Dataset Information

Mass spectrometry analysis of proteins interacting with STAT3

ABSTRACT: Mass spectrometry analysis of proteins interacting with STAT3¬Flag was performed. STAT3-Flag was transiently expressed in HeLa cells and the interactome of STAT3-Flag was studied in lysosomal extracts. proteins co-¬immunoprecipitating with STAT3--Flag identified eight subunits of the vacuolar H+-¬ ATPase (V-¬ATPase) as potential STAT3 binding partners. Validation experiments were performed by antibody pull-down of STAT3.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Hela Cell

SUBMITTER: Johan Palmfeldt

LAB HEAD: Johan Palmfeldt

PROVIDER: PXD006788 | Pride | 2018-08-29

REPOSITORIES: Pride

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Publications

STAT3 associates with vacuolar H<sup>+</sup>-ATPase and regulates cytosolic and lysosomal pH.

Liu Bin B Palmfeldt Johan J Lin Lin L Colaço Alexandria A Clemmensen Knut K B KKB Huang Jinrong J Xu Fengping F Liu Xin X Maeda Kenji K Luo Yonglun Y Jäättelä Marja M

Cell research 20180820 10

Dysregulated intracellular pH is emerging as a hallmark of cancer. In spite of their acidic environment and increased acid production, cancer cells maintain alkaline intracellular pH that promotes cancer progression by inhibiting apoptosis and increasing glycolysis, cell growth, migration, and invasion. Here we identify signal transducer and activator of transcription-3 (STAT3) as a key factor in the preservation of alkaline cytosol. STAT3 associates with the vacuolar H<sup>+</sup>-ATPase in a c ...[more]

PMID: 30127373

Similar Datasets

Project description:Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC) and BarrettM-bM-^@M-^Ys adenocarcinomas (BAC) revealed similar STAT3 expression in ESCCs and BACs, but preferentially activated P-STAT3 in ESCCs. In vitro, strong STAT3 activation was seen by EGF-stimulation in OE21 (ESCC) cells, whilst OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 and OE33 cells and reduced cell migration in OE33, but not in OE21 cells. Transcriptome analysis identified STAT3-knockdown associated down-regulation of cell cycle processes and the selective down-regulation of cyclins and cyclin dependent kinaes associated genes in both OE21 and OE33 cells. Moreover, the transcriptome response showed changes in cell migration/invasion related genes that correlated with the associated phenotype measurements. This study demonstrates the importance of STAT3 expression and activation in esophageal carcinomas, whereby the extent differs between ESCCs and BACs. STAT3 knockdown significantly reduces cell proliferation in both types of esophageal cancer cells and inhibits migration in BAC cells. Thus, STAT3 may be further exploited as potential novel therapeutic target for esophageal cancers. The effect of STAT3 knock-down in OE33 and OE21 cells was calculated from three biologically independent experiments. 3x10e4 cells were seeded in triplicate in 24 well-plates and were transfected with twice 100nM STAT3 siRNA (pool of 4 STAT3 sequences, siGENOMEM-BM-.SMARTpoolM-BM-., Dharmacon RNAi Technologies, Thermo Fisher Scientific, Lafayette, USA) or SilencerM-BM-. negative siRNA control (Invitrogen/Life Technologies GmbH, Darmstadt, Germany) using 1M-BM-5l DharmaFECT (Dharmacon RNAi Technologies, Thermo Fisher Scientific, Lafayette, USA) transfection reagent for OE33 cells or siPORTTM NeoFXTM (Invitrogen/Life Technologies GmbH, Darmstadt, Germany) transfection reagent for OE21 cells. Differential gene regulation was quantified 72 hours after the first transfection by comparing separately for the OE21 and OE33 cells the STAT3 siRNA replicates and the respective cell lines containing the scrambled siRNA vector.

Project description:Signaling by the cytokine LIF and its downstream transcription factor, STAT3, prevents differentiation of pluripotent embryonic stem cells (ESCs) by opposing MAP kinase signaling. This contrasts with most cell types where STAT3 signaling induces differentiation. We find that STAT3 binding across the pluripotent genome is dependent upon Brg, the ATPase subunit of a specialized chromatin remodeling complex (esBAF) found in ESCs. Brg is required to establish chromatin accessibility at STAT3 binding targets, in essence preparing these sites to respond to LIF signaling. Moreover, Brg deletion leads to rapid Polycomb (PcG) binding and H3K27me3-mediated silencing of many Brg-activated targets genome-wide, including the target genes of the LIF signaling pathway. Hence, one crucial role of Brg in ESCs involves its ability to potentiate LIF signaling by opposing PcG. Contrary to expectations, Brg also facilitates PcG function at classical PcG target including all four Hox loci, reinforcing their repression in ESCs. These findings reveal that esBAF does not simply antagonize PcG, but rather, the two chromatin regulators act both antagonistically and synergistically with the common goal of supporting pluripotency. Brg conditional embryonic stem cell (ESC) line was prepared from Brg(lox/lox);Actin-CreER mice that allow conditional deletion of Brg upon addition of 4-hydroxytamoxifen (4OHT, Sigma). Brg conditional ESCs were treated with 4-OHT for 72 hours to delete Brg. Brg protein levels completely abolished only at 48 hours (Brg-KO). RNA from Brg conditional ESCs (WT), Brg-KO ESCs, Brg conditional ESCs starved of LIF (Lif-WD) for 48 hours were extracted using Trizol reagent (Invitrogen), followed by RNeasy kit with DNaseI digestion to eliminate genomic DNA. Purified RNA was processed and hybridized onto Affymetrix Mouse MOE 4.0 3’ expression arrays according to the manufacturer's instructions. ChIP-Seq was used to detect and measure STAT3 occupancy and levels of H3K27me3 in the genome before and after the removal of the SWI/SNF ATPase Brg in mouse ESCs. Total of 4 samples (WT and KO STAT3; WT and KO H3K27me3). Input DNA was sequenced as control.

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	228_Bin-pCDH-flag1.raw	Raw
	229_Bin-hStat-Flag1.raw	Raw
	230_Bin-hStat3-NCI.raw	Raw
	2909-Bin-cross-1A.raw	Raw
	2911-Bin-cross-2A.raw	Raw