Journal: The Journal of Biological Chemistry

Article Title: Adenovirus E4ORF1 activates isoform-specific phosphatidylinositol 3-kinase signaling in human endothelial cells

doi: 10.1016/j.jbc.2025.110947

Figure Lengend Snippet: Ad5E4ORF1 activates PI3K at the plasma membrane . A , subcellular localization of Ad5E4ORF1 via indirect immunostaining and confocal fluorescence microscopy. HUVECs (vector control or expressing HA-tagged WT or ΔC3 mutant Ad5E4ORF1, as indicated on the left of each panel) were grown on glass cover slips, starved for 4 h, immunostained with anti-HA and anti-VE-cadherin (VE-CAD), as indicated on the top of panels. Cell nuclei were stained with DAPI. The scale bar represents 10 μm. Data shown represent experiments using six HUVEC lines. B , coimmunoprecipitation of Ad5E4ORF1 and DLG1 in both the cytosol and the nucleus. Indicated cells were starved for 4 h before lysis. Cell lysates were fractionated into cytosol and nucleus pools and then subjected to anti-HA immunoprecipitation and immunoblot. GAPDH and histone H3 were included as cytosol and nucleus markers, respectively. ( Right panels ) Quantification of Ad5E4ORF1 and DLG1 cytosolic versus nuclear distribution from input (cell lysate) blots. Paired t test (mean ± SD; n = 3 HUVEC lines). C , coaggregation of Ad5E4ORF1 and DLG1 proteins in HUVECs. DLG1 (with insert I3), N terminally fused to ZsGreen and under a tetracycline response element (TRE) promoter, was briefly induced in HUVECs with or without HA-Ad5E4ORF1 coexpression in starving conditions before immunostaining as in ( A ). The scale bar represents 10 μm. Data presented represent experiments with three HUVEC lines. D , subcellular localization of PIP3 revealed by a PIP3-binding GFP fusion protein. The AKT1 PHD domain, N terminally fused to GFP and under a TRE promoter, was briefly induced in HUVECs with or without HA-Ad5E4ORF1 in starving medium. The K14A mutant PHD is defective in PIP3 binding. Immunostaining and microscopy were performed as in ( A ). The scale bar represents 10 μm. Data shown represent results from 5 HUVEC lines. E , identification of IQGAP1 interaction-specific Ad5E4ORF1 mutations. ( Top panel ) HA immunoprecipitation blot analysis of HUVECs expressing indicated Ad5E4ORF1 alleles, performed as in D . ( Lower panels ) Quantification of IQGAP1 and DLG1 IP efficiency. One-way ANOVA with Tukey’s test (mean ± SD; n = 3 HUVEC lines). F , Rac1 activation in HUVECs assayed by Rac1-GTP pull-down and immunoblot. ( Right panel ) Quantification of Rac1-GTP (IP) relative to total Rac1 in input. One-way ANOVA with Tukey’s test (mean ± SD; n = 4 HUVEC lines). G , random cell migration speed measured by sing-cell tracking. Expressed relative to vector control, which is 44.1 ± 6.5 μm/h (mean ± SD). One-way ANOVA with Tukey’s test (mean ± SD; n = 3 HUVEC lines). H , collective cell migration speed measured in gap closure of wounded cell monolayers. Expressed relative to WT Ad5E4ORF1, which is 21.8 ± 6.2 μm/h (mean ± SD). One-way ANOVA with Tukey’s test (mean ± SD; n = 3 HUVEC lines). Ad5E4ORF1, human adenovirus serotype 5 early gene E4ORF1; DAPI, 4′,6-diamidino-2-phenylindole; DLG1, discs large homolg 1; HA, hemagglutinin; HUVEC, human umbilical vein endothelial cell; LDS, lithium dodecyl sulfate; PHD, plant homeodomain; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; Rac1, Ras-related C3 botulinum toxin substrate 1; Rac1-GTP, GTP-loaded Rac1.

Article Snippet: PI3K inhibitors BYL-719 (S2814), PIK-75 (S1205), TGX-221 (S1169), GSK-2269557 (S7937), and CZC-24832 (S7018) were purchased from Selleckchem and dissolved in dimethyl sulfoxide.

Techniques: Clinical Proteomics, Membrane, Immunostaining, Fluorescence, Microscopy, Plasmid Preparation, Control, Expressing, Mutagenesis, Staining, Lysis, Immunoprecipitation, Western Blot, Binding Assay, Activation Assay, Migration, Cell Tracking Assay

Journal: The Journal of Biological Chemistry

Article Title: Adenovirus E4ORF1 activates isoform-specific phosphatidylinositol 3-kinase signaling in human endothelial cells

doi: 10.1016/j.jbc.2025.110947

Figure Lengend Snippet: Ad5E4ORF1 physically and functionally engages PI3K isoform p110α-p85β through multiple-domain interactions . A , coimmunoprecipitation of Ad5E4ORF1 with PI3K proteins. Whole cell lysates (in NP-40 buffer) of vector control (−) and HA-Ad5E4ORF1-expressing (+) HUVECs were subjected to anti-HA immunoprecipitation. HA beads-bound proteins and 0.6% of the corresponding input proteins were analyzed by western blot. ( Right panels ) Relative IP efficiency (IP/Input) of different PI3K subunit isoforms quantified from left immunoblots. Paired t test or one-way ANOVA-Tukey’s test (mean ± SD; n = 5 or 3 HUVEC lines). B , effect of isoform-specific PI3K inhibitors on Ad5E4ORF1-mediated AKT activation. HUVECs as in (A) were starved for 4 h and then treated with indicated PI3K inhibitors (0.5 μM in DMSO) for 1 h before being lysed into LDS loading buffer for western blot. ( Right panel ) Quantification of AKT phosphorylation in Ad5E4ORF1 cells from left immunoblots. One-way ANOVA-Tukey’s test (mean ± SD; n = 3 HUVEC lines). C , effect of shRNA-mediated PI3K regulatory subunit knockdowns on Ad5E4ORF1-mediated AKT activation. Lentiviral shRNAs targeting PIK3R1 (#74, #75), PIK3R2 (#77, #78), or PIK3R3 (#66) were doxycycline-induced in HUVECs for 4 days. After a 4-h starvation, cells were lysed in LDS loading buffer for western blot. ( Right panels ) Quantification of PI3K regulatory subunits knockdown efficiency and AKT phosphorylation in Ad5E4ORF1 cells from left immunoblots. One-way ANOVA-Tukey’s test or paired t test (mean ± SD; n = 3 HUVEC lines). D , interactions of mutant Ad5E4ORF1 proteins with p110α and DLG1 analyzed by coimmunoprecipitation as in ( A ). ( Lower panels) Quantification of p110α and DLG1 IP efficiency (normalized to HA-Ad5E4ORF1 in IP) from upper immunoblots. One-way ANOVA-Tukey’s test (mean ± SD; n = 3 HUVEC lines). E , western blot analyses of indicated Ad5E4ORF1 mutant cell lysates, performed as in ( B ). M, a single methionine added to Ad5E4ORF1 C terminus. ( Lower panel ) Quantification of AKT phosphorylation from top immunoblots. One-way ANOVA-Tukey’s test (mean ± SD; n = 4 HUVEC lines). F , coimmunoprecipitation of Ad5E4ORF1 with p110α. HA-tagged GFP, GFP-p110α fusion, or p85β was coexpressed with Flag-Ad5E4ORF1 (+) or nontagged Ad5E4ORF1 (−) in HEK293T cells. Whole cell lysates (in NP-40 buffer) were subjected to anti-Flag immunoprecipitation; input and beads-bound proteins were analyzed by western blot. ( Right panel ) Quantification of p110α and p85β IP efficiency (normalized to Flag-Ad5E4ORF1 in IP) from left immunoblots. One-way ANOVA-Tukey’s test (mean ± SD; n = 12 independent experiments). G , coimmunoprecipitation of Ad5E4ORF1 with p110α fragments. Performed as in ( A ) on cells coexpressing Ad5E4ORF1 and HA-GFP fusions of full-length (FL) or indicated p110α fragments. The upper scheme shows defined p110α domains and linker regions (L1, L2, and L3). ( Right panel ) Quantification of IP efficiency of different p110α fragments from immunoblots on the left. Where nonspecific binding was observed, signal intensity from the control IP was subtracted from that of the Flag-E4ORF1 IP. IP efficiency of each p110α fragment was further normalized to the amount of Flag-Ad5E4ORF1 in the IP. One-way ANOVA-Tukey’s test (mean ± SD; n = 4 experiments). H and I , fine mapping of Ad5E4ORF1 interactions with p110α L1RL2 and L3H regions by coimmunoprecipitation as in ( G ). Linker regions alone did not bind Ad5E4ORF1 and were used as negative controls in place of HA-GFP. One-way ANOVA-Tukey’s test (mean ± SD; n = 3 experiments). J , schematic model of Ad5E4ORF1-PI3K interaction. ABD, adapter p85-binding domain; Ad5E4ORF1, human adenovirus serotype 5 early gene E4ORF1; AKT, protein kinase B; DLG1, discs large homolg 1; DMSO, dimethyl sulfoxide; HA, hemagglutinin; HEK293T, human embryonic kidney 293T; HUVEC, human umbilical vein endothelial cells; IP, immunoprecipitation; LDS, lithium dodecyl sulfate; PI3K, phosphatidylinositol 3-kinase; RBD, RAS-binding domain.

Article Snippet: PI3K inhibitors BYL-719 (S2814), PIK-75 (S1205), TGX-221 (S1169), GSK-2269557 (S7937), and CZC-24832 (S7018) were purchased from Selleckchem and dissolved in dimethyl sulfoxide.

Techniques: Plasmid Preparation, Control, Expressing, Immunoprecipitation, Western Blot, Activation Assay, Phospho-proteomics, shRNA, Knockdown, Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Adenovirus E4ORF1 activates isoform-specific phosphatidylinositol 3-kinase signaling in human endothelial cells

doi: 10.1016/j.jbc.2025.110947

Figure Lengend Snippet: Mechanistic model of PI3K pathway activation by E4ORF1 . I , Within the p110α–p85β complex, p85β ( purple ) inhibits p110α partly by blocking its PIP2-binding pocket. II , E4ORF1 proteins selectively bind to p110α when complexed with p85β, exerting bifurcating effects depending on the serotype. III , left arm : Ad4 and Ad52 E4ORF1 proteins interact only with the Ras-binding domain (RBD) of p110α, causing limited activation due to continued p85β inhibition. Right arm: in contrast, Ad5, Ad9, and Ad36 E4ORF1s engage multiple domains of p110α, effectively relieving p85β inhibition and resulting in robust activation. IV , the resulting E4ORF1–PI3K complex is recruited to the plasma membrane via DLG1-mediated vesicle trafficking and membrane association. V , at the membrane, p110α converts phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3), in proportion to its activation level. VI , PIP3 accumulation initiates downstream activation of AKT and Rac1, promoting cell survival and angiocrine signaling. Domains of p110α: A, adaptor-binding domain; R, RAS-binding domain; C2, C2 domain; H, helical domain; K, kinase domain. AKT, protein kinase B; DLG1, discs large homolg 1; PI3K, phosphatidylinositol 3-kinase; Rac1, Ras-related C3 botulinum toxin substrate 1.

Article Snippet: PI3K inhibitors BYL-719 (S2814), PIK-75 (S1205), TGX-221 (S1169), GSK-2269557 (S7937), and CZC-24832 (S7018) were purchased from Selleckchem and dissolved in dimethyl sulfoxide.

Techniques: Activation Assay, Blocking Assay, Binding Assay, Inhibition, Clinical Proteomics, Membrane

Journal: PLOS Biology

Article Title: Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy

doi: 10.1371/journal.pbio.3002227

Figure Lengend Snippet: ( A ) Plate colony formation assays and ( B ) soft-agar assays on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl), also with stable knockdown of PDK1 (shPDK1-1 and shPDK1-2) or control (NT). The cells were also treated with PI3K inhibitor Alpelisib (1 μm or 2 μm) or DMSO control. Scale bar: 500 μm. Quantification data as means ± SD. P values by two-way ANOVA followed by Sidak’s multiple comparisons. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ns, not significant. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in . MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.

Article Snippet: The kinase inhibitors used include PI3K inhibitor Alpelisib/BYL-719 (MedChemExpress, Catalog HY-15244), PDK1 inhibitor GSK2334470 (MedChemExpress, Catalog HY-14981), AKT inhibitor MK2206 (Selleckchem, Catalog S1078), and MNK1/2 inhibitors SLV-2436/SEL201 (Medchemexpres, Catalog HY-112113) and EFT-508 (Selleckchem, Catalog S8275).

Techniques: Over Expression, Control, Knockdown

Journal: PLOS Biology

Article Title: Cooperative activation of PDK1 and AKT by MAPK4 enhances cancer growth and resistance to therapy

doi: 10.1371/journal.pbio.3002227

Figure Lengend Snippet: ( A ) Proliferation assays, ( B ) plate colony formation assays, and ( C ) soft-agar assays on the engineered SUM159 and HCC1806 cells with 0.5 μg/ml Dox-induced overexpression of MAPK4 (iMAPK4) or control (iCtrl). The cells were also treated with DMSO control, PDK1 inhibitor GSK2334470 (2 μm), PI3K inhibitor Alpelisib (2 μm), or both inhibitors. Scale bar: 500 μm. Quantification data as means ± SD. P values by two-way ANOVA followed by Sidak’s multiple comparisons. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ns, not significant. Data are representative of at least 3 independent experiments. The numerical values underlying the figures can be found in . MAPK4, mitogen-activated protein kinase 4; PDK1, phosphoinositide-dependent kinase-1.

Article Snippet: The kinase inhibitors used include PI3K inhibitor Alpelisib/BYL-719 (MedChemExpress, Catalog HY-15244), PDK1 inhibitor GSK2334470 (MedChemExpress, Catalog HY-14981), AKT inhibitor MK2206 (Selleckchem, Catalog S1078), and MNK1/2 inhibitors SLV-2436/SEL201 (Medchemexpres, Catalog HY-112113) and EFT-508 (Selleckchem, Catalog S8275).

Techniques: Over Expression, Control

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: Modulation of SOX2 protein stability by canonical PI3K/AKT signaling. A. Increased PI3K activity fosters SOX2 protein expression in LN319 cells. Cell were stably transfected with Doxycycline‐inducible variants of PIK3CA encoding the catalytic subunit of PI3 kinase p110. From left to right: no insert (control), PIK3CA wild type, and the constitutively active mutant alleles H1047R or E545K. Note elevated SOX2 protein expression in response to aggravated PI3K activity, as indicated by increased pAKT (Ser473) and pS6 (Ser235,236) signatures. Actin staining is shown for loading control. B. Effects of 48 h‐exposure to PI3K/AKT/TOR pathway inhibitors on SOX2 protein levels as assessed by Western blot. SOX2 expression is strongly impaired by pan PI3K‐inhibitor (BYL‐719) and AKT‐inhibitors (AKTi1/2, MK‐2206), but not by further downstream inhibitor rapamycin (anti mTORC1). C. Fluorescence microcopy to document nucleo‐cytoplasmatic displacement and reduction of SOX2 protein expression by PI3K inhibitor MK‐2206. D. Impaired invasiveness of MK‐2206‐treated, SOX2‐depleted LN319 cells as assayed by Boyden chamber experiments. E. Schematic illustration of SOX2 protein expression and turnover in dependence of PI3K/AKT signaling.

Article Snippet: PI3K/AKT/TOR signaling pathway inhibitors BYL‐719, MK‐2206, AKTi1/2 and rapamycin were all purchased from Selleckchem (Houston, TX, USA), dissolved and diluted according to manufacturer’s instructions, and applied at the indicated concentrations for 48 h. For induction of mCherry‐SOX2, p110α(WT), p110α(H1047R) and p110α(E545K) protein expression, cells were incubated with the indicated concentrations of doxycycline (D9891, Sigma, St‐Louis, MO, USA) or standardly with 1 µg/ml for a minimum of 24 h.

Techniques: Activity Assay, Expressing, Stable Transfection, Transfection, Mutagenesis, Staining, Western Blot, Fluorescence

Journal: Brain Pathology

Article Title: Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1

doi: 10.1111/bpa.12670

Figure Lengend Snippet: 3q26 gene products modulate glioma cell invasion in various entangled ways. SOX2 balances glioma cell invasion through intrinsically antagonistic mechanisms: While PI3K/AKT/mTOR 41 and PI3K/AKT‐SOX2 loops promote glioma invasion 9, 20, SOX2 trans‐activates OPA1, which results in an effective inhibition of invasion. OPA1 knock‐down mutations resolve this functional antagonism and lead to an effective deregulation of glioma cell invasion.

Article Snippet: PI3K/AKT/TOR signaling pathway inhibitors BYL‐719, MK‐2206, AKTi1/2 and rapamycin were all purchased from Selleckchem (Houston, TX, USA), dissolved and diluted according to manufacturer’s instructions, and applied at the indicated concentrations for 48 h. For induction of mCherry‐SOX2, p110α(WT), p110α(H1047R) and p110α(E545K) protein expression, cells were incubated with the indicated concentrations of doxycycline (D9891, Sigma, St‐Louis, MO, USA) or standardly with 1 µg/ml for a minimum of 24 h.

Techniques: Inhibition, Functional Assay

Journal: Nature communications

Article Title: p85β regulates autophagic degradation of AXL to activate oncogenic signaling.

doi: 10.1038/s41467-020-16061-7

Figure Lengend Snippet: Fig. 1 Oncogenicity of p85β depends on p110 activities but is independent of AKT. a Overall survival (OS) and progression-free survival (PFS) of serous ovarian cancer patients split at the upper tertile of PIK3R2 mRNA level. Data were obtained from Kaplan–Meier Plotter using both GEO and TCGA datasets. Two-sided logrank test P-values of the two-group comparison are shown. b–d Ovarian cancer cells with or without PIK3R2 silencing (R2 siRNA) were examined for (b) BrdU cell proliferation, (c) colony formation, and (d) cell invasion. NS siRNA, nonspecific siRNA. e–g EFO21 cells stably expressing PIK3R2 (R2OX) or empty vector were treated with the indicated inhibitors and subjected to (e) BrdU cell proliferation assay, (f) colony formation assay, and (g) cell invasion assay. h p110α or p110β proteins were immunoprecipitated from protein lysates of cells with or without stable PIK3R2 overexpression. The eluants were subjected to PI3-kinase activity assay. i Protein levels of p85β, p110α, p110β, and Erk2 (a loading control) were examined by western blotting. The western blotting experiment was repeated three times with independent lysates and results were reproducible. Assays in b–h were done in triplicate. Data shown are representative of three independent experiments and presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 using two-tailed t-test. Source data are provided as a Source Data file.

Article Snippet: PI3K inhibitors (GDC-0941, PIK-90, A66, BYL719, and TGX-221), AKT inhibitors (MK-2206 and GDC-0068), and AXL inhibitor (BGB324) were obtained from Selleckchem (Houston, TX).

Techniques: Comparison, Stable Transfection, Expressing, Plasmid Preparation, BrdU Cell Proliferation Assay, Colony Assay, Invasion Assay, Immunoprecipitation, Over Expression, Kinase Assay, Control, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a Percentage of PI3K members ( PTEN , PIK3CA , PIK3CB, and PIK3R1 ) alterations in metastatic castration-resistant prostate cancer cohort (SU2C Cell 2015). b Oncoprint showing PI3K member alterations in all PI3K-altered tumors from the same cohort ( n = 76). c Venn diagram showing the percentage of overlaps among alterations in PIK3CA , PIK3CB , PIK3R1 , and PTEN . d Oncoprint showing PIK3CA , PIK3R1 , and PTEN alterations in a collection of PDOs. e Western blot showing pAKT level and PTEN status in a collection of PDOs. f Western blot showing AR and pAKT levels in PDOs treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. Quantification of pAKT473 and pAKT308 normalized to β-actin. g Western blot showing pAKT levels in isogenic PDO pairs MSK-PCa15 ( PTEN -intact, PTEN -KO) and MSK-PCa16 ( PTEN -intact, PTEN -KO) treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. h Western blot showing pAKT levels in isogenic LNCaP-EV and LNCaP- PTEN pairs treated with doxycycline (500 ng/mL) for 8 h for PTEN induction, followed by either BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. All assays were performed with three biological replicates. Source data are provided as Source data file.

Article Snippet: The PI3K p110α-selective inhibitor BYL719 was purchased from LC Laboratories (cat# A-4477).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a – c Western blot showing pAKT levels in CWR22Pc, LNCaP cells, or MSK-PCa1 organoids with ectopic expression of DDK-tagged p110α E545K or p110β E1051K mutants. d Western blot showing pAKT levels in LNCaP-EV, LNCaP-p110α E545K , or LNCaP-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. e CellTiter-Glo assay showing cell viability in LNCaP-EV, LNCaP-p110α E545K , or LNCaP-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 7 days. f Western blot showing pAKT levels in CWR22Pc-EV, CWR22Pc-p110α E545K , or CWR22Pc-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. g CellTiter-Glo assay showing cell viability in CWR22Pc-EV, CWR22Pc-p110α E545K , or CWR22Pc-p110β E1051K cells treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 7 days. h Western blot showing levels of pAKT and pS6 in LNCaP-EV (empty vector) or LNCaP-p110α WT (ectopic expression of DDK-tagged p110α WT ) cells cultured in regular 10% FBS-RPMI treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. Source data are provided as Source data file. All assays were performed with three biological replicates. **** p < 0.0001, n.s: not significant, e : one-way ANOVA compared to BYL719 group, g : one-way ANOVA compared to AZD8186 group, error bar represents mean values ± SD.

Article Snippet: The PI3K p110α-selective inhibitor BYL719 was purchased from LC Laboratories (cat# A-4477).

Techniques: Western Blot, Expressing, Glo Assay, Plasmid Preparation, Cell Culture

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a , c Western blot showing pAKT levels in PDO models MSK-PCa1 and MSK-PCa3 treated with BYL719 (1 µM), AZD8186 (250 nM), BYL719 + AZD8186, or Veh for 0, 1, 3, 6, 12, and 24 h, respectively. Quantification of pAKT473 and pAKT308 normalized to total AKT. b , d Growth assay of PDO lines MSK-PCa1 and MSK-PCa3 treated with BYL719 (1 µM), AZD8186 (250 nM), BYL719 + AZD8186, or Veh. Cell number was read using CellTiter-Glo assay on Day 1, Day 4, and Day 7, respectively. e , f Western blot showing pAKT levels in CWR22Pc-sg NT or CWR22Pc-sg PTEN isogenic pairs treated with BYL719 (1 µM), AZD8186 (250 nM), BYL719 + AZD8186, or Veh for 0, 1, 3, 6, 12, and 24 h, respectively. Quantification of pAKT473 and pAKT308 normalized to total AKT. g Growth of CWR22Pc tumors in SCID mice treated with BYL719 (25 mg/kg), AZD8186 (75 mg/kg), BYL719 + AZD8186, or Veh. Mice were castrated when tumor reaches ~200 mm 3 . h Western blot showing pAKT and pS6 levels in CWR22Pc tumors from ( g ). Source data are provided as Source data file. All assays were performed with three biological replicates. **** p < 0.0001, * p = 0.0142, n.s: not significant, b , d , g : one-way ANOVA compared to Veh group, error bar represents mean values ± SD, g : error bar represents mean values ± SEM.

Article Snippet: The PI3K p110α-selective inhibitor BYL719 was purchased from LC Laboratories (cat# A-4477).

Techniques: Western Blot, Growth Assay, Glo Assay

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a , b Growth assay of PDO lines MSK-PCa11 and MSK-PCa12 treated with BYL719 (1 µM), AZD8186 (250 nM), BYL719 + AZD8186 or Veh. Cell number was read using CellTiter-Glo assay on Day 1, Day 4, and Day 7, respectively. c Western blot showing levels of IGF1R/INSR and AKT signaling in PDO lines MSK-PCa11 and MSK-PCa12 treated with BYL719 (1 µM) + AZD8186 (250 nM) or Veh for 4 h. d Western blot showing levels of IGF1R and AKT signaling in MSK-PCa12-sh Renilla or MSK-PCa12-shIGF1R lines treated with BYL719 (1 µM) + AZD8186 (250 nM) or Veh for 4 h. e CellTiter-Glo assay showing cell viability of MSK-PCa12-sh Renilla or MSK-PCa12-shIGF1R lines treated with BYL719 (1 µM) + AZD8186 (250 nM) or Veh, Day 7. f Growth of MSK-PCa12-sh Renilla or MSK-PCa12-sh IGF1R tumors in SCID mice treated with BYL719 (25 mg/Kg) + AZD8186 (75 mg/Kg) or Veh ( n = 5 mice per group). g Western blow showing levels of INSR, IGF1R, pAKT, pPRAS40, and pS6 in MSK-PCa12-sh Renilla or MSK-PCa12-shIGF1R tumors from ( f ), 14 days post treatment. h Volume of MSK-PCa12 tumors in SCID mice treated with linsitinib (40 mg/kg), BKM120 (50 mg/kg), linsitinib + BKM120, or Veh, 14 days post treatment, box and whiskers Min to Max.all points ( n = 10 mice per group). i Body weight analysis of mice in ( h ) ( n = 10 mice per group). Source data are provided as Source data file. All assays were performed with three biological replicates. **** p < 0.0001, *** p = 0.0003, ** p = 0.0041, n.s: not significant, a , b : multiple t -test two-sided, e , h : one-way ANOVA compared to Veh group, f : one-way ANOVA compared to shIGF1R group treated with BYL719 + AZD8186, a , b , e : error bar represents mean values ± SD, f , h : error bar represents mean values ± SEM, i : error bar represents ± SD.

Article Snippet: The PI3K p110α-selective inhibitor BYL719 was purchased from LC Laboratories (cat# A-4477).

Techniques: Growth Assay, Glo Assay, Western Blot

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a Western blot showing levels of AKT signaling in MSK-PCa12 organoids treated with ipatasertib (500 nM) or BYL719 (1 µM) + AZD8186 (250 nM) for 4 h. b CellTiter-Glo assay showing cell viability of MSK-PCa12 organoids treated with BYL719 (1 µM) + AZD8186 (250 nM), enzalutamide (10 µM), ipatasertib (500 nM), enzalutamide + ipatasertib, or Veh, Day 7. c Growth of MSK-PCa12 tumors in SCID mice treated with enzalutamide (10 mg/kg), ipatasertib (50 mg/kg), BYL719 (25 mg/kg) + AZD8186 (75 mg/kg), ipatasertib + enzalutamide, BYL719 + AZD8186 + enzalutamide or Veh. Some of mice were castrated when tumor reached ~200 mm 3 ( n = 5 mice per group) *** p -value = 0.0011. d Growth of CWR22Pc tumors in SCID mice treated with BYL719 (25 mg/kg), ipatasertib (50 mg/kg) or Veh. Some of mice were castrated when tumor reaches ~200 mm 3 ( n = 5 mice per group) Castration + BYL719 vs. Castration + Ipatasertinib, week 2: p -value = 0.045, week 3: p -value = 0.0086, week 4: p -value = 0.012, week 5: p -value = 0.046. e Western blot showing PI3K/AKT/MAPK signaling in CWR22Pc tumors from ( d ). f – h Heat map showing drug sensitivity of ipatasertib (500 nM) or BYL719 (1 µM) + AZD8186 (250 nM) on day 7, in cells characterized as PTEN wild type with PIK3CA mutants or RTK hyperactive ( f ), PIK3CA mutants with PTEN -loss ( g ), and PIK3CA wild type with PTEN -loss ( h ). Genetically manipulated lines are isogenic pairs. i Mechanism model of PI3K pathway alterations and response to clinically available therapeutic agents. All assays were performed with three biological replicates. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, n.s: not significant, b : one-way ANOVA compared to Veh group, error bar represents mean values ± SD. c , d : multiple t -test, two-sided error bar represents mean values ± SEM.

Article Snippet: The PI3K p110α-selective inhibitor BYL719 was purchased from LC Laboratories (cat# A-4477).

Techniques: Western Blot, Glo Assay