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Unbiased phosphoproteomics analysis of human myotubes unveils extramitotic CDK1 kinase activity modulates insulin signaling

ABSTRACT: We performed total proteome and phosphoproteome analysis to characterize the role of non-mitotic kinase activity of CDK1 in insulin signaling through pharmacological inhibition and translational repression via siRNA-based knockdown. Specifically, cultured myotubes were treated with RO-3306, a selective ATP competitive inhibitor to inhibit CDK1 activity, or transfected with a CDK1 targeting siRNA pool to decrease CDK1 expression.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: E Shyong Tai

PROVIDER: PXD043414 | JPOST Repository | Thu Jul 04 00:00:00 BST 2024

REPOSITORIES: jPOST

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			Action	DRS
	20230203_JW_Meihui_195_16pc.raw	Raw
	20230203_JW_Meihui_195_19pc.raw	Raw
	20230203_JW_Meihui_195_22pc.raw	Raw
	20230203_JW_Meihui_195_60pc.raw	Raw
	20230203_JW_Meihui_260_11pc.raw	Raw

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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.

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Unbiased phosphoproteomics analysis of human myotubes unveils extramitotic CDK1 kinase activity modulates insulin signaling

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