Journal: Molecular & Cellular Proteomics : MCP
Article Title: PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P 3 Effector Signaling in Colorectal Cancer
doi: 10.1016/j.mcpro.2023.100529
Figure Lengend Snippet: Nuclear PI3Kα signaling is associated with modulation of pre-mRNA splicing. A , schematic highlighting identified nuclear PtdIns(3,4,5)P 3 interactome proteins and their role in the pre-mRNA splicing process. Proteins identified in the interactome are listed in red boxes . B , representative flow cytometry plots for RG6 splicing reporter assays on PIP3 nuc DLD1 and PIP3 mem SKCO1 cells, serum-starved for 1 h, and stimulated with EGF (200 ng/ml) for 24 h ± pretreatment with BYL719 (25 μM) for 1 h. Quantification of dsRed:GFP ratios for flow cytometry–based RG6 splicing reporter assays on PIP3 nuc DLD1, SW480, and CACO2 cells and PIP3 mem SKCO1 and LIM1215 cells. Data show the mean ± SEM for six biological replicates per condition. Statistical significance was determined using Student’s t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. C , representative images of immunofluorescence staining for SC-35 foci in PIP3 nuc DLD1 and PIP3 mem SKCO1 cells, serum starved for 1 h, and stimulated with EGF (200 ng/ml) for 10 min ± pretreatment with BYL719 (25 μM) for 1 h. Scale bar represents 10 μm. Data show the mean ± SEM from five images per condition with n > 20 cells per image. Statistical significance was determined using Student’s t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. D , Agarose gel image of PCR products from in vitro splicing assays with the RG6 probe using nuclear extract from PIP3 mem SKCO1 CRC cells in the absence or presence of soluble PtdIns(3,4,5)P 3 (250 μM) or PtdIns(4,5)P 2 (250 μM). PIP3 mem, PtdIns(3,4,5)P 3 plasma membrane–cytoplasmic localization; PIP3 nuc, PtdIns(3,4,5)P 3 nuclear localization; PtdIns(4,5)P 2, phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5)P 3 , phosphatidylinositol-3,4,5-trisphosphate.
Article Snippet: The RG6 fluorescent splicing reporter plasmid was kindly provided by Thomas Cooper (Addgene plasmid #80167; http://n2t.net/addgene:80167 ; Research Resource Identifier: Addgene_80167) ( ).
Techniques: Flow Cytometry, Immunofluorescence, Staining, Agarose Gel Electrophoresis, In Vitro, Clinical Proteomics, Membrane
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