Journal: bioRxiv

Article Title: Combined inhibition of mTOR and PIKKs exploits replicative and checkpoint vulnerabilities to induce death of PI3K-activated triple-negative breast cancer cells

doi: 10.1101/700625

Figure Lengend Snippet: A . Schematic of the PI3K pathway with select drugs (blue) and measured proteins (red). B . Clustergram heatmap of mean GR max values for 24 drugs in six TNBC cell lines; N=3 experiments. Darker shading denotes GR max <0, which indicates cytotoxicity. Drugs in red are the most effective; the drug in blue is a MAPK inhibitor. Colored circles mark drugs analyzed in panels D-G. C. GR max and GR 50 values for the indicated drugs in a second analysis of 19 TNBC cell lines. *P<0.05, **P<0.01, ***P<0.001, and n.s. (not significant) by Mann-Whitney U test. D . Population growth curves for HCC1806 NLS-mCherry cells. “*” denotes start of treatment. Data points/shading depict mean ± SD of 3 replicates in a single time-lapse experiment; N=3 experiments, representative data shown. E. Mean nuclear F-ara-EdU intensity values in HCC1806 cells after treatment with various drugs for 24h followed by labeling for 75h; N=3 experiments, representative data shown. “a.u.” indicates arbitrary units. Shaded area indicates unlabeled cells. F . C3/C7-positive cell counts over time as detected by live-cell imaging of HCC1806 cells. Shading indicates AUC for 72h of drug exposure. G . C3/C7 activity ( i.e. , mean fold-change in AUC ± SEM for drug vs. DMSO) for PI3K pathway drugs in six TNBC cell lines; N=2 experiments. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA and Tukey’s test (only selected comparisons shown). Drugs were used at GR max doses (1–3.2μM) in D, F and G. In E, Torin2 and omipalisib were used at 0.3xGR max (1μM) due to cell death at higher doses.

Article Snippet: All compounds except Torin2 analogs were obtained from MedChem Express.

Techniques: MANN-WHITNEY, Labeling, Live Cell Imaging, Activity Assay

Journal: bioRxiv

Article Title: Combined inhibition of mTOR and PIKKs exploits replicative and checkpoint vulnerabilities to induce death of PI3K-activated triple-negative breast cancer cells

doi: 10.1101/700625

Figure Lengend Snippet: A . Clustergram heatmaps rank the activity of PI3K pathway drugs based on mean values of the levels and/or localization of nine proteins/phosphoproteins at 24h post-treatment. Data are normalized to DMSO; N≥2 experiments. Drugs were used at GR max doses (1-3.2μM) except for Torin2 and omipalisib, which were used at 0.3xGR max (1μM) in HCC1806 cells due to cell death. B. Mean nuclear intensity values of phospho-pRb S807/811 (p-pRb) in HCC1806 cells at t=T d (28h) post-treatment. C . Cell cycle distributions at t=T d post-treatment for BT-20 (48h), HCC70 (45h) and HCC1806 (28h) based on analysis of nuclear DNA content vs EdU content. Colors indicate cell cycle stage: G1 (black), active S phase (red), S-phase non-replicating (S NR ; yellow), and G2/M (blue). Drugs are ordered from least effective to most effective (top to bottom) based on viable cell counts at t=T d . GR max doses (1–3.2μM) were used for all drugs except Torin2 and omipalisib, which were used at 0.3xGR max dose (BT-20: 1μM; HCC70: 0.32–1μM; HCC1806: 1μM) due to cell death. D . Top row : Nuclear DNA content vs EdU content in HCC70 and HCC1806 cells at t=T d . Data points for DMSO (grey) are shown in background for comparison. Doses were as noted in C. Middle, bottom rows : Nuclear DNA content vs. mean nuclear intensity values for geminin or cyclin A2 at t=T d . Colors indicate different cell cycle stages as determined by DNA content and EdU content values measured in the same cells. In HCC70 only, Torin2 and omipalisib were used at 0.1xGR max dose (0.1–0.32μM) due to cell death. E . Quantification of total nuclear EdU content at time=T d in gated S-phase cells (red cells, panel D, top row; S NR cells excluded). For HCC70 and HCC1806 cells, doses were as noted in C. For Hs 578T cells, GR max doses (1–3.2μM) were used for all except Torin2 and omipalisib, which were used at 0.3xGR max dose (1μM). For BT-549 cells, AZD8055 was used at 0.3xGR max dose (0.32μM), and omipalisib and Torin2 were used at 0.03xGR max dose (0.1μM) due to low numbers of S-phase cells at higher doses. Al:alpelisib, A8:AZD8055, D:DMSO, M:MK-2206, O:omipalisib, R:rapamycin, T2:Torin2. ***, P<0.001 for each drug vs DMSO (Mann-Whitney U test). F . Volcano plots of log 2 (fold change) vs. –log 10 (p-value) for intracellular polar metabolites after exposure of BT-549 cells to GR max doses of rapamycin or AZD8055 or 0.3xGR max dose of Torin2 (1μM for all three drugs) for t=0.15xT d (6h). Downregulated metabolites appear in blue, upregulated metabolites appear in red. Nucleotides and precursors are labeled.

Article Snippet: All compounds except Torin2 analogs were obtained from MedChem Express.

Techniques: Activity Assay, Comparison, MANN-WHITNEY, Labeling

Journal: bioRxiv

Article Title: Combined inhibition of mTOR and PIKKs exploits replicative and checkpoint vulnerabilities to induce death of PI3K-activated triple-negative breast cancer cells

doi: 10.1101/700625

Figure Lengend Snippet: A. Time-lapse imaging of asynchronous HCC1806 cells expressing H2B:mTurquoise and mVenus:hGeminin(1-110). mTurquoise was used to score cell division and death; mVenus intensity levels were used to identify cell cycle stage, as illustrated for a representative cell exposed to DMSO. Heatmaps show the progression of single cells through the cell cycle after exposure to DMSO or a 0.3xGR max dose (1μM) of omipalisib or Torin2 for 48h. Colors denote cell cycle stages and cell fates, as shown in the figure legend. B . Left plots show the frequency of deaths and cell cycle block caused by omipalisib and Torin2; right plots show the frequency of these events by cell cycle stage. C . Frequencies of different classes of responses leading to G1 death or block. Class 1 : a cell in G1 at the start of treatment is killed or blocked in the same G1; class 2 : a cell in S/G2 at start of treatment proceeds to division, and the tracked daughter cell is killed or blocked in the following G1; class 3 : similar to class 2, except the cell starts in G1. D . Phenotypic responses caused by drug exposure. Shown are representative traces of mVenus fluorescence intensity vs. time for single cells (omipalisib in blue, Torin2 in red). For comparison, the trace for a representative cell exposed to DMSO is shown in the background (grey).

Article Snippet: All compounds except Torin2 analogs were obtained from MedChem Express.

Techniques: Imaging, Expressing, Blocking Assay, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Combined inhibition of mTOR and PIKKs exploits replicative and checkpoint vulnerabilities to induce death of PI3K-activated triple-negative breast cancer cells

doi: 10.1101/700625

Figure Lengend Snippet: HCC1806 cells were exposed to 0.3-1xGR max drug doses (1–3.2μM) throughout. A. DNA content vs. intensities of native BrdU, γH2A.X or p-RPA S4/8 after exposure to the indicated drugs for ∼0.2xT d (5h). Cells with increased levels are gated and marked in red. Percentages are of the entire treated population. B . Quantification of nuclear intensity values of native BrdU, γH2A.X, and p-RPA in gated S-phase cells at 5h post-treatment. Boxplots show median and 25th/75th percentiles. D:DMSO, A8:AZD8055, O:omipalisib, T2:Torin2, AZ:AZ20, R:rabusertib. C . Mean percent ± SEM of cells with increased levels of native BrdU, γH2A.X, p-RPA S4/8, p-Chk1 S317 and p-Chk2 T68; N=3 experiments. **P<0.01, ***P<0.001 vs DMSO by one-way ANOVA and Dunnett’s test. D . Mean intensity for EdU vs. γH2A.X after treatment with DMSO or 1μM Torin2 for the indicated times. EdU/γH2A.X double-positive cells (red gate) and EdU-negative/γH2A.X-positive cells (black gate) are quantified as a percent of all EdU-positive (red) or EdU-negative (black) cells, respectively. E . DNA content vs mean EdU intensity at 24h post-treatment with DMSO or 1μM Torin2. Cells with elevated γH2A.X levels (> 10 3 a.u.) are marked in red. F . Nuclear γH2A.X staining pattern in S-phase/S NR cells at 24h post-treatment with drugs at 1μM. Pan-nuclear γH2A.X staining is an indicator of high genotoxic stress. G . Percent of S-phase cells with pan-nuclear γH2A.X staining (red) vs. more focal staining (black) after exposure to DMSO or 1μM Torin2. H. γH2A.X staining pattern in various TNBC cell lines after treatment with 1μM Torin2. Scale bars in F and H=20μm.

Article Snippet: All compounds except Torin2 analogs were obtained from MedChem Express.

Techniques: Staining

Journal: bioRxiv

Article Title: Combined inhibition of mTOR and PIKKs exploits replicative and checkpoint vulnerabilities to induce death of PI3K-activated triple-negative breast cancer cells

doi: 10.1101/700625

Figure Lengend Snippet: A. Dose-response curves (72h) for AZD8055, Torin2, rabusertib, and AZD8055/rabusertib mixed at equimolar concentrations in HCC1806 and HCC70 cells. B . Dose-response curves for AZD8055, Torin1, Torin2 and nine different Torin2 analogs (72h). C . Mean ± SEM of GR max and GR 50 values for Torin1, Torin2, AZD8055 and Torin2 analogs in HCC1806 and HCC70 cells; N=3 experiments. D . Levels of p-AKT S473, p-4E-BP1 T37/46 and γH2A.X at t=0.5xT d (14h) post-treatment vs. GR values computed after 72h of exposure of HCC1806 cells to 0.032–3.2μM of AZD8055, Torin1, Torin2 or Torin2 analogs. Drugs/doses whose GR values correlate with mTORC1/2 signaling activity alone fall on the black regression lines ( left, middle ). Drugs/doses whose activities are discontinuous with the black regression line fall on the vertical red lines ( left, middle ) and cause increased levels of γH2A.X ( right ). E . DNA content vs. nuclear γH2A.X or p-pRb levels at 14h for DMSO (#1) and drugs/doses whose activities correlate with mTORC1/2 signaling activity (#2-4) and those whose activities do not (#5-8). Percentages of gated γH2A.X-high or p-pRb-low cells are relative to entire treated population. F . Mean percent ± SEM of γH2A.X-high cells or p-pRb-low cells at 14h post-treatment with various Torin2 analogues; N=3 experiments. **P<0.01, ***P<0.001 by one-way ANOVA and Dunnett’s test. G . Nuclear γH2A.X staining pattern in S-phase cells after exposure to indicated treatments for 14h. Scale bar=20μm.

Article Snippet: All compounds except Torin2 analogs were obtained from MedChem Express.

Techniques: Activity Assay, Analogues, Staining

Journal: Science Advances

Article Title: Stress granule dynamics govern TOR reactivation and growth recovery during post-heat stress adaptation

doi: 10.1126/sciadv.adv6202

Figure Lengend Snippet: ( A ) TOR activity was strongly inhibited by Torin2, AZD8055, and glucose depletion in 4-day-old 35Spro::S6K1-HA/Col-0 seedlings. TOR activity was assessed by Western blot using an antibody against phosphorylated Thr 449 in S6K1 (S6K1-pT449). Glc, glucose. Torin2 and AZD8055, TOR kinase inhibitors, which were pretreated for 1 hour before sample collection. ( B ) Quantification data of (A). Three biological repeats. Mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA). * indicates P value < 0.05. ( C ) Inhibition of TOR did not trigger SG formation in the roots of PAB4pro::PAB4-GFP and RBP47bpro::GFP-RBP47b seedlings. Scale bars, 10 μm. Torin2 and AZD8055 were pretreated for 1 hour before imaging. ( D ) Heat-induced SG formation was independent of TOR signaling. HS, heat stress treatment. Scale bars, 10 μm. Torin2 and AZD8055 were pretreated for 1 hour before heat treatment.

Article Snippet: Torin2 (1 μM, Tocris Bioscience, catalog no. 4248), AZD8055 (1 μM, Selleckchem, catalog no. S1555), and CHX (200 ng/μl; INALCO, catalog no. 1758-9310) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Activity Assay, Western Blot, Inhibition, Imaging

Journal: iScience

Article Title: OsFKBP12 transduces the sucrose signal from OsNIN8 to the OsTOR pathway in a loosely binding manner for cell division

doi: 10.1016/j.isci.2024.111555

Figure Lengend Snippet:

Article Snippet: Torin2 , Immunoway , Cat# MC0120.

Techniques: Binding Assay, Virus, Recombinant, Magnetic Beads, SYBR Green Assay, Luciferase, Lysis, Mutagenesis, CRISPR, Knock-Out, Over Expression, Knockdown, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: OsFKBP12 transduces the sucrose signal from OsNIN8 to the OsTOR pathway in a loosely binding manner for cell division

doi: 10.1016/j.isci.2024.111555

Figure Lengend Snippet:

Article Snippet: Four inhibitors, AZD8055 (Macklin, A837230), Torin 2 (Immunoway, MC0120), Rapamycin (Biorbyt, orb61092) and FK506 (abcam, ab120223), all were hydrophobic and required to dissolve in dimethyl sulfoxide (DMSO), but concentration of DMSO in the medium greater than 10 μL/mL would inhibit growth of sprout during germination in rice ( ).

Techniques: Binding Assay, Virus, Recombinant, Magnetic Beads, SYBR Green Assay, Luciferase, Lysis, Mutagenesis, CRISPR, Knock-Out, Over Expression, Knockdown, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction