Fig 1: PI3K/AKT inhibitor post-translationally downregulate TRF1 levels. a Quantification of total nuclear TRF1 levels in lung cancer-derived cells (CHA 9-3) treated either with DMSO, AKT inhibitor (AKTi) (10 μM), ETP47037 (10 μM), or ETP47228 (10 μM). A representative western blot image is shown below. b Quantification of total nuclear TRF1 levels in in immortalized p53 −/− MEFs treated either with DMSO, AKT inhibitor (AKTi) (10 μM), or ETP47037 (10 μM). A representative western blot image is shown below. c Quantification of total nuclear TRF1 levels in P110α lox/lox MEFs cells transduced with pBabe-GFP or with pBabe-Cre recombinase. A representative WB images is shown below. d Quantification by immunofluorescence of mean TRF1 foci intensity in CHA 9-3 cells treated either with DMSO, DMSO plus bortezomib (50 nM), ETP47037 (10 μM) plus bortezomib (50 nM), ETP47228 (10 μM) plus bortezomib (50 nM), and AKTi (10 μM) plus bortezomib (50 nM). Student’s t test was used for statistical analysis; P values are shown. Error bars represent standard error. n number of independent experiments
Fig 2: Chemical inhibition of TRF1 binding to telomere by PI3K inhibitors. a Structures of ETP-47037, ETP-47228, and their corresponding “inactive analogs” ETP-51259 and ETP-50952. b PI3K/mTOR IC50 data generated internally and reported for the inhibitors used in the study. c Time course Inhibition of AKT phosphorylation at Ser473 by ETP-47037, ETP-51259, ETP-47228, and ETP-50952 at 10 μM in CHA-9.3 cell line. d Percent inhibition of TRF1 foci by immunofluorescence in CHA-9.3 mouse lung tumor cell line treated with 10 μM of either ETP-47037, ETP-47228, or their corresponding inactive analogs (ETP-51259 and ETP-50952, respectively) relative to TRF1 levels with DMSO treatment. The ETP compound inhibitory activity on PI3K pathway is stated at the bottom of the graph. Error bars represent standard deviation. N/D not determined, n number of independent experiments
Fig 3: TRF1 regulation by PI3K and AKT inhibitors. a Structurally diverse PI3K and PI3K/mTOR inhibitors used in the study. b PI3K and mTOR data generated internally and reported in literature. c Representative western blot images of phosphorylated AKT-Ser473 and total AKT in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with PI3K, AKT, and mTOR inhibitors as indicated. d Percent inhibition of TRF1 foci by immunofluorescence and of AKT phosphorylation at S473 (pAKT) in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with the indicated inhibitors relative to TRF1 levels and to pAKT levels in control cells treated with DMSO. The inhibitors were tested at 10 μM except GSK-2126458 that was used at 1.0 μM (in c and d). Error bars represent standard deviation. The Student’s t test was used for statistical analysis; P values are shown. n number of independent experiments
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