, Sequencing, Tandem Mass Spectroscopy, Modification

Journal: bioRxiv

Article Title: Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

doi: 10.1101/2025.02.28.639952

Figure Lengend Snippet: Spectra showing b- and y-ion coverage of citrullinated peptides, as well as XICs retention times for unmodified and citrullinated peptides treated with PAD2 and PAD4, corresponding to a - c, citR165 (intact peptide monoisotopic m/z (+2) 665.8214), d - f, citR191(intact peptide monoisotopic m/z (+2) 672.8469), g - i, citR268/272 (intact peptide monoisotopic m/z (+2) 728.3560), j - l, citR293 (intact peptide monoisotopic m/z (+2) 638.29).

Article Snippet: PAD2 and PAD4 primary antibodies were purchased from Proteintech, US (#12110-1-AP and 17373-1-AP) and diluted at 1:10000 vs. 1:1000, respectively.

Techniques:

Journal: bioRxiv

Article Title: Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

doi: 10.1101/2025.02.28.639952

Figure Lengend Snippet: a, Schematic representation of seven polyclonal antibodies raised against the citrullinated arginine epitopes. Epitope positions within each TDP-43 functional domain is indicated. b, Antibody specificity was determined by western blot via PAD4-mediated citrullinated TDP-43 protein probed with citR83 (NLS), citR165 and citR191 (RRM1, RRM2), citR268/272, citR275 (CTD) antibodies. c, Immunohistochemical images of TAR4/4 and non-Tg littermates cortical tissue labeled against citR TDP-43 antibody panel. Increased neuronal expression of citR83, citR165, citR191, citR268/272 and citR275 in TAR4/4 tissue compared to Non-Tg tissue. Arrows indicate the citR TDP-43 morphologies observed with the citR268/272 and citR275 antibodies raised against TDP-43 C-terminal domain (arrows). Insets represent magnified neuronal citR TDP-43 morphological properties recognized from each antibody. d, Images of cortical tissue incubated with secondary antibody only - “no-primary” control. n = 3, scale bar is 50µm and 200 µm.

Article Snippet: PAD2 and PAD4 primary antibodies were purchased from Proteintech, US (#12110-1-AP and 17373-1-AP) and diluted at 1:10000 vs. 1:1000, respectively.

Techniques: Functional Assay, Western Blot, Immunohistochemical staining, Labeling, Expressing, Incubation, Control

Journal: bioRxiv

Article Title: Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

doi: 10.1101/2025.02.28.639952

Figure Lengend Snippet: a, Immunohistochemical images of Non-Tg, TAR4 and TAR4/4 cortex labeled with PAD2 and PAD4 antibodies. b, Fold change of PAD2 and PAD4 (% area mean of the values ± SEM) normalized to the Non-Tg control. One-way or Two-way ANOVA, followed by Tukey’s post hoc multiple comparisons tests, n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. c, Schematic overview of the TAR TDP-43 mouse model and the fractionation of cortical tissue into RIPA-soluble and (7 M) Urea-soluble fractions. d, The expression levels of the human TDP-43 in the TAR model. e, Cortical RIPA soluble and f, Urea soluble fraction analyzed by Western blotting and probed for citR TDP-43 antibody panel (citR83, 165, 191, 268/272, 275), pTDP-43 409/410 and total human TDP-43 protein. g, Quantification of citR TDP-43 43 kDa protein levels, proteolytic fragments (17, 25 & 35 kDa) and intermediate high molecular species (72, 98 & 120k Da) normalized to GAPDH in RIPA and h, Urea fraction normalized to the total protein loaded signal. Data represent the mean of the values ± SEM; One-way ANOVA, followed by Tukey’s or Šidák post hoc multiple comparisons tests, n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: PAD2 and PAD4 primary antibodies were purchased from Proteintech, US (#12110-1-AP and 17373-1-AP) and diluted at 1:10000 vs. 1:1000, respectively.

Techniques: Immunohistochemical staining, Labeling, Control, Fractionation, Expressing, Western Blot

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: E. coli O175:H7 strains used for antibacterial experiments.

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Isolation

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: Antibacterial activity of synthesized AgNPs at 1000 μg/mL concentrations in the water against E. coli O157:H7 strains ( A – I ).

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Activity Assay, Synthesized

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: Antibacterial efficacy of probiotic-mediated synthesized AgNPs against E. coli O157:H7 strains.

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Synthesized, Inhibition

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: Antibacterial activity of some commercial antibiotics against nine E. coli O157:H7 strains ( A – I ). Abbreviations: P (penicillin G, 10 μg/disc), E (erythromycin, 15 μg/disc), and VA (vancomycin, 30 μg/disc).

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Activity Assay

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: Antibacterial efficacy of some commercial antibiotics against E. coli O157:H7 strains—no inhibition zone.

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Inhibition

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: Growth curves of nine E. coli O157:H7 strains ( A – I ) cultured in LB broth with various concentrations of synthesized AgNPs to determine MIC. Control (●); 200 μg/mL (◦); 100 μg/mL (▼) 50 μg/mL (△); 25 μg/mL (§); 12.5 μg/mL (□); 6.25 μg/mL (w); 3.12 μg/mL (◊).

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Cell Culture, Synthesized, Control

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: MBC of probiotic-mediated synthesized AgNPs against E. coli O157:H7 strains ( A – I ).

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Synthesized

Journal: Polymers

Article Title: Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7

doi: 10.3390/polym14091834

Figure Lengend Snippet: FE-SEM images of normal E. coli O157:H7 (ATCC 35150) ( A , B ) and 1 × MBC probiotic-mediated synthesized AgNPs treated E. coli O157:H7 (ATCC 35150) ( C , D ).

Article Snippet: Minimum bactericidal concentrations (MBCs) were 25 μg/mL for E. coli O157:H7 ATCC 35150, E. coli O157:H7 ATCC 43895, E. coli O157:H7 ATCC 43890, E. coli O157:H7 ATCC 43889, and E. coli ATCC 25922; and 50 μg/mL for E. coli O157:H7 2257, E. coli O157: NM 3204-92, E. coli O157:H7 8624 and E. coli O157:H7 ATCC 43894.

Techniques: Synthesized

Journal:

Article Title: Induction of kinase suppressor of RAS-1(KSR-1) gene by1, α 25-dihydroxyvitamin D 3 in human leukemia HL60 cells through a vitamin D response element in the 5′-flanking region

doi: 10.1038/sj.onc.1209697

Figure Lengend Snippet: 1,25D selectively increases the expression of KSR-1 gene. HL60 cells were treated with the indicated differentiation agents for 48 h. (a) Only 1,25D (1 nm) significantly increased KSR-1 gene expression. (b) Ratios of optical density of each RNA band to β-actin (mean±s.d., n = 3). *P<0.01 compared to the untreated control. Other groups P>0.05. (c) Myelo-monocytic differentiation markers CD11b and CD14 show that all agents used here except ceramide induced differentiation. TPA = 12-O-tetradecanoylphorbol-13-acetate, (2 nm); RA = all-trans retinoic acid, (1 μm); DMSO = dimethyl sulfoxide, (1.5%); Ceramide = C2-ceramide (100 μm).

Article Snippet: ChIP assays ChIP assays were performed essentially as described ( Wang et al. , 2005b ) with HL60 cell lysates immunoprecipitated with either normal rabbit IgG or anti-VDR (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Gene Expression, Control

Journal:

Article Title: Induction of kinase suppressor of RAS-1(KSR-1) gene by1, α 25-dihydroxyvitamin D 3 in human leukemia HL60 cells through a vitamin D response element in the 5′-flanking region

doi: 10.1038/sj.onc.1209697

Figure Lengend Snippet: 1,25D antagonist ZK159222 (ZK) inhibits 1,25D-induced expression of KSR-1 gene in parallel with inhibition of monocytic differentiation. (a) HL60 cells were pretreated for 1 h with 100 nm ZK before adding 1 nm 1,25D for the indicated times. The numbers below the bar graph also refer to the groups shown in (b and c). (b) The ratios of optical density of each KSR-1 mRNA band to the constitutively expressed β-actin mRNA (mean±s.d., n = 3). ZK inhibited 1,25D-induced KSR-1 mRNA expression, P<0.05. (c) The myelo-monocytic markers CD11b and CD14 were determined by two-parameter flow cytometery. The numbers below the bar graph refer to the groups shown in (a). The experimental groups marked with an asterisk were significantly decreased compared to groups treated with 1,25D at that time point (P<0.05). As ZK is also a weak agonist, there was some differentiation at 48 h in cells treated with ZK alone.

Article Snippet: ChIP assays ChIP assays were performed essentially as described ( Wang et al. , 2005b ) with HL60 cell lysates immunoprecipitated with either normal rabbit IgG or anti-VDR (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Inhibition

Journal:

Article Title: Induction of kinase suppressor of RAS-1(KSR-1) gene by1, α 25-dihydroxyvitamin D 3 in human leukemia HL60 cells through a vitamin D response element in the 5′-flanking region

doi: 10.1038/sj.onc.1209697

Figure Lengend Snippet: KSR-1 is an immediate-early gene target of 1,25D. (a) HL60 cells were pretreated for 1 h with 200 μg/ml CHX, a protein synthesis inhibitor, before induction of differentiation with 1 nm 1,25D. The KSR-1 mRNA levels were determined by real-time PCR. Note that the 1,25D-induced KSR-1 gene expression was not affected by the presence of CHX. (b). To monitor cytotoxicity of CHX, cell viability of HL60 cells was determined by Trypan blue exclusion. The data shown are means±s.d., n = 3.

Article Snippet: ChIP assays ChIP assays were performed essentially as described ( Wang et al. , 2005b ) with HL60 cell lysates immunoprecipitated with either normal rabbit IgG or anti-VDR (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal:

Article Title: Induction of kinase suppressor of RAS-1(KSR-1) gene by1, α 25-dihydroxyvitamin D 3 in human leukemia HL60 cells through a vitamin D response element in the 5′-flanking region

doi: 10.1038/sj.onc.1209697

Figure Lengend Snippet: 1,25D increases the binding of VDR and RXRα to the putative VDRE in the promoter region of KSR-1 gene. (a) Gel-shift analysis of the VDRE binding by proteins in nuclear extracts in HL60 cells treated for 48 h with the indicated (in nm) concentrations of 1,25D. VDRE binding increased in 1,25D-dependent manner. (b) Western blot analysis of the expression of VDR and RXR isoforms in cells treated with graded concentrations of 1,25D in parallel with the experiment shown in (a). Note the correspondence between gel shift band intensity and protein abundance. (c) The specificity of VDRE binding was shown by its marked inhibition by a 50-fold excess of the unlabeled VDRE oligonucleotide, but not by an excess of the unlabeled mutated VDRE oligonucleotide. The mutated oligonucleotide did not bind nuclear proteins. (d) Different antibodies were added to the reaction mixture for complex blocking assay. Both VDR and RXRa antibodies blocked the VDRE binding. The control antibody was to c-fos.

Article Snippet: ChIP assays ChIP assays were performed essentially as described ( Wang et al. , 2005b ) with HL60 cell lysates immunoprecipitated with either normal rabbit IgG or anti-VDR (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Binding Assay, Gel Shift, Western Blot, Expressing, Quantitative Proteomics, Inhibition, Blocking Assay, Control

Journal:

Article Title: Induction of kinase suppressor of RAS-1(KSR-1) gene by1, α 25-dihydroxyvitamin D 3 in human leukemia HL60 cells through a vitamin D response element in the 5′-flanking region

doi: 10.1038/sj.onc.1209697

Figure Lengend Snippet: Parallel, 1,25D-dependent, increases in VDR/VDRE and Pol II/KSR-1 transcription start site binding. ChIP analyses of binding of VDR in intact HL60 cells to the region of the KSR-1 gene containing the VDRE, and of Pol II to the transcription start site. Regions amplified are indicated in gray, along with the positions of the VDRE. αVDR, αPol II = immunoprecipitation with the corresponding antibody. The arrow indicates the transcription start site of KSR-1. The table presents results of OD scanning of signals in regions 1 and 2. No signals were detected in region 3, a randomly chosen genomic sequence without a VDRE-like motif.

Article Snippet: ChIP assays ChIP assays were performed essentially as described ( Wang et al. , 2005b ) with HL60 cell lysates immunoprecipitated with either normal rabbit IgG or anti-VDR (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Binding Assay, Amplification, Immunoprecipitation, Sequencing

Journal:

Article Title: Induction of kinase suppressor of RAS-1(KSR-1) gene by1, α 25-dihydroxyvitamin D 3 in human leukemia HL60 cells through a vitamin D response element in the 5′-flanking region

doi: 10.1038/sj.onc.1209697

Figure Lengend Snippet: Schematic representation of the postulated signaling by 1,25D of HL60 cell differentiation. VDR liganded by 1,25D heterodimerizes with RXR and binds to VDREs in the promoters of 1,25D-responsive genes, thus transactivating their expression. The functional VDRE identified here heteromerizes specifically with RXRα and may connect VDR signaling to the MAPK pathways by upregulating the expression KSR-1, shown here at the mRNA level, and previously at the protein level (Wang and Studzinski, 2004). The KSR-1 protein then acts as a scaffold to increase the efficiency of Raf-1 signaling to downstream transcription factors.

Article Snippet: ChIP assays ChIP assays were performed essentially as described ( Wang et al. , 2005b ) with HL60 cell lysates immunoprecipitated with either normal rabbit IgG or anti-VDR (C-20) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Cell Differentiation, Expressing, Functional Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) is highly expressed in oesophageal squamous cell carcinoma (OSCC) tissues and linked to tumour stage and poor prognosis. (A) Volcano plot showing gene expression differences between cisplatin‐resistant and parental groups in GSE169337. (B) Kaplan–Meier survival analysis of OSCC patients with different PADI4 levels using The Cancer Genome Atlas (TCGA) data. (C) Immunohistochemical (IHC) analysis comparing PADI4 expression in OSCC tissues to normal oesophageal tissues. (D) IHC analysis of PADI4 expression across TNM stages in OSCC. (E, F) Kaplan–Meier analysis of overall and recurrence‐free survival in OSCC patients with varying PADI4 expression from IHC data. (G) PADI4 knockdown markedly inhibited the proliferation ability of ECA109 and KYSE150 cells indicated by colony formation assay. (H) PADI4 knockdown markedly inhibited the migration ability of ECA109 and KYSE150 cells indicated by transwell assay.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Gene Expression, Immunohistochemical staining, Expressing, Knockdown, Colony Assay, Migration, Transwell Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Clinical characteristics of patients and expression of peptidylarginine deiminase 4 (PADI4).

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Expressing

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Regression analysis of oesophageal squamous cell carcinoma (OSCC) patients.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques:

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Cancer stem cell (CSC)‐like phenotypes and cisplatin resistance of oesophageal squamous cell carcinoma (OSCC) cells were suppressed by peptidylarginine deiminase 4 (PADI4) knockdown. (A) Comprehensive analysis of PADIs and the stemness across 33 tumour types within the The Cancer Genome Atlas (TCGA) database. (B) Correlation between PADI4 and stemness markers (CD133, LGR5, Nanog, OCT4 and CD24) in GSE23400. (C, D) qPCR showed PADI4 knockdown significantly reduced CD133 and Nanog mRNA levels in ECA109 (C) and KYSE150 (D) cells. (E, F) Western blot revealed PADI4 knockdown significantly decreased CD133 and Nanog protein levels in ECA109 (E) and KYSE150 (F) cells. (G, H) Western blot showed PADI4 plasmids significantly increased CD133 and Nanog protein levels in ECA109 (G) and KYSE150 (H) cells. (I) PADI4 knockdown significantly reduced sphere formation in ECA109 and KYSE150 cells. (J, K) Downregulation of PADI4 enhanced the resistance of ECA109 (J) and KYSE150 (K) cells to cisplatin through CCK8 assay. (L, M) Limiting dilution assays showing the self‐renewing capacity of PADI4 downregulated in CD133‐positive cells of ECA109 and KYSE150.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Knockdown, Western Blot, CCK-8 Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) interacts with protein arginine methyltransferase 2 (PRMT2) and maintains stability of PRMT2. (A) Mass spectrometry analysis identified PRMT2 bound with PADI4. (B) Immunostaining of ECA109 cells transfected with PADI4‐HA and PRMT2‐Flag was performed using anti‐HA and anti‐Flag antibodies, with confocal microscopy revealing the subcellular localization of PADI4‐Flag (green), PRMT2 (red) and DAPI (blue, nucleus marker). (C, D) Endogenous cimmunoprecipitation was used to study the interaction between PADI4 and PRMT2 in ECA109 and KYSE‐150 cells after transformed into plasmid of PADI4‐HA. (E, F) PRMT2 protein level in PADI4‐knockdown cell lines. (G, H) PRMT2 protein level in PADI4‐upregulated cell lines. (I) PRMT2 protein degradation levels were measured in treat with cycloheximide (CHX) (10 µg/mL) over specified time intervals in PADI4‐KO ECA109 cells. (J) Lysates from cells co‐transfected with His‐Ub, Flag‐PRMT2 and PADI4‐HA underwent immunoprecipitation with a Flag antibody, followed by immunoblotting with an anti‐His antibody in HEK 293T cells. DAPI, 4′,6‐Diamidino‐2‐phenylindole.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Mass Spectrometry, Immunostaining, Transfection, Confocal Microscopy, Marker, Transformation Assay, Plasmid Preparation, Knockdown, Immunoprecipitation, Western Blot

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) citrullinates protein arginine methyltransferase 2 (PRMT2) to maintain the stability of PRMT2. (A) PADI4‐HA overexpression was immunoprecipitated using Flag‐PRMT2 antibodies, and immunoblotted with modified‐citrulline antibodies. (B) Recombinant PADI4 was immunoprecipitated with a Flag‐PRMT2 antibody in calcium presence, and reaction products were analysed by western blot with modified‐citrulline antibody. (C) The amino acid sequence of PRMT2 in different species is highly conserved at R312 and R397. (D) Two PRMT2 mutant plasmids and transfected into HEK 293T cells to perform immunoprecipitation experiments to investigate citrullination. (E) ECA109 cells were analysed by western blot after 8‐h treatment with varying doses of BB‐Cl‐Amidine. (F) In ECA109 cells treated with GSK484, PRMT2's half‐life was shown by using cycloheximide (CHX) to block protein synthesis. (G) Immunoprecipitating PRMT2 with an anti‐PRMT2‐Flag antibody and probing for polyubiquitination with an anti‐ubiquitin antibody revealed a significant increase in polyubiquitinated PRMT2 protein after GSK484 treatment. (H, I) KYSE150 (H) and ECA109 (I) cells were analysed of cancer stem cell (CSC) markers by western blot after 24‐h treatment with varying doses of GSK484.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Over Expression, Immunoprecipitation, Modification, Recombinant, Western Blot, Sequencing, Mutagenesis, Transfection, Blocking Assay, Ubiquitin Proteomics

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) acts as a positive regulator of inhibitors of DNA binding (IDs). (A, B) The mRNA and protein levels of cancer stem cell (CSC) markers in PRMT2 knockdown ECA109 cells were also assessed by qPCR and western blot. (C) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of CSC markers were measured using western blot. (D, E) The mRNA and protein levels of ID1, ID2 and ID3 in PRMT2 knockdown ECA109 cells were assessed using qPCR and western blot. (F) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of ID1, ID2 and ID3 were measured using western blot. (G, H) The mRNA and protein levels of ID1, ID2 and ID3 in peptidylarginine deiminase 4 (PADI4) knockdown and ECA109 cells were assessed using real‐time PCR and western blot. (I) The protein levels of ID1, ID2 and ID3 in ECA109 cells transfected with PADI4 plasmids were measured. (J) H3R8me2a expression was analysed via western blot in PADI4‐transfected ECA109 cells. (K, L) ChIP assay revealed H3R8me2a enrichment at the human ID1 and ID2 promoter in ECA109 cells. (M, N) Cell viability was assessed using a CCK8 assay after 48‐h treatment with cisplatin in ECA109 (M) and KYSE150 cells (N). (O) PRMT2‐KO cells showed reduced sphere formation in ECA109 and KYSE150 cells. (P, Q) GSK484 reduction in the expression levels of ID1, ID2 and ID3 with different concentration in ECA109 and KYSE150 cells. (R, S) Limiting dilution assays showing the self‐renewing capacity of PRMT2 downregulated in ECA109 (R) and KYSE150 (S) CD133‐positive cells. (T) PRMT2 knockdown markedly inhibited the proliferation ability of ECA109 and KYSE150 cells indicated by colony formation assay. (U) Immunihistochemical (IHC) analysis comparing PRMT2 expression in oesophageal squamous cell carcinoma (OSCC) patients tumour tissues to adjacent normal oesophageal tissues. (V) Correlation between PADI4 and PRMT2 in OSCC patients tumour tissues.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Binding Assay, Knockdown, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, CCK-8 Assay, Concentration Assay, Colony Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) citrullinate disrupts the interaction between USP7 and PRMT2. (A) HEK 293T cells were co‐transfected with PRMT2‐Flag and PADI4‐HA plasmids, and USP7 levels were assessed via western blot. (B) Co‐transfect HEK 293T cells with PADI4‐Flag and USP7‐HA, treat with GSK484 (20 µM) for 24 h, and use western blot to evaluate PRMT2 levels. (C) HEK 293T cells were co‐transfected with PRMT2‐WT‐Flag, PRMT2‐R312E‐Flag and PRMT2‐R397E‐Flag plasmids, and USP7 levels were assessed via western blot. (D) HEK 293T cells were co‐transfected with HA‐USP7, His‐Ub, PRMT2‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, then treated with MG132 (20 µM) for 5 h before harvesting to assess PRMT2‐Flag ubiquitination levels. (E) HEK 293T cells were co‐transfected with HA‐USP7, PRMT2‐WT‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, and then use western blot to evaluate Flag levels. (F) Western blot was used to analyse ID1, ID2 and ID3 expression in HEK 293T cells transfected with PRMT2‐WT, PRMT2‐R397E and PRMT2‐R312E. (G) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate resistance of ECA109 cells to cisplatin. (H) Peptidylarginine deiminase 4 (PADI4) knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (I) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate the sphere formation. (J) PADI4 knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (K) Downregulation of PADI4 enhanced the resistance of ECA109 cells to cisplatin through CCK8 assay and PRMT2 restores this effect.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Knockdown, CCK-8 Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) facilitates tumour growth and metastasis in vivo, while GSK484 exhibits a synergistic effect with cisplatin in vivo. (A) A schematic diagram illustrating the treatment protocol for mice in this study is presented. (B, C) Images of excised subcutaneous tumours from mice treated with shPADI4, GSK484 and cisplatin, and the combination of GSK484 and cisplatin are shown. (D) Immunohistochemical (IHC) analysis of PADI4 protein expression in excised tumours from the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is presented. (E) The volume of subcutaneous tumours in the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is depicted. (F) The weight of subcutaneous tumours in the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is reported. (G) HE staining revealed oesophageal squamous cell carcinoma (OSCC) metastases in the lung tissues. (H) The schematic diagram of the underlying mechanism in this study was shown.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: In Vivo, Immunohistochemical staining, Expressing, Staining