anti ack685 stat3  (Cell Signaling Technology Inc)

Journal: ACS Omega

Article Title: Nicotinamide N -Methyltransferase Remodeled Cell Metabolism and Aggravated Proinflammatory Responses by Activating STAT3/IL1β/PGE 2 Pathway

doi: 10.1021/acsomega.2c04286

Figure Lengend Snippet: NNMT activated STAT3 and proteomic analysis was performed for Ctrl and NNMT-OE cells. (A) Western blotting analysis for STAT3, STAT3 AcK685, STAT3 pS727, and β-actin expression levels in Ctrl and NNMT-OE cells. (B) Abundance distribution of proteins from all the biological replicates. (C) Volcano plots revealed significantly changed proteins between Ctrl and NNMT-OE cells, in which blue dots in upper left and upper right sections represent proteins with fold-change <0.77 or >1.3 and p values <0.05 from t test statistics. (D) Heatmap clustering analysis showed hierarchical patterns and differentially expressed proteins (top 100) in Ctrl and NNMT-OE A549 cells. (E) IPA analysis for those differentially expressed proteins.

Article Snippet: Primary anti-NNMT, anti-ZO-1, anti-Snail, anti-Cytokeratin-18, anti-Stat3, anti-Stat3 pS727, anti-Stat3 AcK685, anti-TGFβ2 (Proteintech, Rosemont, USA), anti-HPGD (Bioworld), anti-COX2, anti-E-cadherin, anti-N-cadherin (Cell Signaling Technology), and secondary antirabbit HRP-IgG antibodies (CST, Danvers, MA) were used for immunoblotting.

Techniques: Western Blot, Expressing

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a , b The protein levels of CREPT and p-STAT3 in human breast cancers ( a ) and colon cancers ( b ). N refers to paired normal tissue and T refers to tumour tissue from the same patient. The bands were quantified using ImageJ and presented as a value normalised according to the moderate level of a band in each blot. c A graph presentation of CREPT/actin and p-STAT3/STAT3 in adjacent normal and tumour tissues. The grey values in a , b were used to calculate the ratio of CREPT/actin or p-STAT3/STAT3. Each sample was dotted by closed circle (normal tissue for CREPT/actin), square (tumor tissue for CREPT/actin), triangle (normal tissue for p-STAT3/STAT3) and inverted triangle (tumor tissue for p-STAT3/STAT3), and the average was subject to a statistic analysis between normal and tumour tissues (* p < 0.05). d , e Representative images for CREPT and p-STAT3 levels using immunohistochemical staining in breast ( d ) and colon ( e ) cancers with an antibody against CREPT or p-STAT3(Y705). f , g Representative images for CREPT and p-STAT3 in breast ( f ) and colon ( g ) tumour tissue and adjacent normal tissue.

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Immunohistochemical staining, Staining

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a – d Overexpression of CREPT promotes the APRE-luciferase activity. Luciferase assay was performed in wild-type or CREPT transiently overexpressing cells in the absence or presence of LIF (20 ng/ml) (** p < 0.01; *** p < 0.001). e – h Depletion of CREPT using a mixture of siRNA#1 and siRNA#2 resulted in a decreased transcriptional activity of STAT3 (* p < 0.05; ** p < 0.01; *** p < 0.001). i CREPT promotes the expression of STAT3-targeted genes. Flag-CREPT plasmid was transfected into MCF7 cells with or without LIF (20 ng/ml) for 4 h. The mRNA levels of STAT3-targeted genes were examined by qRT-PCR. j Depletion of CREPT using a mixture of siRNA#1 and siRNA#2 impairs the expression of STAT3-targeted genes in response to LIF. k Overexpression of CREPT enhanced the protein levels of STAT3-targeted genes. Flag-CREPT plasmid was transfected into MCF7 (left) or SW480 (right) cells with or without LIF (20 ng/ml) for 12 h. The protein levels of STAT3-targeted genes were examined by Western blots. l Depletion of CREPT decreased the protein levels of STAT3-targeted genes.

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Over Expression, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a Myc-CREPT interacts with Flag-STAT3 in response to LIF. b The interaction of CREPT and STAT3 occurs in the nucleus. The nuclear marker JunB and the cytoplasmic marker tubulin were used to demonstrate the purity of fractions. c Interaction of CREPT with STAT3 depends on STAT3 phosphorylation rather than its nuclear localisation. d , e CREPT interacts with STAT3 physically. A GST pull-down assay was performed with purified GST or GST-CREPT protein and Flag-STAT3 from HEK293T cell lysates ( d ). A reciprocal GST pull-down assay was performed with purified GST or GST-STAT3 protein and Flag-CREPT from HEK293T cell lysates ( e ). f , g Endogenous interaction between CREPT and STAT3. Nuclear extracts from MCF7 ( f ) or SW480 ( g ) cells were immunoprecipitated with an anti-CREPT antibody. h The RPR domain, but not CCT domain of CREPT interacts with Myc-STAT3. i CREPT preferentially interacts with the SH2-CT domain of STAT3. j CREPT co-localises with STAT3 upon LIF treatment. MCF7 cells were treated with LIF (20 ng/ml) or a control medium for 30 min, followed with immunostaining using an anti-CREPT antibody and an anti-STAT3 antibody. k CREPT and STAT3 co-occupied at the promoter region of STAT3-targeted genes. ChIP assay was performed with the CREPT or STAT3 antibody in MCF7 cells with or without the treatment of LIF (20 ng/ml) for 30 min.

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Marker, Pull Down Assay, Purification, Immunoprecipitation, Immunostaining

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a , b Deletion of CREPT decreases the level of ac-H3K27 ( a ) or ac-H3K18 ( b ) on the APRE region. c – e Loss of CREPT decreases chromatin accessibility at the promoter region of STAT3. ATAC-seq peaks at the promoter regions of the STAT3-targeted genes from wild-type (blue) or CREPT depletion (green) MCF7 cells were shown. The STAT3 binding consensus sequence was labelled at the corresponding locus. f , g The endogenous interaction between CREPT and p300. Nuclear extracts from MCF7 ( f ) or SW480 ( g ) cells were immunoprecipitated with an anti-CREPT antibody. h CREPT, STAT3 and p300 formed a ternary complex in MCF7 cells. Cell lysates were precipitated by an anti-CREPT antibody. i , j CREPT enhances the interaction of STAT3 and p300. Flag-STAT3 was co-expressed with Myc-CREPT ( I ) or siRNAs against endogenous CREPT ( j ) in MCF7 cells. k Overexpression of CREPT hardly affected the interaction between STAT3 and GCN5. l , m Deletion of CREPT attenuates p300 ( l ) or RNAPII ( m ) occupancy on the promoters of STAT3-targeted genes. The quantity indicating the presence of p300 ( l ) or RNAPII ( m ) on APRE was normalised with respective inputs. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Over Expression

Journal: British Journal of Cancer

Article Title: CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner

doi: 10.1038/s41416-021-01269-1

Figure Lengend Snippet: a , b Depletion of p300 impaired the enhanced effect of CREPT on STAT3 transcriptional activity. c – g Depletion of p300 impaired the STAT3-targeted gene expression promoted by CREPT both at mRNA ( c – e ) and protein ( f , g ) levels examined by QRT-PCR and western blot. h – k CREPT promotes colony formation dependent on p300. Colony formation assays were performed in MCF7 ( h , i ) or SW480 ( j , k ) cells. A representative colony is shown in ( h , J ) and the quantitative numbers are shown in ( i , k ). l A model indicating that highly expressed CREPT in tumour cells facilitates the recruitment of p300 to the promoter of STAT3-targeted genes (* p < 0.05; ** p < 0.01; *** p < 0.001; n.s., no statistical difference).

Article Snippet: Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology.

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

Journal: Cell Death Discovery

Article Title: Acetylation-dependent glutamate receptor GluR signalosome formation for STAT3 activation in both transcriptional and metabolism regulation

doi: 10.1038/s41420-020-00389-6

Figure Lengend Snippet: a 293 T cells were transfected with GluR1 or GluR1 plus GluR2. Cells were subjected to immunoprecipitation with Flag antibody followed by western blot with GluR2 and Flag antibodies. b 293 T cells were transfected with indicated plasmids followed with or without treatment of glutamate. Cells were subjected to immunoprecipitation with Flag antibody followed by western blot with GluR2 and Flag antibodies. c , d 293 T cells were transfected with indicated plasmids. Cells were subjected to immunoprecipitation with Flag antibody followed by western blot with Myc and Flag antibodies. e , f Cells were treated with either glutamate or AMPK for different time, cells were subjected to immunoprecipitation with Flag antibody followed by western blot with Myc and Flag antibodies. Data shown are representative of three independent experiments. g , h Mutation assay of acetylation sites that affected interaction between GluR1 and arrestin1/2. 293 T cells were transfected with indicated plasmids and then were treated with or without glutamate. Cells were subjected to immunoprecipitation with Myc antibody followed by western blot with Myc and Flag antibodies. i 293 T cells were transfected with indicated plasmids and then were treated with or without glutamate. Cells were subjected to immunoprecipitation with STAT3 antibody followed by western blot with Myc and Flag antibodies. Data shown are representative of two independent experiments. j 293 T cells were transfected with GluR2-GFP and Stat-RFP. Localization of GluR2 and STAT3 was observed by microscopy. Scale bar: 10 μm. Data shown are representative of three independent experiments.

Article Snippet: Acetylated-lysine antibody (9441), acK685-STAT3 antibody (2523), pS727-STAT3 antibody (9136), P70/85 S6 kinase antibody (9202), pERK1/2 antibody (9106), and ERK1/2 antibody (9102) were from Cell Signaling Technology (Boston).

Techniques: Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Microscopy

Journal: Cell Death Discovery

Article Title: Acetylation-dependent glutamate receptor GluR signalosome formation for STAT3 activation in both transcriptional and metabolism regulation

doi: 10.1038/s41420-020-00389-6

Figure Lengend Snippet: a , b U87 a and HepG2 b cells were treated with glutamate, followed by western blot with indicated antibodies. Data shown are representative of two independent experiments. c 293 T cells were transfected with STAT3 and treated with either calcium or glutamate, or both. Cells were lysed and blotted with indicated antibodies. Data shown are representative of three independent experiments. d C6 cells were treated with or without glutamate and followed by fractionation of cell. Cells were lysed and blotted with indicated antibodies. e C6 cells were treated with glutamate for different time, and then cells were immunostained with anti-STAT3 antibody. Cells were countstained with DAPI. Scale bar: 10 μm. Data shown are representative of two independent experiments. f C6 cells were treated with different combination of glutamate and PhTX-74. Cells were lysed and blotted with indicated antibodies. g C6 cells were treated with different combination of glutamate and PhTX-74. Cells were then lysed and fractionated, and blotted with indicated antibodies.

Article Snippet: Acetylated-lysine antibody (9441), acK685-STAT3 antibody (2523), pS727-STAT3 antibody (9136), P70/85 S6 kinase antibody (9202), pERK1/2 antibody (9106), and ERK1/2 antibody (9102) were from Cell Signaling Technology (Boston).

Techniques: Western Blot, Transfection, Fractionation

Journal: Cell Death Discovery

Article Title: Acetylation-dependent glutamate receptor GluR signalosome formation for STAT3 activation in both transcriptional and metabolism regulation

doi: 10.1038/s41420-020-00389-6

Figure Lengend Snippet: a C6 and Hela cells were treated with glutamine and cell proliferation was observed by microscopy. Scale bar: 50 μm. Data shown are representative of three independent experiments. b SH-SY5Y cells were with either glutamate or AMPK, cells were lysed and blotted with indicated antibodies. Data shown are representative of two independent experiments. c 293 T cells were transfected with indicated plasmids and then were treated with or without glutamate. Cells were lysed and blotted with indicated antibodies. d 293 T cells were transfected with GluR1 followed by treatment with glutamate or serum starvation. Cells were lysed and blotted with indicated antibodies. e 293 T cells were transfected with GluR2 followed by treatment with glutamate for different time. Cells were lysed and blotted with indicated antibodies. f Wild-type and STAT3 −/− MEF cells were treated with glutamate. Cell proliferation was observed by microscopy. Scale bar: 50 μm. Data shown are representative of two independent experiments.

Article Snippet: Acetylated-lysine antibody (9441), acK685-STAT3 antibody (2523), pS727-STAT3 antibody (9136), P70/85 S6 kinase antibody (9202), pERK1/2 antibody (9106), and ERK1/2 antibody (9102) were from Cell Signaling Technology (Boston).

Techniques: Microscopy, Transfection

Journal: Scientific Reports

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

doi: 10.1038/srep39517

Figure Lengend Snippet: ( a ) Time course of induction of STAT3 acetylation by serum starvation and reintroduction (SSR) (left) or serum starvation and insulin treatment (right) in PC3 cells transfected with STAT3. Pan acetyl-lysine antibody was used to detect acetyl-STAT3. ( b ) Alignment of C-terminal dimerization domain sequences from STAT1, STAT3, and STAT5b. K685, K707, and K709 are acetylation sites identified by mass spectrometry analysis of STAT3 prepared from PC3 cells transfected with STAT3. Y705 is phosphorylated after cytokine treatment. ( c ) SSR for 30 min (left) or insulin (5 μg/ml) for 15 min (right) induced STAT3 post-translational modifications, as indicated in MEFs that were serum-starved (−) for at least 6 hrs. ( d ) 293T cells were transfected with STAT3 or STAT3 and CBP, and this was followed by a 6-hr treatment with NAM (100 μM, 200 μM) or TSA (1 μM, 5 μM). Whole cell lysates were prepared and subjected to western blotting with the indicated antibodies. Acetyl-STAT3 was detected with anti-pan acetyl-lysine antibody. ( e ) CBP was depleted with siRNA in 293T cells. After insulin treatment for 30 min, WCL were prepared, and acetyl-STAT3 was blotted with the anti-pan acetyl-lysine antibody. ( f ) In 293T cells, CBP was transfected for 36 hrs before harvesting. Cytoplasmic, mitochondrial, and nuclear fractions were prepared for CBP and mitochondrial marker porin analysis with specific antibodies using western blotting. ( g ) MEFs were treated with insulin for 30 min or were left untreated. CBP was immunoprecipitated and subjected to blotting with anti-insulin receptor or anti-CBP antibodies. ( h ) In 293T cells, CBP was transfected for 36 hrs before harvest. Cytoplasmic, mitochondrial and nuclear fractions were prepared for STAT3 analysis.

Article Snippet: The anti-H3 antibody (3638), anti-pS727-STAT3 antibody (9136) and anti-acK685-STAT3 antibody (2523) were from Cell Signaling Technology (Boston).

Techniques: Transfection, Mass Spectrometry, Western Blot, Marker, Immunoprecipitation

Journal: Scientific Reports

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

doi: 10.1038/srep39517

Figure Lengend Snippet: ( a ) All MEFs were serum starved for 6 hrs before treatment with insulin (right) or SSR (left) for 30 min. Cell lysates of the cytoplasm (Cyt), mitochondria (Mit) and nuclei (Nuc) were prepared for western blotting analysis of STAT3 and other subcellular fraction markers. ( b ) MEFs were pretreated with cycloheximide or MG132, then subjected to insulin treatment for the indicated times. STAT3 or β-actin levels were analysed in the whole cell lysates by using western blotting. ( c ) Mass spectrometric analysis of STAT3 immunoprecipitated from STAT3-expressing 293T cells treated with insulin or left untreated. K87 was identified as the lysine residue of STAT3 that was acetylated after insulin treatment. ( d ) The mitochondrial fraction was prepared from MEFs treated with insulin or left untreated. Mitochondrial STAT3 was then analysed by western blotting. ( e ) Myc-tagged STAT3 was transfected alone or along with HDCA6 in 293T cells. Immunoprecipitated STAT3 was blotted with anti-aK87-STAT3 or anti-STAT3 antibodies, and HDAC6 was detected in the input. ( f ) Immunoprecipitated STAT3 was blotted with anti-aK87-STAT3 or anti-STAT3 in mitochondrial lysates from the above types of 293T cells. ( g ) 293T cells were transfected with an EV, wild-type Myc-STAT3, Myc-STAT3-Y705F and S727A mutants. Mitochondrial lysate was prepared under conditions of SSR for 30 min. STAT3 was analysed by western blotting with an anti-Myc antibody. ( h ) Myc-tagged full-length STAT3 or different domains, as indicated, were transfected alone or along with CBP in 293T cells. Mitochondrial lysates were prepared for STAT3 and porin blotting analysis. ( i ) PC3 cells were transfected with an EV, wild-type Myc-STAT3 or Myc-STAT3-3KR mutant, and treated with SSR for 30 min. STAT3 in cytoplasmic and mitochondrial lysates fractions was analysed by western blotting with an anti-Myc antibody. ( j ) 293T cells were transfected with wild-type Myc-STAT3 and Myc-STAT3-K87R mutant. Mitochondrial lysate was prepared under conditions of SSR or insulin treatment for 30 min.

Article Snippet: The anti-H3 antibody (3638), anti-pS727-STAT3 antibody (9136) and anti-acK685-STAT3 antibody (2523) were from Cell Signaling Technology (Boston).

Techniques: Western Blot, Immunoprecipitation, Expressing, Transfection, Mutagenesis

Journal: Scientific Reports

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

doi: 10.1038/srep39517

Figure Lengend Snippet: ( a ) Wild-type and STAT3−/− MEFs were treated with insulin for 30 min, and this was followed by immunoprecipitation of PDC E1. PDC E1 precipitates were subjected to western blotting for STAT3 co-immunoprecipitation. PDC E2 and PDC E3 were detected in the cell lysates. ( b ) Myc-STAT3 full length (FL) and Myc-STAT3 domains, as indicated, were cotransfected with PDC E1 in 293T cells, which were then subjected to by anti-Myc immunoprecipitation. STAT3 FL and STAT3 685-770 precipitated STAT3. ( c ) PDC E1 activity was measured in WT and STAT3−/− MEFs treated with insulin or left untreated. ( d ) 293T cells were transfected with an EV or Myc-tagged STAT3 fused with Cox4 MLS (Cox4-STAT3). Cytoplasmic (cyt) and mitochondrial lysates (mit) were analysed for Cox4-STAT3 acetylation and Cox4-STAT3 (anti-Myc) via western blotting. ( e ) Mitochondrial PDC E1 activity was measured in STAT3−/− MEFs transfected with an EV, Myc-STAT3, or Myc-Cox4-STAT3. ( f ) Mitochondrial PDC E1 activity was measured in STAT3−/− MEFs transfected with STAT3 WT, STAT3-3KR, STAT3-Y705F, or STAT3-S727A mutant. ( g ) Flag-STAT3 WT and Flag-STAT3 mutants, as indicated, were transiently transfected along with Myc-tagged PDC E1 in 293T cells. Anti-Myc immunoprecipitates were analysed for Flag-STAT3. ( h ) Acetyl-CoA content was measured in WT or STAT3−/− MEFs treated with insulin.

Article Snippet: The anti-H3 antibody (3638), anti-pS727-STAT3 antibody (9136) and anti-acK685-STAT3 antibody (2523) were from Cell Signaling Technology (Boston).

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Transfection, Mutagenesis

Journal: Scientific Reports

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

doi: 10.1038/srep39517

Figure Lengend Snippet: ( a ) In HeLa cells SIRT3, SIRT4, and SIRT5 were depleted with siRNA. Immunoprecipitated STAT3 was detected with anti-pan-acetyl-lysine antibody via western blotting. ( b ) A549 cells were transfected with control or SIRT5 shRNA. Elevated STAT3 acetylation was detected in cytoplasmic, mitochondrial, and nuclear fractions prepared from these cells. Tubulin, porin, and histone H3 were used as cytoplasmic, mitochondrial, and nuclear controls, respectively. ( c ) In 293T cells, STAT3 full length, 1–130, and 465–770 were cotransfected with Flag-SIRT5. STAT3 association was detected in Flag-SIRT5 immunoprecipitates via western blotting. ( d ) In 293T cells, Flag-STAT3 was cotransfected with an EV, CBP, CBP and SIRT3 or CBP and SIRT5. Acetylation of immunoprecipitated STAT3 was then detected with a pan-acetyl-lysine antibody. ( e ) In MEFs, STAT3 was cotransfected with Flag-SIRT5 WT or Flag-SIRT5 H158Y mutant, then subjected to insulin treatment for 30 min. STAT3 acetylation or phosphorylation was detected with the indicated antibodies. ( f ) A synthetic acetyl-peptide covering the STAT3 K685 site was incubated with Flag-SIRT5 or Flag-SIRT3 protein purified from 293T cells and NAD. Mass spectrometry analysis was then performed. ( g ) MEFs were transiently transfected with SIRT3 or SIRT5. STAT3 C-terminal acetylation induced by insulin was analysed with specific antibodies against STAT3 acetylation by western blotting, as indicated. SIRT3 and SIRT5 were blotted with anti-Flag; STAT3 was detected in the input.

Article Snippet: The anti-H3 antibody (3638), anti-pS727-STAT3 antibody (9136) and anti-acK685-STAT3 antibody (2523) were from Cell Signaling Technology (Boston).

Techniques: Immunoprecipitation, Western Blot, Transfection, shRNA, Mutagenesis, Incubation, Purification, Mass Spectrometry

Journal: Scientific Reports

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

doi: 10.1038/srep39517

Figure Lengend Snippet: ( a ) Wild-type or STAT3−/− MEFs were treated with insulin (5 μg/ml, 30 min) or NAM (100 μM, 6 hrs) and subjected to mitochondrial membrane potential analysis with JC-1 staining. ( b ) The mitochondrial membrane potential was measured in STAT3−/− MEFs transfected with STAT3 WT or mutants as indicated. ( c ) STAT3 WT or 3KR mutant MEFs were transfected with an EV, CBP and CBP together with SIRT5, and the mitochondrial membrane potential was measured. ( d ) PDC E1 activity (unit/10 4 cells) was measured in STAT3−/− MEFs transfected with an EV, CBP and CBP together with SIRT5. ( e ) ATP production was compared between STAT3 WT and STAT3−/− MEFs in response to treatment with insulin at the indicated concentrations. ( f ) In STAT3−/− MEFs, different forms of STAT3 were transiently transfected, and ATP levels were analysed.

Article Snippet: The anti-H3 antibody (3638), anti-pS727-STAT3 antibody (9136) and anti-acK685-STAT3 antibody (2523) were from Cell Signaling Technology (Boston).

Techniques: Staining, Transfection, Mutagenesis, Activity Assay

Journal: Scientific Reports

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

doi: 10.1038/srep39517

Figure Lengend Snippet: ( a ) Cellular glucose levels were measured in control or STAT3−/− MEFs receiving insulin (5 μg/ml) treatment for 30 min or no treatment. ( b ) Seahorse extracellular acidification rates (ECAR) were measured in quadruplicate wells containing equal numbers of wild-type or STAT3−/− MEFs treated with insulin (5 μg/ml). ( c ) The Seahorse oxygen consumption rate (OCR) was measured in quadruplicate wells containing equal numbers of wild-type or STAT3−/− MEFs treated with insulin (5 μg/ml). ( d ) α-ketoglutaric acid concentrations were measured in control or STAT−/− MEFs receiving insulin (5 μg/ml) treatment for 30 min or no treatment. ( e ) Cytoplasmic and mitochondrial Acetyl-CoA levels were measured in MEFs treated with insulin for various times as indicated. ( f ) Oil red O staining of control MEFs and STAT3−/− MEFs, treated with or without insulin (5 μg/ml, 30 min). Lipid droplet accumulation was visualized with a microscope. ( g ) The IOD (integrated optical density) of the lipid droplets from ( c ) was measured with IPP software ( d ). ( h ) The IOD (integrated optical density) of lipid droplets was measured in STAT3−/− MEFs stained with Oil red O after SSR (90% DMEM and 10% FBS, 30 min), insulin (5 μg/ml, 30 min) and NAM (100 μM, 6 hrs) treatment.

Article Snippet: The anti-H3 antibody (3638), anti-pS727-STAT3 antibody (9136) and anti-acK685-STAT3 antibody (2523) were from Cell Signaling Technology (Boston).

Techniques: Staining, Microscopy, Software

Journal: Scientific Reports

Article Title: STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

doi: 10.1038/srep39517

Figure Lengend Snippet: ( a ) PC3 cells (STAT3-null) were transfected with an EV, STAT3 or Cox4-STAT3 and visualized with the mitochondrial membrane potential dye JC-1. ( b ) Mitochondrial lysates from the above three types of PC3 cells were prepared for STAT3 acetylation analysis. ( c ) Mitochondrial membrane potential (left), mitochondrial PDC E1 activity (middle) and ATP production levels (right) were measured in the above three types of PC3 cells. ( d ) Parental PC3 cells (STAT3-null), PC3 cells expressing STAT3 WT, or PC3 cells expressing STAT3-3KR mutant were transfected with GFP-LC3 (green), stained with DAPI (blue), and subjected to confocal microscopy. ( e ) A549 cells were treated with or without insulin. STAT3 acetylation was detected with anti-pan-acetyl-lysine antibody in cytoplasmic and mitochondrial fractions. ( f ) Mitochondrial PDC E1 activity was measured in A549 cells transfected with an EV or Flag-SIRT5. Flag-SIRT5 levels (right) were detected via western blotting in these cells. ( g ) Lactate levels were measured in parental PC3 (green), PC3-STAT3 (blue) or A549 cells (red) after insulin treatment for the indicated times. ( h ) The absorbance of adipose droplets stained by Oil red O was measured in parental PC3 (green), PC3-STAT3 (blue) or A549 cells (red) at 490 nm in extracts prepared with isopropanol. ( i ) Lung tissue samples (cancer sections and adjacent normal sections) obtained from non-small cell adenocarcinoma and squamous carcinoma lung cancer patients were immunostained with polyclonal antibody against K685-acetylated STAT3. ( j ) Mitochondrial and cytoplasmic fractions were prepared from tumour tissue samples (T) and adjacent normal tissue samples (N) from 4 lung cancer patients. aK685-STAT3, STAT3, SIRT5 and porin were analysed in the mitochondrial fraction (upper panel), aK685-STAT3, STAT3 and tubulin were analysed in the cytoplasmic fraction (lower panel). ( k ) ATP production was compared between tumour tissue samples (T) and adjacent normal tissue samples (N) from 3 lung cancer patients.

Article Snippet: The anti-H3 antibody (3638), anti-pS727-STAT3 antibody (9136) and anti-acK685-STAT3 antibody (2523) were from Cell Signaling Technology (Boston).

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