Journal: bioRxiv

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1101/2020.08.31.275909

Figure Lengend Snippet: A-C) Indicated cell lines were transfected with vector, AKT1, AKT2, or AKT3 along with the HSE luciferase reporter for 48 hrs. Firefly/Renilla ratios were compared across groups. D) Recombinant proteins were incubated at 30°C for 2 hrs in the presence of ATP and subsequently subjected to SDS-PAGE and immunoblotting with indicated antibodies. E) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. F-G) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 in the presence or absence of rapamycin (10-20 nM). Cell lysates were subjected to firefly reporter assay (F) or immunblotting with indicated antibodies (G). Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.

Article Snippet: The pcDNA3 Myr HA AKT1 plasmid was purchased from Addgene (ID 9008, RRID:Addgene_9008), which was originally established by Dr William Sellers ( ).

Techniques: Transfection, Plasmid Preparation, Luciferase, Recombinant, Incubation, SDS Page, Western Blot, Reporter Assay

Journal: bioRxiv

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1101/2020.08.31.275909

Figure Lengend Snippet: A-B) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS-PAGE and immunoblotting for HSF1 trimers (A) and AKT isoforms (B). C) HEK-293 cells were transfected with vector, AKT1, AKT2, or AKT3 for 48 hrs. Cells were fixed with formaldehyde, lysed, and sonicated prior to ChIP with either control IgG or HSF1 antibodies. ChIP product was subjected to qPCR for indicated gene promoters. Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.

Article Snippet: The pcDNA3 Myr HA AKT1 plasmid was purchased from Addgene (ID 9008, RRID:Addgene_9008), which was originally established by Dr William Sellers ( ).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Sonication, Control

Journal: bioRxiv

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1101/2020.08.31.275909

Figure Lengend Snippet: A) HEK-293 cells were transfected with the HSE luciferase reporter for 48 hrs and cells were then pre-treated with MK-2206 at the indicated concentrations for 1 hr and cells were then incubated at 42°C for 1 hr. Firefly/Renilla ratios were compared across groups. B-D) HEK-293 cells were transfected with non-targeting (NT), AKT1, or AKT2 siRNA for 48 hrs followed by incubation at 42°C for 1 hr. Cells were then collected and subjected to immunoblotting (B), luciferase reporter assay (C), or qPCR (D). Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.

Article Snippet: The pcDNA3 Myr HA AKT1 plasmid was purchased from Addgene (ID 9008, RRID:Addgene_9008), which was originally established by Dr William Sellers ( ).

Techniques: Transfection, Luciferase, Incubation, Western Blot, Reporter Assay

Journal: bioRxiv

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1101/2020.08.31.275909

Figure Lengend Snippet: A) Recombinant proteins were incubated at 30°C for 2 hrs in the presence of ATP and subsequently subjected to SDS-PAGE and immunoblotting with indicated antibodies. B-C) HEK-293 cells were transfected with vector, AKT1, mTOR, MEK1, or p38 for 48 hrs. Cells were collected and subjected to immunoblotting with indicated antibodies (B) or reporter luciferase assay (C). D) HEK-293 cells were transfected with non-targeting control siRNA or siRNA directed to AKT1, mTOR, MEK1, or p38δ along with an HSE luciferase reporter. Cells were then incubated at 42°C for 1 hr and subsequently collected and subjected to the luciferase reporter assay. E) Gene set enrichment analysis (GSEA) was performed using data from TCGA for breast (upper row) and colorectal cancers (lower row). Patients from these datasets were separated into high and low HSF1 activity groups using previously published gene signature for HSF1 activity. GSEA was then performed on these two groups using activity signatures for AKT1, AKT2, p38, mTORC1, or MEK1. Normalized enrichment score (NES) is reported and calculated GSEA p-value. Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05. Data in D has normalized enrichment score (NES) reported and calculated GSEA p-value.

Article Snippet: The pcDNA3 Myr HA AKT1 plasmid was purchased from Addgene (ID 9008, RRID:Addgene_9008), which was originally established by Dr William Sellers ( ).

Techniques: Recombinant, Incubation, SDS Page, Western Blot, Transfection, Plasmid Preparation, Luciferase, Control, Reporter Assay, Activity Assay

Journal: bioRxiv

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1101/2020.08.31.275909

Figure Lengend Snippet: A) Workflow for identification of kinase-specific phosphorylation sites. B) Pattern of HSF1 phosphorylation for each kinase tested by mass spectrometry. C) Conservation of four sites phosphorylated by AKT1 across mammalian species. D) HEK-293 cells were transfected with WT-HSF1 or single phospho-null HSF1 mutants for AKT1 phosphorylation sites along with the HSE reporter for 48 hrs followed by incubation at 42°C for 1 hr. Firefly/Renilla ratios were compared across groups. E) HEK-293 cells were transfected with WT-HSF1 or an HSF1 mutant with all four AKT1 phosphorylation sites with phospho-null mutations for 48 hrs followed by incubation at 42°C for 1 hr. Firefly/Renilla ratios were compared across groups. Experiments were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.

Article Snippet: The pcDNA3 Myr HA AKT1 plasmid was purchased from Addgene (ID 9008, RRID:Addgene_9008), which was originally established by Dr William Sellers ( ).

Techniques: Phospho-proteomics, Mass Spectrometry, Transfection, Incubation, Mutagenesis

Journal: bioRxiv

Article Title: AKT1 mediates multiple phosphorylation events that functionally promote HSF1 activation

doi: 10.1101/2020.08.31.275909

Figure Lengend Snippet: A-C) HEK-293 cells were transfected with WT-HSF1 or T142A-HSF1 in the presence or absence of AKT1 (A, C) or CK2 (B) for 48 hrs. Cells were fixed with 1 mM EGS for 30 min and lysates were subjected to SDS-PAGE and immunoblotting for HSF1 trimers. Samples in C were subjected to size exclusion chromatography and fractions underwent immunoblotting with HSF1 antibodies to identify HSF1 complex sizes according to the molecular weight of the extracted fractions. A and B were completed in triplicate and analyzed using one-way ANOVA and Tukey’s post-hoc test. Data is presented as mean ± SEM. *P<0.05.

Article Snippet: The pcDNA3 Myr HA AKT1 plasmid was purchased from Addgene (ID 9008, RRID:Addgene_9008), which was originally established by Dr William Sellers ( ).

Techniques: Transfection, SDS Page, Western Blot, Size-exclusion Chromatography, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion *

doi: 10.1074/jbc.M116.742742

Figure Lengend Snippet: S6K1 phosphorylates PIPKIγ90 at Thr-553 and Ser-555. A, alignment of the Akt/S6K1 consensus sequences from different species. B, transfection of constitutively active Akt1 or S6K1 promoted PIPKIγ90 phosphorylation. FLAG-PIPKIγ90 was co-transfected with an empty vector, Myr-Akt1, and S6K1-F5A-E389-R3A into CHO-K1 cells. FLAG-PIPKIγ90 was immunoprecipitated, and PIPKIγ90 phosphorylation was detected with an anti-RXRXXpS/T motif antibody. a.u., arbitrary unit; *, p < 0.05. C, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in vitro. Recombinant GST-PIPKIγ90501–668, -PIPKIγ90501–668T553A, -PIPKIγ90501–668S555A, and -PIPKIγ90501–668T553A,S555A were phosphorylated with constitutively active S6K1 that was immunoprecipitated from CHO-K1 cells. D, S6K1 phosphorylated PIPKIγ at Thr-553 and Ser-555 in CHO-K1 cells. HA-S6K1-F5A-E389-R3A was co-transfected with FLAG-PIPKIγ90, -PIPKIγ90T553A, -PIPKIγ90S555A, and -PIPKIγ90T553A,S555A into CHO-K1 cells. Data are presented as mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 versus WT. E, EGF and HGF stimulated PIPKIγ phosphorylation. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved and stimulated with EGF (20 ng/ml), HGF (50 ng/ml), SCF (20 ng/ml), and PDGF (20 ng/ml) for 20 min. FLAG-PIPKIγ90 was immunoprecipitated (IP), and phosphorylation was detected with an anti-RXRXXpS/T motif antibody. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 versus control (Ctrl). F, HGF-stimulated PIPKIγ phosphorylation was inhibited by Akt and S6K1 inhibitors. MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 were serum-starved, treated with Akt inhibitor VIII and the S6K1 inhibitors DG2 (10 μm) or PF4708671 (10 μm), and then stimulated with HGF for 20 min. Data are presented as mean ± S.E. of four independent experiments. *, p < 0.05 versus HGF. G, HGF-stimulated PIPKIγ phosphorylation was suppressed by depletion of S6K1. S6K1 in MDA-MB-231 cells stably expressing FLAG-PIPKIγ90 was depleted using lentiviruses that express S6K1 shRNAs. Cell were serum-starved and then stimulated with HGF (20 ng/ml) for 20 min. Data are presented as mean ± S.E. of three independent experiments. *, p < 0.05.

Article Snippet: The Akt1 shRNA clones were TRCN0000010174 and TRCN0000039793. pBabe-Puro-Myr-FLAG-AKT1 was a gift from William Hahn (Addgene plasmid 15294). pRK7-HA-S6K1-F5A-E389 was a gift from John Blenis (Addgene plasmid 8988).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Stable Transfection, Expressing

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet:

Article Snippet: AKT pT308 , Santa Cruz Biotechnology , Cat# sc-271966; RRID: AB_10715102.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Membrane, Protease Inhibitor, Extraction, cDNA Synthesis, Reporter Assay, Viability Assay, Plasmid Preparation, Software