Journal: PLoS ONE

Article Title: S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton

doi: 10.1371/journal.pone.0004881

Figure Lengend Snippet: A–B), S9 depresses EGF-triggered activation of PI3K-Akt-mTOR signaling pathway. Serum-deprived Rh30 cells (A) and SK-OV-3 cells (B) were treated with indicated concentrations of S9 for 1 h followed by EGF (50 ng/ml) stimulation for 10 min. Cells were harvested for Western blot analysis with antibodies specific for p-PDK1 (S241), PDK, p-Akt (T308), p-Akt (S473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP1(T37/64), p-4E-BP1 (T70), 4E-BP1 and actin. Arrows indicate p70 isoform of S6 kinase protein. C) S9 blocks Akt membrane translocation and membrane ruffling. CHO (pCORON1000-EGFP-Akt) cells seeded on chamber were starved for 2 h then treated with 5 or 10 µM S9 for 1 h followed by IGF stimulation for 5 min. Fluorescent pictures were captured with confocal fluorescent microscopy. White arrows indicate cell membrane ruffling. Blue arrows indicate fluorescent foci. D) Total Akt granule intensity in each CHO (pCORON1000-EGFP-Akt) cell was counted with statistic module by IN Cell Analyzer 1000. Data shown are representative from two independent experiments.

Article Snippet: Effect of S9 on PI3K activity was measured by PI3K ELISA kit (Echelon Biosciences Inc., Salt Lake City, UT) according to the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Translocation Assay, Microscopy

Journal: PLoS ONE

Article Title: S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton

doi: 10.1371/journal.pone.0004881

Figure Lengend Snippet: A–B) Active kinases were immunoprecipitated from exponentially growing Rh30 cells and kinase assays were performed. In the presence of indicated concentrations of S9, phosphorylation PIP2 to PIP3 were measured by ELISA (A). Phosphorylation of 4E-BP1 (B) was detected by Western blot. Band intensity was quantitated by optical densitometric analysis and normalized to vehicle control. Representative images were presented and relative kinase activity was plotted as mean±SD of three independent experiments. C) S9 abrogates rapamycin-induced hyperphosphorylation of Akt (Ser 473). Rh30 cells were starved overnight and treated with 10 µM of S9 and/or rapamycin (Rapa) for 1.5 h on the next day, followed by stimulation with 50 ng/ml EGF for 10 min. Phosphorylated Akt at Serine 473 and Threonine 308 and total Akt were detected by Western blot. D) Rh30 cells were exposed to 10 µM camptothecin (CPT) for 1 h after pre-incubation with indicated concentrations of S9 or wortmannin (Wort) for 30 min. Cells were collected and γ-H2AX levels were detected with Western blot analysis. E–F) The binding mode of S9 within PI3K (E), mTOR (F). The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. All of the structural diagrams were prepared using PyMOL ( http://pymol.sourceforge.net ).

Article Snippet: Effect of S9 on PI3K activity was measured by PI3K ELISA kit (Echelon Biosciences Inc., Salt Lake City, UT) according to the manufacturer's instructions.

Techniques: Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

doi: 10.1016/j.jbc.2022.102187

Figure Lengend Snippet: VPS34 activity is elevated upon PIKfyve inhibition in Inpp4b -deficient cells. A , Inpp4b +/+ or Inpp4b −/− MEFs treated with vehicle or apilimod 10 nM for 48 h, followed by immunoblotting against Inpp4b, VPS34, or Beta Actin. B , Inpp4b +/+ or Inpp4b −/− MEFs treated with vehicle or apilimod 10 nM for 48 h, followed by VPS34 immunoprecipitation and kinase assay to monitor VPS34 activity. C , Inpp4b +/+ MEF transiently expressing LAMP1-mCherry and treated with vehicle or 500 nM VPS34-IN1 for 48 h followed by PtdIns(3)P immunostain. D , quantification from ( C ) of total cell PtdIns(3)P fluorescence signal or ( E ) PtdIns(3)P fluorescence signal overlayed on LAMP1-mCherry positive regions within a cell. The scale bar represents 20 μm, zoomed inset: 5 μm. F , Inpp4b +/+ or Inpp4b −/− MEF transiently expressing LAMP1-mCherry and treated with vehicle or apilimod 10 nM or VPS34-IN1 500 nM at various combinations for 48 h. G , quantification of LAMP1-positive vacuoles greater than 1.5 μm in diameter per cell and ( H ) mean vacuole diameter (μm). The scale bar represents 20 μm. Data represent ± SD from three independent experiments with 25 to 30 cells assessed per treatment condition per experiment. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ ( p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PtdIns, phosphoinositide; PtdIns(3)P, phosphatidylinositol-3-monophosphate; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.

Article Snippet: Kinase assay performed on immunoprecipitated VPS34 using Class III PI3K ELISA Kit (K-3000, Echelon) through the protocol recommended for beads conjugated to enzyme.

Techniques: Activity Assay, Inhibition, Western Blot, Immunoprecipitation, Kinase Assay, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

doi: 10.1016/j.jbc.2022.102187

Figure Lengend Snippet: Model for lysosome enlargement upon PIKfyve inhibition in Inpp4b deficient cells. A , in the steady state, cycling of phosphoinositides involve VPS34-dependent conversion of phosphatidylinositol (PtdIns) to PtdIns(3)P, PtdIns(3,4)P 2 conversion by Inpp4b to PtdIns(3)P, and PtdIns(3)P conversion by PIKfyve to PtdIns(3,5)P 2 . Apilimod-induced PIKfyve inhibition deplete PtdIns(3,5)P 2 to elevate the substrate PtdIns(3)P, and simultaneous PIKfyve and Inpp4b suppression further augment PtdIns(3)P levels. B , PIKfyve inhibition in Inpp4b +/+ MEF induce moderate lysosome enlargement, where lysosomes coalesce to become fewer in number and greater in individual lysosome volume. PIKfyve inhibition in Inpp4b −/− MEF exacerbate lysosome enlargement, where lysosomes are much greater in individual volume and this enlargement is facilitated by enrichment of VPS34-induced membrane PtdIns(3)P. INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PtdIns, phosphoinositide; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns(3)P, phosphatidylinositol-3-monophosphate; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing.

Article Snippet: Kinase assay performed on immunoprecipitated VPS34 using Class III PI3K ELISA Kit (K-3000, Echelon) through the protocol recommended for beads conjugated to enzyme.

Techniques: Inhibition