Journal: Nucleic Acids Research

Article Title: Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor

doi: 10.1093/nar/gks764

Figure Lengend Snippet: PRMT6 −/− MEFs harbor H3R2me2 hypomethylation and display growth defects. ( A ) Total cellular RNA was isolated from primary MEFs of the indicated genotype and subjected to semi-quantitative RT–PCR. The migration of the DNA fragments for PRMT6 and GAPDH is shown. The molecular weight marker is the 1 kb ladder and the gel was visualized using ethidium bromide. No RT denotes the absence of reverse transcriptase and is a control used to show that RNA was indeed amplified and not DNA. ( B ) Total cellular proteins from PRMT6 +/ + and PRMT6 −/− MEFs were separated by SDS–PAGE and subsequently immunoblotted with anti-PRMT6, anti-H3R2(me2a), anti-H3 and anti-α-Tubulin antibodies. ( C ) PRMT6 +/ + and PRMT6 −/− MEFs growing in log phase were trypsinized, fixed in 75% MeOH, stained with PI and analyzed by flow cytometry.

Article Snippet: Immunoblotting was performed as described previously ( ) and antibodies used were the following: α-PRMT6 (A300-929 A, Bethyl laboratories, 1:1000); α-tubulin (B-5-1-2, Sigma, 1:50 000); α-H3R2(me2a) (NB21-1002, Novus Biologicals, 1:2000); α-H3 (ab1791, Abcam, 1:1000); α-Cyclin D1 (06-137, Millipore, 1:2000); α-p53 (2524, Cell Signaling, 1:1000); α-PML(36.1-104, Millipore, 1:1000); and α-p21 (05-345, Millipore, 1:1000).

Techniques: Isolation, Quantitative RT-PCR, Migration, Molecular Weight, Marker, Reverse Transcription, Control, Amplification, SDS Page, Staining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor

doi: 10.1093/nar/gks764

Figure Lengend Snippet: PRMT6 associates with the promoter region of Trp53 to regulate H3R2 methylation. ( A ) A schematic representation of the murine Trp53 gene with the primers used for ChIP analysis. The transcription start site is shown with the numbering upstream of this site. ChIP-qPCR analysis was performed using wild-type MEFs and the immunoprecipitated DNA enriched using anti-PRMT6, -PRMT1, -CARM1 or -PRMT5 antibodies was normalized with an internal IgG control. ( B ) ChIP-qPCR analysis using anti-PRMT6 antibodies from PRMT6 +/ + and PRMT6 −/− primary MEFs at −1322 of the Trp 53 promoter is shown. PRMT6 occupancy was normalized to an IgG control. ( C ) The presence of H3R2(me2a), H3K4(me3), H3R26(me2a) and H3R17(me2a) at the −1322 upstream region of Trp 53 promoter was determined using ChIP-qPCR analysis. The values for the histone marks were normalized to total histone H3 levels. ( D ) SA-β-Gal staining of PRMT6 −/− , p53 −/− , PRMT6 −/− ; p53 −/− and wild-type primary MEFs is shown. The numbers represent the percentage of SA-β-Gal positive cells. ( E ) Whole-cell extracts obtained from PRMT6 −/− ; p53 −/− MEFs or controls were analyzed by immunoblotting using anti-p53, -PML, -PRMT6 antibodies. Anti-α-tubulin was used as a loading control. Molecular mass markers for PML isoforms are shown on the right.

Article Snippet: Immunoblotting was performed as described previously ( ) and antibodies used were the following: α-PRMT6 (A300-929 A, Bethyl laboratories, 1:1000); α-tubulin (B-5-1-2, Sigma, 1:50 000); α-H3R2(me2a) (NB21-1002, Novus Biologicals, 1:2000); α-H3 (ab1791, Abcam, 1:1000); α-Cyclin D1 (06-137, Millipore, 1:2000); α-p53 (2524, Cell Signaling, 1:1000); α-PML(36.1-104, Millipore, 1:1000); and α-p21 (05-345, Millipore, 1:1000).

Techniques: Methylation, ChIP-qPCR, Immunoprecipitation, Control, Staining, Western Blot

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

Article Title: Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

doi: 10.4049/jimmunol.1301913

Figure Lengend Snippet: FIGURE 1. Identification of Arg2 mRNA as a novel miR155-regulated mRNA in DCs. (A) Schematic representation of the identification of potential miR155 targets in BM-cDCs and BM-pDCs. (B) Signal intensities observed for Arg2 mRNA in microarray experiments are represented for BM-cDCs and BM-pDCs from WT and miR1552/2 mice. DCs were unstimu- lated (0) or activated for 4 or 24 h with LPS (BM-cDCs) or imiquimod (BM-pDCs). Results are represented as fold change relative to un- stimulated WT DCs. Means and SDs were de- rived from three microarray experiments. (C) qRT-PCR was used to analyze Arg2 and BIC (miR155 precursor) expression in WT and miR1552/2 BM-cDCs and BM-pDCs stimu- lated for 0, 4, and 24 h with LPS (BM-cDCs) or imiquimod (BM-pDCs). IL-6 and IL12-p40 mRNAs were used as controls for maturation. Results are represented as fold change relative to unstimulated WT DCs. Means and SDs were derived from three experiments. **p , 0.01, ***p , 0.001.

Article Snippet: Protein extracts were fractionated by SDS-PAGE, and Western blotting was performed using anti-mouse Arg2 (sc-20151; Santa Cruz Biotechnology), anti-mouse Arg1 (sc-20150; Santa Cruz Biotechnology), and anti-tubulin (B-5-1-2; Sigma-Aldrich).

Techniques: Microarray, Quantitative RT-PCR, Expressing, Derivative Assay

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

Article Title: Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

doi: 10.4049/jimmunol.1301913

Figure Lengend Snippet: FIGURE 2. Deregulated Arg2 expression in DCs from miR1552/2 mice. (A) Expression of Arg2 and Arg1 proteins was analyzed by Western blotting in WT and miR1552/2 BM- cDCs and BM-pDCs stimulated for 0, 24, and 48 h with LPS (BM-cDCs) or imiquimod (BM- pDCs). Tubulin was used as internal control. Results are representative of two independent experiments. (B) Arginase activity was mea- sured in WT and miR1552/2 BM-cDCs and BM-pDCs stimulated for 0 and 48 h with LPS (cDCs) or 0, 24, and 48 h with imiquimod (pDCs). (C) Arg2 mRNA expression was analyzed by qRT-PCR in immature WT and miR1552/2 BM-cDCs treated for 0, 24, and 48 h with poly I/C, PGN, imiquimod, PAM3CSK4, or CpG; total splenic DCs treated for 0, 4, and 24 h with LPS, and CD82 and CD8+ splenic DCs treated for 0 and 24 h with CpG. Results in (B) and (C) are represented as fold change relative to unstimulated WT cells. Means and SDs were derived from three inde- pendent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Protein extracts were fractionated by SDS-PAGE, and Western blotting was performed using anti-mouse Arg2 (sc-20151; Santa Cruz Biotechnology), anti-mouse Arg1 (sc-20150; Santa Cruz Biotechnology), and anti-tubulin (B-5-1-2; Sigma-Aldrich).

Techniques: Expressing, Western Blot, Control, Activity Assay, Quantitative RT-PCR, Derivative Assay

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

Article Title: Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

doi: 10.4049/jimmunol.1301913

Figure Lengend Snippet: FIGURE 3. Deregulated Arg2 expression in the lungs of miR1552/2 mice. (A) Masson Trichrome staining of sections showing lung bronchioles of WT and miR1552/2 mice. Collagen is stained in blue. Results are representative of five independent experi- ments. (B) Total soluble lung collagen was quantified for WT and miR1552/2 mice. (C) Arginase activity and Arg2 protein were analyzed in total lung cells from WT and miR1552/2 mice. Means and SDs in (B) and (C) were derived from at least five mice per group. (D) BIC RNA (miR155 precursor), mature miR155, Arg2 mRNA, Arg2 protein, and arginase activity were measured in lung myofibroblasts from WT and miR1552/2 mice. Tubulin was used as internal control. Means and SDs were derived from at least six mice per group. *p , 0.05, **p , 0.01.

Article Snippet: Protein extracts were fractionated by SDS-PAGE, and Western blotting was performed using anti-mouse Arg2 (sc-20151; Santa Cruz Biotechnology), anti-mouse Arg1 (sc-20150; Santa Cruz Biotechnology), and anti-tubulin (B-5-1-2; Sigma-Aldrich).

Techniques: Expressing, Staining, Activity Assay, Derivative Assay, Control

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

Article Title: Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

doi: 10.4049/jimmunol.1301913

Figure Lengend Snippet: FIGURE 4. Arg2 is a direct target of miR155. (A) A schematic repre- sentation of mouse Arg2 mRNA is shown: sizes in nucleotides of the 59- UTR, open reading frame (ORF), and 39-UTR are indicated. The 39-UTR contains one predicted binding site (black box) for miR155. The sequence of mouse miR155 is shown aligned with its predicted target site in the 39- UTR of Arg2 mRNA. A-U and G-C bp are represented by solid lines. G-U bp are represented by dotted lines. The miR155 seed region is enclosed by a box. The sequence of the mutated (Mut) 39-UTR of mouse Arg2 mRNA is indicated. (B) Luciferase reporter constructs containing the WT 39-UTR of mouse Arg2 mRNA (Arg2), the mutated 39-UTR of mouse Arg2 mRNA (Mut-Arg2), and the WT 39-UTR of mouse Arg1 mRNA (Arg1) were transfected into 293T cells, alone or together with the indicated amounts of mouse miR155 precursor (top panel) or BIC (miR155 precursor) expres- sion vector (bottom panel). Luciferase activity was measured 24 h after transfection, normalized with respect to activity obtained with the empty reporter vector and expressed as relative luciferase activity. The average and SDs were derived from three independent experiments.

Article Snippet: Protein extracts were fractionated by SDS-PAGE, and Western blotting was performed using anti-mouse Arg2 (sc-20151; Santa Cruz Biotechnology), anti-mouse Arg1 (sc-20150; Santa Cruz Biotechnology), and anti-tubulin (B-5-1-2; Sigma-Aldrich).

Techniques: Binding Assay, Sequencing, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay, Derivative Assay

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

Article Title: Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

doi: 10.4049/jimmunol.1301913

Figure Lengend Snippet: FIGURE 5. Arg2 expression is downregulated by miR155. (A) Expression of mature miR155 was ana- lyzed by qRT-PCR in 24-h LPS-treated BM-cDCs and in BM-cDCs transduced with a BIC expression vector (BIC) or a mutated BIC control vector (Mut). Expres- sion of Arg2 mRNA was analyzed by qRT-PCR in unstimulated and LPS-treated BM-cDCs from WT or miR1552/2 mice transduced with a BIC expression vector (BIC) or the mutated control vector (Mut). Results are represented as fold change in miR155 or Arg2 mRNA expression relative to control vector– transduced WT cells. (B) Left panel, The expression of mature miR155 and Arg2 mRNA was analyzed in im- mature DC2114 cells transduced with the BIC expres- sion vector (BIC) or mutated control vector (Mut) under the control of two different promoters (EF and CMV). The results are represented as fold change in mature miR155 or Arg2 mRNA expression relative to the control vector–transduced cells. Right panel, Time-course experiments were performed to analyze the expression of mature miR155 and Arg2 mRNA in immature and CpG-stimulated mouse DC2114 cells transduced with the BIC expression vector (BIC) or a control vector. Results are provided as fold change relative to unstimulated DC2114 cells. The means and SDs were derived from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Protein extracts were fractionated by SDS-PAGE, and Western blotting was performed using anti-mouse Arg2 (sc-20151; Santa Cruz Biotechnology), anti-mouse Arg1 (sc-20150; Santa Cruz Biotechnology), and anti-tubulin (B-5-1-2; Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Transduction, Plasmid Preparation, Control, Derivative Assay

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

Article Title: Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

doi: 10.4049/jimmunol.1301913

Figure Lengend Snippet: FIGURE 6. Deregulated Arg2 expression in DCs impairs T cell proliferation in vitro. (A) Analysis of GFP expression by flow cytometry in DC2114 cells transduced with control or Arg2 expression vectors. (B) Arg2 expression was analyzed by immunofluorescence staining (red) in unstimulated (2CpG) and CpG- stimulated (+CpG) Arg2-transduced and nontransduced (NT) DC2114 cells. Arg2-transduced cells were costained with Tom-20 (blue) to visualizing mitochondria. Merged images indicate colocalization of Arg2 and Tom-20 staining. Original magnification 3630. (C) The expression of Arg2 protein was analyzed in unsti- mulated control and Arg2-transduced DC2114 cells. (D) Arginase activity was measured in unstimulated control and Arg2-transduced DC2114 cells. (E) The con- centrations of the indicated amino acids (single letter code) were determined in supernatants from unstimulated control and Arg2-transduced DC2114 cells. Results are presented as percentage of the concentration in the control. The average and SD were derived from two independent experiments. (F) Activated CD4+ T cells were cultured in supernatants from unstimulated control and Arg2-transduced (Arg2) DC2114 cells. As controls, the cells were cultured in the presence of fresh medium (+Arg) or medium lacking arginine (2Arg). T cell activation was induced with anti-CD3/CD28 and assessed by CD69 staining. Proliferation was assessed by analyzing Ki67 expression. Representative flow cytometry profiles are shown for Ki67 staining. In control samples, the Ki67 Ab was replaced with an isotype control. The percentages of CD69+ and Ki67+CD4+ T cells are indicated. Means and SDs were derived from three experiments, each with three mice per group. (G) Activated CD4+ T cells were cultured in media from WT and miR1552/2 BM-cDCs. Control cells were cultured in fresh medium (+Arg) or medium lacking arginine (2Arg). Proliferation was assessed by analyzing Ki67 expression. Representative flow cytometry profiles are shown: percentages of Ki67+CD4+ T cells are indicated. In control samples, the Ki67 Ab was replaced with an isotype control. Means and SDs were derived from three experiments, each with three mice per group. (H) Activated CD4+ T cells were cultured in supernatants from unstimulated control (vector) and Arg2-transduced (Arg2) DC2114 cells. Supernatants were supplemented (+) or not (2) with arginine. As controls, cells were cultured in the presence of fresh medium (full), medium lacking arginine (2Arg), or arginine- lacking medium supplemented (+) or not (2) with arginine. T cell activation was induced with anti-CD3/CD28. Proliferation was assessed by analyzing Ki67 expression. Means and SDs were derived from three experiments, each with three mice per group. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Protein extracts were fractionated by SDS-PAGE, and Western blotting was performed using anti-mouse Arg2 (sc-20151; Santa Cruz Biotechnology), anti-mouse Arg1 (sc-20150; Santa Cruz Biotechnology), and anti-tubulin (B-5-1-2; Sigma-Aldrich).

Techniques: Expressing, In Vitro, Cytometry, Transduction, Control, Staining, Activity Assay, Concentration Assay, Derivative Assay, Cell Culture, Activation Assay, Plasmid Preparation

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

Article Title: Repression of arginase-2 expression in dendritic cells by microRNA-155 is critical for promoting T cell proliferation.

doi: 10.4049/jimmunol.1301913

Figure Lengend Snippet: FIGURE 7. Deregulated Arg2 expression im- pairs T cell proliferation in vivo. CD4+ T cells from OTII mice and OVA-loaded Arg2 or control (vector)- transduced DC2114 cells were transferred into WT recipients. (A) Proliferation of splenic Va2+ OTII CD4+ T cells was assessed by analyzing Ki67 staining after 48 and 72 h. Representative flow cytometry profiles are shown: percentages of Ki67+Va2+CD4+ T cells are indicated. The means and SEM were derived from two experiments in- cluding six to seven mice per group. (B) Splenic Va2+ OTII CD4+ T cells were stained with anti- CD3z after 48 and 72 h. Representative flow cytometry profiles are shown. Means and SDs for the mean fluorescence intensity (MFI) were derived from two experiments with six to seven mice per group. (C) Proliferation of splenic CD45.1+ OTII CD4+ T cells was assessed by analyzing CFSE dilution after 72 h. Representative flow cytometry profiles and histograms are shown. Means and SDs for CFSE mean fluorescence intensity (MFI) were derived from three experiments with three mice per group. Percentages of AnnexinV+7AAD+CD4+

Article Snippet: Protein extracts were fractionated by SDS-PAGE, and Western blotting was performed using anti-mouse Arg2 (sc-20151; Santa Cruz Biotechnology), anti-mouse Arg1 (sc-20150; Santa Cruz Biotechnology), and anti-tubulin (B-5-1-2; Sigma-Aldrich).

Techniques: Expressing, In Vivo, Control, Plasmid Preparation, Staining, Cytometry, Derivative Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Construction and evaluation of endometriosis diagnostic prediction model and immune infiltration based on efferocytosis-related genes

doi: 10.3389/fmolb.2023.1298457

Figure Lengend Snippet: qPCR primers.

Article Snippet: To block nonspecific antibody binding, paraffin sections were incubated with normal goat serum for 90 min, and then the primary antibodies of GAS6 (Abcam, ab264098, 1:200), C3 (Abcam, ab200999, 1:200), and ARG2 (Affinity Biosciences, AF0738-200, 1:200) were added dropwise and incubated overnight at 4°C.

Techniques: Sequencing

Journal: Frontiers in Molecular Biosciences

Article Title: Construction and evaluation of endometriosis diagnostic prediction model and immune infiltration based on efferocytosis-related genes

doi: 10.3389/fmolb.2023.1298457

Figure Lengend Snippet: diagnostic markers selection: (A) Receiver operating characteristic (ROC) curves for the 8 EFRDEGs are displayed. The x -axis denotes the false-positive rate, while the y -axis represents the true-positive rate, quantified by sensitivity. The area under the ROC curve (AUC) measures the intensity of connection between the gene and the disease, with a higher AUC indicating a pretty association. (B) Box plots depict the expression levels of the eight chosen genes (CLU, C3, CLU, FGL2, PROS1, GAS6, C1QA, ARG2, and PECAM1) in both eutopic and ectopic endometrial tissues. Green represents eutopic endometria, while red represents ectopic endometria. *** p < 0.001 signifies a statistically great difference in gene expression between the two types of endometria.

Article Snippet: To block nonspecific antibody binding, paraffin sections were incubated with normal goat serum for 90 min, and then the primary antibodies of GAS6 (Abcam, ab264098, 1:200), C3 (Abcam, ab200999, 1:200), and ARG2 (Affinity Biosciences, AF0738-200, 1:200) were added dropwise and incubated overnight at 4°C.

Techniques: Diagnostic Assay, Selection, Expressing, Gene Expression

Journal: Frontiers in Molecular Biosciences

Article Title: Construction and evaluation of endometriosis diagnostic prediction model and immune infiltration based on efferocytosis-related genes

doi: 10.3389/fmolb.2023.1298457

Figure Lengend Snippet: Diagnostic biomarkers selection using two machine learning methods. (A) The least absolute shrinkage and selection operator (LASSO) algorithm results are presented in two plots. In the left plot, the horizontal axis symbolizes log(λ) values and the vertical axis symbolizes regression cross-validation errors. The right plot displays the ln-transformed minimum log(λ) values along the horizontal axis and the corresponding coefficients on the vertical axis. Six genes whose coefficients were not 0 when lambda = 0.037 were screened out. (B) Support vector machine recursive feature elimination (SVM-RFE) regression model algorithm identified seven diagnostic biomarkers. The right plot illustrates the ranking of these seven feature genes according to their importance from highest to lowest as follows: PECAM1, GLU, GAS6, ARG2, PROS1, FGL2, and C3.

Article Snippet: To block nonspecific antibody binding, paraffin sections were incubated with normal goat serum for 90 min, and then the primary antibodies of GAS6 (Abcam, ab264098, 1:200), C3 (Abcam, ab200999, 1:200), and ARG2 (Affinity Biosciences, AF0738-200, 1:200) were added dropwise and incubated overnight at 4°C.

Techniques: Diagnostic Assay, Selection, Biomarker Discovery, Transformation Assay, Plasmid Preparation

Journal: Frontiers in Molecular Biosciences

Article Title: Construction and evaluation of endometriosis diagnostic prediction model and immune infiltration based on efferocytosis-related genes

doi: 10.3389/fmolb.2023.1298457

Figure Lengend Snippet: Multivariate stepwise logistic regression.

Article Snippet: To block nonspecific antibody binding, paraffin sections were incubated with normal goat serum for 90 min, and then the primary antibodies of GAS6 (Abcam, ab264098, 1:200), C3 (Abcam, ab200999, 1:200), and ARG2 (Affinity Biosciences, AF0738-200, 1:200) were added dropwise and incubated overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Construction and evaluation of endometriosis diagnostic prediction model and immune infiltration based on efferocytosis-related genes

doi: 10.3389/fmolb.2023.1298457

Figure Lengend Snippet: Establishment and assessment of diagnostic prediction model: (A) A nomogram of diagnostic biomarkers, where “Point” represents individual scores on the scale; ARG2, GAS6, and C3 correspond to the scores of each gene; “Total Point” represents the combined score of the three hub genes. (B) Decision curve analyses (DCAs) for the nomogram, show that the model curves are above the high-risk threshold curve. (C) Calibration curves of the hub genes, demonstrating good calibration of the combined model after bias correction. (D) ROC curve of the nomogram model with an AUC of 0.978, and the test sets GSE37837 and GSE6364, with an AUC of 0.627 and 0.635, respectively.

Article Snippet: To block nonspecific antibody binding, paraffin sections were incubated with normal goat serum for 90 min, and then the primary antibodies of GAS6 (Abcam, ab264098, 1:200), C3 (Abcam, ab200999, 1:200), and ARG2 (Affinity Biosciences, AF0738-200, 1:200) were added dropwise and incubated overnight at 4°C.

Techniques: Diagnostic Assay