Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-c , CD8 + T cells were treated with EPZ004777 for 48 hours. Western Blot showed H3K79me2 in CD8 + T cells ( a ). FACS demonstrated CD8 + T cell apoptosis ( b ) and cytokines ( c ). d , Western Blot showed H3K79me2 in Dot1l f/f and Dot1l −/− CD8 + T cells. e, f , Effect of DOT1L deletion on CD8 + T cells apoptosis ( e ) and cytokines ( f ). g-i , Effect of T cell DOT1L deficiency on MC38 growth ( g, h ) and T cell viability ( i ). j, k , Effect of methionine supplementation on Dot1l f/f and Dot1l −/− CD8 + T cells apoptosis ( j ) and cytokines ( k ). l , GSEA plot showed enriched apoptotic pathway in Dot1l −/− CD8 + T cells. m , Heat map showed Jak-Stats mRNA levels in mouse Dot1l −/− vs Dot1l f/f CD8 + T cells. n , Western blot showed STAT5 and p-STAT5 in Dot1l f/f ( f/f ) and Dot1l −/− (−/−) CD8 + T cells. o , Western blot showed STAT5 and p-STAT5 in CD8 + T cells cultured with fresh medium (FM), Supernatant (Sup), and Sup supplemented with different metabolites (Sup+). p , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells. q , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells cultured with FM, Sup, or Sup+Met. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Western Blot, Cell Culture

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a, b , H3K79me2 ( a ) and STAT5 ( b ) levels in CD8 + T cells from tumor draining lymph node (dLN) and tumor in B16F10 bearing mice. c, d , H3K79me2 ( c ) and STAT5 ( d ) levels in CD8 + T cells from spleen and tumor ascites in ID8 bearing mice. e , H3K79me2 levels in CD8 + T cells from healthy peripheral blood and human ovarian cancers ascites. f, g , H3K79me2 ( f ) and STAT5 ( g ) levels in CD8 + T cells from healthy human blood and human ovarian cancer omentum tissues. h, i , FACS showed H3K79me2 and STAT5 levels in human tumor infiltrating CD8 + T cells. j-m , Effect of methionine on human tumor infiltrating CD8 + T cells. Human colorectal cancer infiltrating CD8 + T cells were cultured with or without methionine. T cell cytokine production ( j, k ), H3K79me2 ( l ), and STAT5 ( m ) were analyzed by FACS. One representative of four is shown. n , Effect of methionine supplementation on apoptosis of tumor infiltrating CD8 + T cells and ID8 tumor cells in vivo. ID8 tumor bearing mice were treated with methionine or PBS. T cell and tumor cell apoptosis was determined by FACS. o , Methionine levels in ID8 tumor after methionine or PBS treatment. p-r: Effect of anti-PD-L1 on methionine-affected CT26 tumor progression. Mice bearing CT26 tumor were treated with anti-PD-L1, methionine, and their combination. Tumor volume ( p ), T cell tumor infiltration ( q ) and apoptosis ( r ) were assessed. Data are mean ± s.e.m. Information on sample sizes, experimental number, times, biological replicates, statistical tests, and P values is available in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Cell Culture, In Vivo

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-e , Methionine supplementation restored T cell immunity in B16F10-bearing mice. Tumor growth ( a ), tumor infiltrating CD8 + T cell H3K79me2 ( b ) and STAT5 ( c ) were monitored. FACS showed intratumor CD8 + T cell apoptosis ( d ) and cytokines ( e ) . f-h , Methionine supplementation restored T cell immunity in ID8-bearing mice. Tumor growth was monitored by bioluminescence imaging ( f ). FACS showed ascites CD8 + T cell ( g ) and intratumor CD8 + T cell cytokines ( h ) . i-l , Studies on colorectal cancer patients treated with methionine. Western blot showed p-STAT5 and H3K79me2 in peripheral CD8 + T cells prior and post methionine treatment ( i ). FACS showed IL-2 + T cells ( j ), CD8 + T cell effector cytokines ( k ) and apoptosis ( l ) in patients prior and post methionine treatment. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Imaging, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding

Journal: Journal of Biological Chemistry

Article Title: Interleukin-2 Receptor β Thr-450 Phosphorylation Is a Positive Regulator for Receptor Complex Stability and Activation of Signaling Molecules

doi: 10.1074/jbc.m115.660654

Figure Lengend Snippet: FIGURE6.IL-2RThr-450phosphorylationisapositiveregulatorforactivationofdownstreamsignalingmolecules,receptorcomplexformation,and STAT5B transcriptional activity in a reconstituted HEK293 cell system. HEK293 cells were transfected with c, JAK3, STAT5B, and IL-2R (WT or T450A) and incubated for 48 h in complete medium, followed by 48 h of incubation in 1% FBS medium. Transfected cells were stimulated without () or with IL-2 () for 10 min. A, whole cell lysates were separated by SDS-PAGE and Western-blotted as indicated. B, IL-2R was immunoprecipitated (IP), separated by SDS-PAGE, and analyzed by WB for coimmunoprecipitation of JAK3, STAT5, and c as indicated. Input controls for immunoprecipitated JAK3 and STAT5 are indicated. Representative data from three independent experiments are shown. C, HEK293 cells were transfected with IL-2R (WT or T450A), c, JAK3, STAT5B, -casein- luciferase, and pCMV--galactosidase. 48 h post-transfection, cells were incubated with or without IL-2 for an additional 48 h, and luciferase activity was measured and normalized to -gal activity. Each treatment was performed in triplicate. Results are presented as the mean S.D. of three independent experiments. Statistical significance was determined using Student’s t test. *, p 0.05.

Article Snippet: The pcDNA3.1 human JAK3 and STAT5B cDNAs (OriGene) were obtained as described previously (31, 34).

Techniques: Activity Assay, Transfection, Incubation, SDS Page, Western Blot, Immunoprecipitation, Luciferase

Journal: Nature Communications

Article Title: Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation

doi: 10.1038/s41467-022-33733-8

Figure Lengend Snippet: a RT-PCR analysis and b Western blotting of Stat5, DBP and E2F8 in WT and Tgfbr1 KO CD4 + T cells cultured with TGF-β, IL-4 or TGF-β plus IL-4. Expression of Stat5a, DBP and E2F8 during TGF-β and IL-4 stimulation in WT, S213A- or EPSM-Smad3 mutants transfected CD4 + T cells c , d or p38 inhibitor-pretreated CD4 + T cells e , f as in a , b . a , c , e The data were representative of four independent experiments or b , d , f three experiments. Data were analyzed by two-way ANOVA with Tukey’s test. Graphs show the mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: For overexpression, pCMV6- Stat5a (Origene), pPM-C-HA- Dbp (Abcam) and Myc-DDK-tagged E2f8 (Origene) were used.

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

Journal: Nature Communications

Article Title: Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation

doi: 10.1038/s41467-022-33733-8

Figure Lengend Snippet: a Identification of Stat5a, DBP and E2F8 binding elements in the Il9 gene. b – d ChIP assay analysis for binding of Stat5a, DBP and E2F8 to the promoter region of the Il9 during Th9 differentiation. e , g Expression of Il9 mRNA and f , h ELISA of IL-9 and in CD4 + T cells transfected with Stat5a , Dbp or E2f8 specific shRNA e , f or overexpressing virus g, h followed by stimulation with TGF-β plus IL-4. O/E, Overexpressed. b – d , f These data were representative of three independent experiments or e , g , h four experiments. These data were analyzed by b , d two-tailed unpaired Student’s t test or e – h one-way ANOVA with Tukey’s test. Graphs show the mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: For overexpression, pCMV6- Stat5a (Origene), pPM-C-HA- Dbp (Abcam) and Myc-DDK-tagged E2f8 (Origene) were used.

Techniques: Binding Assay, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, shRNA, Virus, Two Tailed Test

Journal: Nature Communications

Article Title: Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation

doi: 10.1038/s41467-022-33733-8

Figure Lengend Snippet: a RT-PCR analysis and b Western blotting of STAT5, DBP and E2F8 in human CD4 + T cells cultured with TGF-β plus IL-4 for 5 days. c Expression of Il9 mRNA after 3 days and d ELISA of IL-9 after 5 days in human CD4 + T cells transfected with STAT5A , DBP or E2F8 specific siRNA, followed by stimulation with TGF-β plus IL-4. e Western blotting of STAT5, DBP and E2F8 and f IL-9 protein were measured by ELISA in human CD4 + T cells cultured with TGF-β plus IL-4 with or without p38 inhibitor for 5 days. a , c , f These data are representative of four independent experiments or b , d , e three experiments. These data were analyzed by a two-tailed unpaired Student’s t -test or c , d , f one-way ANOVA with Tukey’s test. Graphs show the mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: For overexpression, pCMV6- Stat5a (Origene), pPM-C-HA- Dbp (Abcam) and Myc-DDK-tagged E2f8 (Origene) were used.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test