Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 regulates STAT3 activation. ( A ) NIH-3T3 cells were transfected with either the mock or Ssu72 overexpression vector and stimulated with IL-6 (20 ng/ml) for 1 h. Cells were used to detect the p-STAT3 Tyr705 and p-STAT3 Ser727 levels. ( B ) NIH-3T3 cells were stained with DAPI (blue) and antibodies against tubulin (green) and p-STAT3 Tyr705 (red). Confocal scanning microscopy was performed to detect the p-STAT3 Tyr705 and p-STAT3 Ser727 levels in the transfected cells. ( C ) NIH-3T3 cells were transfected with the expression vector and stimulated with IL-6 (20 ng/ml) for 1 h. ( D ) Cells were transfected with STAT3 overexpression vectors and either the mock or Ssu72 overexpression vector. The expression levels of the Il17a mRNA were measured using real-time PCR. ( E ) NIH-3T3 cells were transfected with the Il17a promoter construct and either mock or Ssu72 expression vectors. Luciferase activity was then detected. ( F ) Lysates from the transfected NIH-3T3 cells were immunoprecipitated with the anti-FLAG antibody and immunoblotted with anti-p-STAT3 Tyr705, anti-p-STAT3, and anti-Ssu72 antibodies. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, ***P < 0.01).

Article Snippet: An siRNA construct targeting the Ssu72 mRNA and a non-targeting siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with the transfection system to knockdown Ssu72 expression.

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Staining, Microscopy, Expressing, Real-time Polymerase Chain Reaction, Construct, Luciferase, Activity Assay, Immunoprecipitation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 controls inflammatory responses in vitro . ( A ) NIH-3T3 cells were transfected with a control siRNA or Ssu72 siRNA and stimulated with IL-6 (20 ng/ml) for 0.5 h. Cells were used to examine the p-STAT3 Tyr705 and p-STAT3 Ser727 levels. ( B ) Expression levels of the Ssu72 mRNA in cells transfected with the siRNAs were measured by real-time PCR. ( C ) NIH-3T3 cells were transfected with the Il17a promoter construct and either the siRNA control or siRNA Ssu72 to detect luciferase activity. ( D ) NIH-3T3 cells were transfected with siRNAs and stimulated with IL-6 (20 ng/ml) for 0.5 h. Real-time PCR was performed to measure the expression levels of the IL-1β , IL-17A , IL-21 , TBK1 , and IKBKE mRNAs. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01).

Article Snippet: An siRNA construct targeting the Ssu72 mRNA and a non-targeting siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with the transfection system to knockdown Ssu72 expression.

Techniques: In Vitro, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction, Construct, Luciferase, Activity Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 ameliorates the development of CIA. ( A ) The Ssu72 overexpression or mock vector was administered systemically to mice with CIA once per week. The clinical scores and incidence of CIA were measured in these mice (***P < 0.01, n = 10). ( B ) Total IgG levels were measured in each group (***P < 0.01, n = 10). ( C ) The levels of p-STAT3 Tyr705 and p-STAT3 Ser727 in splenocytes from mice with CIA (mock or Ssu72-overexpressing) that had been stimulated with IL-6 (20 ng/ml) for 1 h were examined by western blot analysis. ( D ) Joint tissues from mice with CIA were stained with hematoxylin and eosin (original magnification, 40× or 200×, n = 6) and Safranin O (original magnification, 40×, n = 6). ( E ) Immunohistochemical staining was used to detect IL-6, IL-21, IL-17, IL-1β, TNF-α and RANKL in the synovium of mice with CIA (mock or Ssu72-overexpressing; (original magnification, 200× or 400×, n = 6). The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01).

Article Snippet: An siRNA construct targeting the Ssu72 mRNA and a non-targeting siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with the transfection system to knockdown Ssu72 expression.

Techniques: Over Expression, Plasmid Preparation, Western Blot, Staining, Immunohistochemical staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 inhibits the progression of osteoclastogenesis. ( A ) TRAP expression in the synovium of mice with CIA (mock or Ssu72-overexpressing) was observed using immunohistochemical staining (original magnification, 200× or 400×, n = 6). ( B and C ) Bone marrow cells from mice with CIA (mock or Ssu72-overexpressing) were cultured with macrophage colony-stimulating factor (M-CSF) (10 ng/ml) and RANKL (50 ng/ml). Cells were fixed, stained for TRAP, and the number of TRAP + cells was counted using a light microscope (original magnification 100×, n = 6). Real-time PCR was performed to measure the relative mRNA levels of osteoclastogenic markers (n = 6). The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01).

Article Snippet: An siRNA construct targeting the Ssu72 mRNA and a non-targeting siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with the transfection system to knockdown Ssu72 expression.

Techniques: Expressing, Immunohistochemical staining, Staining, Cell Culture, Light Microscopy, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 regulates the reciprocal balance between Th17 and Treg cells in mice with CIA. ( A ) Populations of IL-17-, CD25-, and Foxp3-producing CD4 + T cells were analyzed by intracellular flow cytometry. ( B ) Spleens from mice with CIA (mock or Ssu72-overexpressing) were subjected to immunostaining to detect the CD4 + IL-17 + , CD4 + CD25 + FOXP3 + and CD4 + Ssu72 + cells (scale bar, 10 μm). Cell numbers were counted in four independent quadrants. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01, n = 6).

Article Snippet: An siRNA construct targeting the Ssu72 mRNA and a non-targeting siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with the transfection system to knockdown Ssu72 expression.

Techniques: Flow Cytometry, Immunostaining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 reduces STAT3 activation and Th17 cell differentiation. ( A ) Splenocytes from mice with CIA were stimulated with IL-6 (20 ng/ml) for 1 h and then treated with either GST or GST-Ssu72. ( B ) Splenocytes from mice with CIA were cultured under Th17 conditions for 72 h and then the number of CD4 + IL-17 + cells was quantified. Statistical analyses were conducted using one-way ANOVA with Bonferroni’s post hoc test. ( C ) IL-17 and TNF-α expression in culture supernatants was measured using ELISA. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01, n = 5).

Article Snippet: An siRNA construct targeting the Ssu72 mRNA and a non-targeting siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with the transfection system to knockdown Ssu72 expression.

Techniques: Activation Assay, Cell Differentiation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 downregulates Th17 cell differentiation and IL-17 level. ( A ) CD4 + T cells from CIA mice splenocytes were cultured under Th17 conditions for 72 h and then the number of CD4 + IL-17 + cells was quantified. Statistical analyses were conducted using one-way ANOVA with Bonferroni’s post hoc test. ( C ) Concentration of IL-17 and TNF-α in culture supernatants was measured using ELISA. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01, n = 5).

Article Snippet: An siRNA construct targeting the Ssu72 mRNA and a non-targeting siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used with the transfection system to knockdown Ssu72 expression.

Techniques: Cell Differentiation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: HIV-1 Tat interacts directly with the human Ssu72 CTD phosphatase. ( A ) MudPIT analysis of affinity-purified HA-Tat101-associated protein complexes from a stable Tet-OFF/Tat-ON HeLa cell line. ( B ) Immunoblot analysis of anti-HA immunoprecipitates from HA-Tat HeLa stable cells with the indicated antibodies. ( C ) Immunoblot analysis of anti-Tat serum immunoprecipitates from 2D10 Jurkat cells before and after TNF-α treatment. ( D ) Recombinant S-tagged Ssu72 and S-tagged CycT1 (amino acids 1–303) were incubated with recombinant GST-Tat101. The recovered pull-down products (PD) were detected by immunoblot analysis with anti-GST antibody (to detect GST and GST-Tat proteins) and HRP-conjugated S-protein (to detect Ssu72 and Cyclin T1). ( E ) The pull-down assays were performed as in D for S-tagged Ssu72, with indicated GST-Tat fragments. The amounts of GST and GST-Tat proteins were monitored by Ponceau staining (PS). The regions of Tat binding to Ssu72 are shown in the schematic diagram at the bottom of the panel. ( F ) Recombinant full-length and truncated GST-Ssu72 were incubated with recombinant Flag-Tat101 (GST was cleaved after purification). The recovered pull-down products were detected by immunoblot with anti-Flag (to detect Flag-Tat101) and anti-GST (to detect GST and GST-Ssu72) antibodies. Arrowheads indicate GST-Ssu72 fusion proteins. ( G ) HA-Tat HeLa stable cells were transfected with Flag-tagged full-length or coiled-coil domain truncated (∆CC) Ssu72. The immunoprecipitation experiments were performed as in B . The region of Ssu72 binding to Tat is shown in the schematic diagram at the bottom of the panel. ( H ) Recombinant GST-CycT1 (amino acids 1–303) was incubated with recombinant S-tagged Ssu72 in the absence or presence of recombinant Flag-Tat101. The pull-down products were detected by immunoblot with the indicated antibodies.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Affinity Purification, Western Blot, Recombinant, Incubation, Staining, Binding Assay, Purification, Transfection, Immunoprecipitation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Ssu72 synergizes with Tat to activate HIV-1 transcription. ( A ) Luciferase activities were measured in extracts of HIV-1:Luc HeLa cells transfected with the indicated constructs. The protein expression was monitored by immunoblot analysis. ( B ) Luciferase activities were measured in extracts transfected with the indicated amounts of Ssu72 expression constructs in the presence or absence of Tat. The protein lysates were analyzed by immunoblot with the indicated antibodies. ( C ) Luciferase assays were performed as in B to compare the activity of wild type or catalytically inactive mutants (C12S and D143A) of Ssu72. ( D ) Immunoblot analysis of anti-HA immunoprecipitates from HA-Tat HeLa stable cells transfected with Flag-tagged Ssu72 (wild type, C12S, and D143A). ( E ) Luciferase assays were performed as in B to analyze the activity of full-length Ssu72 and the Ssu72∆CC mutant. ( F ) Wild-type Ssu72 or Ssu72 C12S expression constructs were transfected in the HIV-1:Luc HeLa cells in the absence or presence of Tat. Nuclear run-on analysis was performed as described in the Materials and Methods. Error bars represent the standard deviation obtained from three independent experiments.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Luciferase, Transfection, Construct, Expressing, Western Blot, Activity Assay, Mutagenesis, Standard Deviation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Tat stimulates Ssu72 CTD phosphatase activity in vitro. ( A ) TFIIH-phosphorylated GST-CTD was incubated with the indicated amounts of recombinant human GST-Ssu72 or catalytically inactive GST-Ssu72 (C12S) in the presence of GST or GST-Tat86. The phosphatase assay products were detected with the indicated antibodies by immunoblot analysis. ( B ) Kinetics of Tat-stimulated Ssu72 phosphatase activity were monitored for the indicated times. The intensity of the S5P bands was quantitated and plotted. The half-time for reduction of S5P levels in the absence or presence of GST-Tat86 was calculated as described in the Supplemental Material. ( C ) The phosphatase assays were performed as in A except that the indicated amounts of GST-Tat86 were included. ( D ) In vitro purified recombinant S-tagged Ssu72 was incubated with GST, wild-type GST-Tat86, or mutant GST-Tat86 proteins as indicated. The recovered pull-down products were subjected to immunoblot analysis with anti-GST (to detect GST or GST-Tat86) or anti-Ssu72 antibodies. The amounts of GST and GST-Tat86 proteins were monitored by Commassie staining. ( E ) The phosphatase assays were performed as in A for GST-Ssu72 except that the wild-type or mutant GST-Tat86 proteins were used, as indicated.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Activity Assay, In Vitro, Incubation, Recombinant, Phosphatase Assay, Western Blot, Purification, Mutagenesis, Staining

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Ssu72 is essential for Tat:P-TEFb to phosphorylate the S5P-CTD in vitro. ( A ) The GST-CTD (52 repeats) substrate was incubated with indicated amounts of P-TEFb in the absence or presence of Tat (GST-Tat86). CTD phosphorylation was monitored by immunoblot with the indicated antibodies. ( B ) In vitro phosphorylation of the TFIIH:CDK7-phosphorylated GST-CTD substrate by recombinant Tat:P-TEFb complexes was monitored in the presence or absence of recombinant Ssu72, as indicated. The experimental procedure is outlined above the panel, and CTD phosphorylation was monitored by immunoblot with the indicated antibodies. Note that each reaction contained identical levels of the S5P-CTD substrate.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: In Vitro, Incubation, Phospho-proteomics, Western Blot, Recombinant

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Ssu72 promotes Tat transactivation and regulates S5P-CTD levels at the HIV-1 promoter in vivo. ( A ) Luciferase activities were measured in extracts of HIV-1:Luc HeLa cells transfected with different siRNAs against Ssu72, Cyclin T1, or AFF1 in the presence or absence of Tat101. The efficiency of siRNA-mediated knockdown was monitored by immunoblot. ( B ) HIV-1:Luc HeLa cells were transfected with siRNAs specific for Ssu72, RPAP2, or SCP1 in the presence or absence of Tat, as indicated. Luciferase assays were performed as in A . ( C ) HIV-1:Luc HeLa cells were first transfected with control or Ssu72 siRNAs for 24 h and then were transfected with 5 µg of EGFP or EGFP-Tat101 for another 24 h. The cell extracts were subjected to ChIP analysis with the indicated antibodies. Error bars represent the standard deviation obtained from three independent experiments. Protein lysates were monitored by immunoblot with the indicated antibodies.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: In Vivo, Luciferase, Transfection, Knockdown, Western Blot, Control, Standard Deviation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: Tat recruits Ssu72 to the integrated HIV-1 proviral promoter in 2D10 T cells. ( A ) Immunoblot analysis of Tat and cellular protein expression in TNF-α treated 2D10 cells for the indicated times. ( B ) Immunoblot analysis of protein expression of 2D10 cells transfected with Ssu72 or Cyclin T1 siRNAs with the indicated antibodies. ( C ) ChIP analysis of the recruitment of virus-encoded Tat and host cell factors to the integrated provirus in TNF-α-induced 2D10 cells. Schematic representation of the genomic organization of the lentiviral vector and the relative locations of the primers used for ChIP are shown at the top of the panel. ( D ) Model depicting three different steps regulated by Tat to facilitate the transition from promoter clearance to early elongation at the HIV-1 promoter. See the Discussion for details.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: Western Blot, Expressing, Transfection, Virus, Plasmid Preparation

Journal: Genes & Development

Article Title: A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

doi: 10.1101/gad.250449.114

Figure Lengend Snippet: ChIP-seq and GRO-seq analysis of Ssu72 in hESCs. ( A , left ) Genomic distribution of Ssu72 ChIP-seq peaks. ( Right ) Metagene representation of the Ssu72 ChIP-seq profile. ( B ) The graph at the left shows a statistically significant (CC = 0.88; P -value < 1 × 10 −100 ) correlation between the S5P–RNAPII and Ssu72 ChIP-seq peaks around gene TSSs. The right panel shows a heat map analysis of the correlation of Ssu72 and S5P–RNAPII occupancy. ( C ) Ssu72 and S5P–RNAPII binding at the POU5F1 and NANOG genes, as captured from the Integrative Genomics Viewer genome browser. Images show visualization of WIG files. Gene diagrams are shown at the bottom , with scale bars above . ( D ) Immunoblot from H1 hESCs transfected with control (Ctrl) or Ssu72 siRNAs for 48 h. DDX39 antibody was used as a loading control. ( E ) GRO-seq read count covering 10 kb from the gene TTS in siCtrl and siSsu72 hESCs, as indicated. The read counts from both the sense and antisense strands are plotted, as indicated in the legend.

Article Snippet: Five micrograms of Ssu72 (Genetex, GTX116436) or 3 μg of S5P-RNAPII (Millipore, 05-623) antibody was added, and the samples were incubated overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Transfection, Control