Journal:

Article Title: pp32 Reduction Induces Differentiation of TSU-Pr1 Cells

doi:

Figure Lengend Snippet: Expression of IL-6 in TSU-Pr1 cells. TSU-Pr1 cells stably transfected with pp32 anti-sense express higher levels of IL-6 message as compared to parental TSU-Pr1 cells and vector-only control by RT-PCR analysis, which validates the cDNA microarray analysis (see Figure 6).

Article Snippet: Microarray Analysis of TSU-Pr1 Cell Lines This procedure was performed at The Johns Hopkins University Oncology Microarray facility by using a human glass 12K cDNA chip.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Reverse Transcription Polymerase Chain Reaction, Microarray

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Ascites in peritoneal carcinomatosis (PC) is biologically active (A) Kaplan-Meier curve showing survival of PC patients who underwent cytoreductive surgery (n = 121), stratified by the presence or absence of ascites. (B and C) Proliferation curves of (B) Colo-205 and (C) SNU-C1 cells treated with varying concentrations of ascites, measured by CellTitre-Glo assay. Data are represented as cell viability at day 5 relative to day 0. Dotted line represents cell viability of Colo-205 treated with 10% fetal bovine serum (FBS) at day 5 relative to day 0. Graph shows mean ± SD. (D and E) Migration of (D) Colo-205 and (E) SNU-C1 cells pre-treated with serum-free media (SFM), 10% FBS medium or 5% cell-free ascites (CFA) medium, assessed by transwell migration assay. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with SFM and 10% FBS or 5% CFA was denoted by ∗. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with 10% FBS and SFM or 5% CFA was denoted by # . # p < 0.05, ## p < 0.01, ### p < 0.001. Graph shows mean ± SD. (F) Enriched signaling pathways in colorectal PC cell lines upon treatment with CFA, identified by comparing gene expressions of cells treated with 5% versus 0.1% CFA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G and H) Treatment of (G) Colo-205 and (H) SNU-C1 cells with 5% CFA activated STAT3 signaling pathway through phosphorylation at Tyr705.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Glo Assay, Migration, Transwell Migration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Identification and validation of prognostic putative activators of STAT3 signaling in ascites (A) Workflow to identify clinically significant putative secreted STAT3 activators. (B) Kaplan-Meier survival curve illustrating poorer survival in patients expressing three biomarkers as compared to patients expressing 0–2 biomarkers. (C) Characterization of cytokine profiles in colorectal PC ascites and benign ascites. Top 25% most highly abundant cytokines in colorectal PC ascites (by mean abundance) are shown. Heatmap displays Z score of normalized mean pixel density of duplicate cytokine spots on the array. (D) Downregulated signaling pathways upon PAI-1 inhibition in Colo-205 cells exposed to CFA with high PAI-1 levels (PC085), CFA with low PAI-1 levels (PC249) and no PAI-1 (FBS control), identified using RNA microarray. Only signaling pathways with differential downregulation are presented. IL6-JAK-STAT3 signaling pathway was significantly downregulated in high PAI-1 CFA-treated cells upon PAI-1 inhibition. Normalized enrichment scores <0 indicate pathway suppression and scores >0 indicate pathway activation. (D) is representative of two independent biological experiments. ∗p < 0.05.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Expressing, Inhibition, Microarray, Activation Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Correlating PAI-1 level in ascites and intracellular STAT3 activation in cancer cells revealed distinct subgroups associated with differing susceptibility to PAI-1 inhibition (A) PAI-1 prevalence in colorectal PC ascites (n = 54). (B) Correlation between colorectal PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.4691, p = 0.0003). (C) PAI-1 prevalence in ascites of various histological PC subtypes (n = 156). † indicates benign ascites. (D) Correlation between various histological PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.6476, p < 0.0001). (A–D) PAI-1 concentrations are plotted on a log 2 scale to transform skewed data to normal distribution. p-STAT3(Y705) level was shown as optical density reading at 450 nm (OD450). (E) Untransformed values of PAI-1 and p-STAT3(Y705) levels from (D) were used for stratification strategy to identify patient subpopulations who might benefit from PAI-1 inhibition. Using 20 ng/mL PAI-1 level and 0.2 OD450 p-STAT3(Y705) level as cut-off values, three distinct subgroups of samples were observed: (i) high PAI-1 and high p-STAT3 levels, termed PAI-1 paracrine addicted (PPA) group (yellow region), (ii) low PAI-1 and high p-STAT3 levels, termed co-activators predominant (CAP) group (pink region) and (iii) low PAI-1 and low p-STAT3 levels, termed alternative pathways activation (APA) group (blue region). Each dot represents one patient ascites. Colors in each panel represent the various histological PC subtypes. All histological subtypes with less than five samples are grouped into other histological subtypes.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Activation Assay, Inhibition

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Cells dependent on PAI-1 to activate STAT3 are most susceptible to PAI-1 inhibition (A) Effect of TM5441 (PAI-1 inhibitor) on CFA-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow), CAP group (pink), APA group (blue) and FBS (control, black) demonstrated a left shift, indicating responsiveness to PAI-1 inhibition. (B) Differential sensitivity to TM5441 corresponding to the three subgroups, with PPA (n = 18) being the most sensitive to PAI-1 inhibition, followed by CAP (n = 59) and APA (n = 17). Graph shows mean ± SD. (C–E) Effect of (C) Napabucasin (STAT3 inhibitor), (D) BEZ235 (dual PI3K/mTOR inhibitor) and (E) Mitomycin C (conventional chemotherapeutic agent – DNA crosslinker) on the three subgroups of ascites-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow; n = 3), CAP group (pink; n = 3), APA group (blue; n = 1) and FBS (control, black). Targeting PAI-1, a dominant paracrine factor in ascites, was more effective than targeting downstream signaling pathway activated by ascites, proliferation pathway or DNA synthesis. (F) Evaluation of STAT3 suppression in Colo-205 cells treated with PPA CFA (PC085 and PC383), CAP CFA (PC249), APA CFA (PC010) and various concentrations of TM5441 by ELISA. STAT3 activation was shown as p-STAT3(Y705) and total STAT3 ratio at the indicated concentration relative to DMSO vehicle. Cells exposed to PPA CFA relied on PAI-1 to activate STAT3 as they required lower concentrations of TM5441 to suppress STAT3 activation. Graph shows mean ± SEM. Data in (A-E) are representative of at least three independent biological experiments and (F) is representative of two independent biological experiments. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Inhibition, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet:

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Sequencing, Microarray, Cell Culture, Software

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD4 + CD28 null T cell clones express CD158b/j, but do not express KARAP/DAP12. CD4 + CD28 null T cells were sorted from patients with RA, and clones were established by limiting dilution. Clones were analyzed by flow cytometry for expression of CD28 and CD158b/j. Four representative clones (#1 through #4) are shown. All clones expressed CD4 (unpublished data; A). RT-PCR was used to amplify transcripts for KARAP/DAP12 and β-actin from PBMCs (lane 1), Jurkat T cells (lane 2), and CD4 + CD28 null T cell clones #1–#4 (lanes 3–6, respectively). cDNA was omitted for the negative control (lane 7) (B). Western blotting was used to detect KARAP/DAP12 and β-actin protein (bottom panels) in Jurkat T cells (lane 1), Jurkat T cells transfected with KARAP/DAP12 + vaccinia virus (lane 2), and CD4 + CD28 null T cell clones (lanes 3–7) (C).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Clone Assay, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Transfection, Virus

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Stimulation through CD158b/j results in an up-regulation of ATF-2 and HSP27 transcripts. The PathwayFinder cDNA Array is spotted in duplicate with 23 cDNAs. Represented on the membrane are the ERK (egr-1 and c-fos), JNK (ATF-2, hsf1, HSP27, and HSP90), NF-κB (iNos, NF-κB, and IκBα), NFAT (IL-2, Fas, and CD5), TGF-β (p16, p21, and p57 Kip2 ), Wnt (c-myc), p53 (p21, gadd45, pig7, pig8, mdm2, and bax), and CREB pathways (egr-1, CYP19, and c-fos). The membrane also included a negative control (pUC18) and two positive controls (β-actin and GAPDH) (A). A CD4 + CD28 null CD158b/j + T cell clone was stimulated with control mouse IgG or anti-CD158j mAb and cross-linked with rabbit anti–mouse IgG Ab. Total RNA was harvested and used to probe the PathwayFinder cDNA Array (B).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Membrane, Negative Control, Control

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Phosphorylation of JNK is initiated by stimulation specifically through CD158j. Two CD4 + CD28 null CD158j + T cell clones (top panels) and a CD4 + CD28 null CD158b1 + T cell clone (bottom panels) were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels; A). Jurkat T cells were infected with either wild-type vaccinia virus (WR) or vaccinia virus containing CD158j cDNA and were analyzed for expression of CD158j by flow cytometry (B). Jurkat T cells infected with WR vaccinia virus or CD158j + vaccinia virus were stimulated with anti-CD3 and/or anti-CD158b/j mAbs and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (top left panels) and MKK4 (bottom left panel). The blots were stripped and reprobed with Abs against β-actin (top right panels) or MKK4 (bottom right panel) (C).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Phospho-proteomics, Clone Assay, SDS Page, Membrane, Infection, Virus, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: Mutation of transmembrane lysine residue in CD158j abolishes ability to induce JNK phosphorylation. Jurkat T cells were transiently transfected with constructs containing the CD158j cDNA or the CD158j233I cDNA. Cell-surface expression was confirmed by flow cytometry (A). Jurkat T cells transfected with either CD158j or CD158jK233I were stimulated with anti-CD3 or anti-CD158b/j mAb and cross-linked with rabbit anti–mouse IgG Ab. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against total JNK (right panels) (B).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Mutagenesis, Residue, Phospho-proteomics, Transfection, Construct, Expressing, Flow Cytometry, SDS Page, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Selective Activation of the c-Jun NH 2 -terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4 + T Cells

doi: 10.1084/jem.20020383

Figure Lengend Snippet: CD158j and DAP10 do not associate. RT-PCR was used to amplify transcripts for DAP10 from PBMCs (lane 1) and CD4 + CD28 null T cell clones (lanes 2–5). cDNA was omitted for the negative control (lane 6) (A). CD4 + CD28 null CD158b/j + T cell clones were stimulated with anti-CD3 or anti-CD158b/j in the presence or absence of 2.0 μM wortmannin. After SDS-PAGE and transfer to a nitrocellulose membrane, the cell lysates were analyzed for phosphorylation of JNK (left panels). The blots were then stripped and reprobed with Abs against β-actin (right panels). Results from two T cell clones are shown (B). DAP10-expressing RBL cells (left panel) were stably transfected with CD158j alone (middle panel) or with CD158j and KARAP/DAP12 (right panel). Cell surface expression of CD158j was confirmed by flow cytometry (top panels). DAP10 or KARAP/DAP12 was immunoprecipitated from lysates of biotinylated transfected RBL cells. After SDS-PAGE and transfer to nitrocellulose membranes, coimmunoprecipitated cell-surface proteins were detected by streptavidin-HRP (middle panels). Immunoprecipitation of DAP10 and KARAP/DAP12 was confirmed by immunoblot with anti-DAP10 or anti-KARAP/DAP12 Ab (bottom panels) (C). DAP10 or KARAP/DAP12 was immunoprecipitated from Jurkat T cells (lanes 1–3) or RBL cells (lanes 4–6). After SDS-PAGE and transfer to a nitrocellulose membrane, samples (protein-G preclear, lanes 1 and 4; DAP10 immunoprecipitate, lanes 2 and 5; KARAP/DAP12 immunoprecipitate, lanes 3 and 6) were analyzed by Western blot using DAP10 Ab (D).

Article Snippet: After 4 h, total RNA was harvested. cDNA was synthesized and used to probe the PathwayFinder cDNA Array (SuperArray) according to the manufacturer's instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Negative Control, SDS Page, Membrane, Phospho-proteomics, Expressing, Stable Transfection, Transfection, Flow Cytometry, Immunoprecipitation, Western Blot

Journal: Current Biology

Article Title: Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a ( Ink4a/Arf )

doi: 10.1016/j.cub.2017.09.007

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (clone 197G2) , Cell Signaling Technology , Cat# 4377; RRID: AB_331775.

Techniques: Recombinant, Produced, Reverse Transcription, Microarray, Labeling, DNA Purification, Methylation, TA Cloning, Sequencing, SYBR Green Assay, Software

Journal: Cell host & microbe

Article Title: Reshaping of the Dendritic Cell Chromatin Landscape and Interferon Pathways During HIV Infection

doi: 10.1016/j.chom.2018.01.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Blots were incubated with primary antibodies from Cell Signaling: IRF3 (Cat# 4302S, RRID:AB_1904036); p-IRF3 (Cat# 4947S, RRID:AB_823547); STAT1 (Cat# 9172P, RRID:AB_10831362); p-STAT1 (Cat# 9167S, RRID:AB_561284); IRF7 (Cat# 13014); TBK1 (Cat# 3504, RRID: AB_2255663); p-TBK1 (Cat# 5483, RRID:AB_10693472); IRF1 (Cat# 8478, RRID:AB_10949108); STING (Cat# 13647); p-STING (Cat# 85735); IRF5 (Cat# 13496); MAVS (Cat# 3993, RRID:AB_823565); MYD88 (Cat# 4283S, RRID:AB_10547882); IFI16 (Cat# 14970); cGAS (Cat# 15102); p-NF-kB S536 (Cat #3033, RRID: AB_331284); p-NF-kB S468 (Cat# 3039, RRID:AB_330579); NF-kB (Cat #8242, RRID: AB_10859369); Histone H3 (Cat# 4499, RRID:AB_10544537); and GAPDH (Cat# 5174, RRID:AB_10622025).

Techniques: Recombinant, Molecular Weight, Electron Microscopy, SYBR Green Assay, Staining, Next-Generation Sequencing, Fractionation, Plasmid Preparation, DNA Purification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Infection, Sequencing, Clone Assay, shRNA, Software, Flow Cytometry, Expressing, Microarray

Journal: Cancers

Article Title: Clinical Significance and Regulation of ERK5 Expression and Function in Cancer

doi: 10.3390/cancers14020348

Figure Lengend Snippet: ERK5 expression in human cancers and clinical outcome.

Article Snippet: 306 , IHC–TMA , anti-pERK5 (Thr218/Tyr220, mRb; CST) , T+; LN+.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Microarray, DNA Methylation Assay, Immunohistochemistry

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 potentiates STAT3 activation via Smad7. A) PRMT5 depletion dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were harvested and analyzed by using western blotting with indicated antibodies. B) Knockdown of PRMT5 attenuates IL‐6‐induced STAT3 phosphorylation in MCF10A cells. MCF10A cells were transfected with 40 pm siRNA against PRMT5. 36 h later, cells were treated with IL‐6 (10 ng mL −1 ) for the indicated time and harvested for western blotting analysis with appropriate antibodies. C) PRMT5 inhibition attenuates endogenous activation of STAT3 in H358 cells. H358 cells were treated with 20 × 10 −6 m of PRMT5 inhibitors EPZ015666 or GSK591 for the indicated time. Cell lysates were collected and subject to western blotting analysis. SDMA indicates global arginine di‐methylation. D) Smad7 potentiates STAT3 activation in A549 cells. A549 cells stably expressing FLAG‐GFP or FLAG‐Smad7 were harvested and subject to Western blotting analysis using appropriate antibodies. E) Stable knockdown of Smad7 dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shSmad7 or shCtrl were harvested and subject to western blotting analysis using appropriate antibodies. F) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF7 cells. Cells were transfected with siSmad7 (40 pm) and treated with IL‐6 (10 ng mL −1 ) for the indicated time. Cells were harvested and analyzed by western blotting with appropriate antibodies. G) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF10A cells. Cell transfection, treatment, and Western blotting were done as described in Panel F. H) PRMT5 potentiates STAT3 activation dependent of Smad7. MCF10A cells were transduced with lentiviral particles expressing HA‐PRMT5 or HA‐G367A/R368A. After 24 h, cells were transfected with 40 pm siSmad7. 12 h later, cells were stimulated with IL‐6 (2 ng mL −1 ) for the indicated time. Cell lysates were harvested and subject to Western blotting analysis using appropriate antibodies.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Activation Assay, Stable Transfection, Expressing, Control, Western Blot, Knockdown, Phospho-proteomics, Transfection, Inhibition, Methylation, Transduction

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 methylates Smad7 on R57. A) PRMT5 methylates Smad7. HEK293T cells were transfected with expression plasmids carrying MYC‐PRMT5/MEP50 and an SFB‐tagged construct, including gp130, JAK2, STAT3, Smad7, SHP2, and SOCS3. Cell lysates were harvested and precipitated with streptavidin beads. The retrieved complexes and input were analyzed by Western blotting with indicated antibodies. B) PRMT5 methylates the R57 residue on Smad7. HEK293T cells were transfected with MYC‐PRMT5/MEP50 and an SFB‐tagged Smad7 construct, i.e., wildtype Smad7 (WT) or a R‐to‐K substitution of Smad7 as indicated above the blots. Cell lysate was precipitated with streptavidin beads. Arginine di‐methylation of Smad7 was detected by Western blotting analysis. C) Mass spectrum of Smad7 Arg‐57 dimethylated peptide. Mass spectrometry identified Arg‐57 dimethylation of Smad7 in HEK293T cells expressing MYC‐PRMT5/MEP50 and SFB‐Smad7. Mass spectrometry profile of Smad7 sequence covering residue 47–64 is shown, and the dimethylated arginine side chains are indicated. D) PRMT5/MEP50 methylate Smad7, but not the R57K mutant. HEK293T cells were transfected MYC‐PRMT5/MEP50 and SFB‐Smad7 or Smad7 R57K mutant for 36 h. Cell lysates were harvested and immunoprecipitated with SYM10 antibody. The immunocomplexes and inputs were analyzed by Western blotting with indicated antibodies. E) PRMT5 methylates Smad7 in vitro. MYC‐PRMT5 or MYC‐G367A/R368A together with MYC‐MEP50 were immunopurified using anti‐MYC antibody from transfected HEK293T cells. Purified recombinant GST‐Smad7, GST‐Smad7 R57K mutant, and GST‐Smad4 were produced in E. coli . GST proteins and MYC‐PRMT5/MEP50 proteins were incubated in the presence of S‐adenosyl‐methionine to allow methylation reaction. Dimethylated Smad7 on R57 was detected by using Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Transfection, Expressing, Construct, Western Blot, Residue, Methylation, Mass Spectrometry, Sequencing, Mutagenesis, Immunoprecipitation, In Vitro, Purification, Recombinant, Produced, Incubation

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: Arg methylation enhances Smad7 binding to gp130. A) Smad7 methylation increases its association with gp130. HEK293T cells were transfected with SFB‐Smad7 or Smad7 R57K mutant and HA‐gp130, together with MYC‐PRMT5/MEP50. Cell lysates were harvested and immunoprecipitated with Streptavidin beads. Western blotting analysis was done with appropriate antibodies. B) PRMT5 depletion blocks Smad7 methylation and its interaction with endogenous gp130. A549 tet‐on cells expressing SFB‐Smad7 were cultured with or without 1 µg mL −1 Dox for 3 d and then transfected with 40 × 10 −12 m siPRMT5. Cell lysates were harvested and immunoprecipitated with streptavidin beads. Endogenous gp130 was detected from the immunoprecipitates by using Western blotting analysis. C) Methylated Smad7 binds more tightly to gp130. HEK293T cells were transfected with indicated expression plasmids for MYC‐PRMT5, MYC‐G367A/R368A, and MYC‐MEP50 as well as SFB‐Smad7 or SFB‐R57K. Dimethylated Smad7 was immunopurified using SYM10 antibody, while total Smad7 was retrieved using an‐FLAG antibody. Bacterially expressed GST‐gp130‐ICD was purified using glutathione‐sepharose and eluted with elution buffer (10 × 10 −3 m glutathione, pH 8.0). In the in vitro binding experiments for evaluating the Smad7‐gp130 interaction, recombinant GST‐gp130‐ICD was added to the immunopurified Smad7. gp130‐ICD binding to immobilized Smad7 was analyzed by using Western blotting. D) Unmethylatable Smad7 R57K mutant loses its ability to potentiate STAT3 activation. MCF10A tet‐on cells stably expressing SFB‐Smad7 or Smad7 R57K were induced with 10 ng mL −1 Dox for 3 d, and treated with indicated concentrations of IL‐6. Cell lysates were collected and subject to Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Methylation, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Cell Culture, Purification, In Vitro, Recombinant, Activation Assay, Stable Transfection

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes STAT3 transcriptional and growth‐promoting responses. A) PRMT5 inhibition attenuates CDC25C expression in A549 cells. EPZ015666 or GSK591 (20 × 10 −6 m ) were added to A549 cells for 48 h. Cell lysates were harvested and analyzed by using qRT‐PCR to examine CDC25C mRNA levels. Data are shown as mean ± SD; n = 3. *** P < 0.001. B) PRMT5 inhibition attenuates CCNB1 expression in A549 cells. Cell treatment, harvest, and qRT‐PCR analysis were done as described in Panel A. Data are shown as mean ± SD; n = 3. *** P < 0.001. C) PRMT5 deficiency disables IL‐6/STAT3 responsiveness. GSEA showed that downregulated genes in PRMT5‐depleted A549 cells (shPRMT5‐2) were highly enriched in the IL‐6/STAT3 signaling gene set. Red, upregulated genes; blue, downregulated genes. NES = ‐1.73, FDR q value = 0.002. D) Heatmap showing expression levels (log 2 FPKM; left) and relative expression changes (log 2 (shPRMT5‐2/shCtrl); right) of the IL‐6/STAT3 signaling genes. E) Depletion of PRMT5 reduces DNA synthesis. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were subject to EdU staining to determine DNA incorporating rate (RiboBio),20x. F) Statistic analysis of the result in panel E. Data are shown as mean ± SD; n = 3. 0.01 < * P < 0.05. G) Inhibition of PRMT5 attenuates invasiveness in A549 cells. A549 cells were treated with PRMT5 inhibitors EPZ015666 or GSK591 (20 × 10 −6 m ) for 2 d, starved overnight in FBS‐free medium. 1 × 10 5 cells were plated in a transwell chamber and stained with crystal violet after 12 h. Purple color indicates crystal violet staining of the invaded cell population. H) PRMT5 depletion blocks colony formation. A549 stable cells were subject to crystal violet staining and photography.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, DNA Synthesis, Stable Transfection, Control, Staining

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes lung tumorigenesis. A) Depletion of PRMT5 attenuates tumorigenesis. LLC cells stably expressing shControl or mouse sh‐mPRMT5‐1 or sh‐mPRMT5‐2 were subcutaneously injected into female nude mice. Ten days after implantation, tumors were dissected and photographed. B) Measurement of tumor weight in Panel A. Data are shown as mean ± SD; n = 5 for each group. 0.01 < * P < 0.05. C) PRMT5 depletion impairs STAT3 signaling in tumors. Tumor samples were analyzed by Western blotting to examine phosphorylated STAT3 (p‐STAT3) and STAT3 target gene products such as c‐Myc and Survivin. D) PRMT5 is highly expressed in nonsmall cell lung cancer tissues (NSCLC). NSCLC tissue microarray (Alenabio) was subject to immunohistochemistry (Servicebio) using PRMT5 antibody. E) Statistic analysis of IHC score in Panel D. Statistical analysis was performed using a two‐tailed Student's t ‐test. Data are shown as mean ± SD. Lung cancer samples = 45. Normal lung tissue samples = 55. *** P < 0.001. F) A working model for PRMT5‐mediated STAT3 activation.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Stable Transfection, Expressing, Injection, Western Blot, Microarray, Immunohistochemistry, Two Tailed Test, Activation Assay