p-f4516  (Echelon Biosciences)

Journal: Molecular and Cellular Biology

Article Title: INPP5E Preserves Genomic Stability through Regulation of Mitosis

doi: 10.1128/MCB.00500-16

Figure Lengend Snippet: Stable INPP5E knockdown weakens the SAC in HeLa cells and primary human fibroblasts. (A) INPP5E levels in cell lines stably expressing the indicated shRNAs. (B) Accumulation of an INPP5E substrate, PI(4,5)P 2 , in INPP5E knockdown HeLa cells. (C) Representative images of the indicated cell lines treated with paclitaxel for 22 h. Note multinucleation reflecting a weakened SAC in INPP5E-deficient HeLa cells and fibroblasts. (D) Quantification of SAC assay results. (E) Representative time-lapse images of Inpp5e flox/flox MEFs transduced with negative-control GFP lentivirus (top panel) and GFP-Cre recombinase (bottom panel) following paclitaxel exposure. Note accelerated SAC escape in the Inpp5e flox/flox cell. (F) Quantification of the length of time between NEB and SAC escape. The P value was calculated with an unpaired t test. For both cell types, n = 30 (two pooled experiments). (G) Western blot of whole-cell lysates from Inpp5e flox/flox MEFs transduced with lentivirus encoding GFP control or GFP-fused Cre recombinase.

Article Snippet: For paclitaxel-PI(4,5)P 2 treatment, PI(4,5)P 2 [metabolically stable PI(4,5)P 2 P-F4516; Echelon Biosciences] was combined with unlabeled Shuttle PIP Carrier 3 (P-9C3; Echelon Biosciences) per the manufacturer's instructions, and the complex was diluted to a final concentration of 10 μM in growth medium.

Techniques: Stable Transfection, Expressing, Transduction, Negative Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: INPP5E Preserves Genomic Stability through Regulation of Mitosis

doi: 10.1128/MCB.00500-16

Figure Lengend Snippet: Excess PI(4,5)P 2 impairs the SAC. (A) Assay schematic. Prolonged SAC arrest triggers cell death unless checkpoint escape occurs. (B) Representative time-lapse images of cells treated with paclitaxel alone versus paclitaxel plus PI(4,5)P 2 . (C) Cells treated with paclitaxel plus PI(4,5)P 2 are less likely to die and more likely to escape SAC upon prolonged arrest within 24 h of mitotic entry than cells treated with paclitaxel alone ( n = 100 arrested cells tracked via time-lapse imaging per condition; P values were calculated with Fisher's exact test). Percentages of categorical values are shown. (D) Cumulative incidence of SAC escape in cells treated with paclitaxel plus DMSO versus paclitaxel plus PI(4,5)P 2 . The P value for risk of SAC escape was calculated with the log rank Mantel-Cox test. (E) Representative images of HeLa cells stably expressing shRNA against INPP5E stained with a PI(4,5)P 2 -specific antibody after a 24-hour exposure to DMSO (top) or the PIP5K1C/PIP4K2C inhibitor UNC3230 (100 nM) (bottom). Note decreased nuclear PI(4,5)P 2 in UNC3230-treated cells. (F) Cumulative incidence of SAC escape in stable INPP5E knockdown HeLa cells treated with paclitaxel plus DMSO versus paclitaxel plus UNC3230. The P value for risk of SAC escape was calculated with the log rank Mantel-Cox test. For DMSO- and UNC3230-treated cells, n = 53 and 60, respectively.

Article Snippet: For paclitaxel-PI(4,5)P 2 treatment, PI(4,5)P 2 [metabolically stable PI(4,5)P 2 P-F4516; Echelon Biosciences] was combined with unlabeled Shuttle PIP Carrier 3 (P-9C3; Echelon Biosciences) per the manufacturer's instructions, and the complex was diluted to a final concentration of 10 μM in growth medium.

Techniques: Imaging, Stable Transfection, Expressing, shRNA, Staining

Journal: Molecular and Cellular Biology

Article Title: INPP5E Preserves Genomic Stability through Regulation of Mitosis

doi: 10.1128/MCB.00500-16

Figure Lengend Snippet: Phosphoinositides localize to mitotic centrosomes. (A) HeLa cells stained with a PI(4,5)P 2 -specific antibody and anti-phospho-Aurora to mark mitotic centrosomes. (B) Representative images of HeLa cells transiently expressing an RFP-fused construct of the PI(4,5)P 2 - and PI(3,4,5)P 3 -binding domain of phospholipase C (PLCδ-PH). Cells were costained with antipericentrin to mark centrosomes.

Article Snippet: For paclitaxel-PI(4,5)P 2 treatment, PI(4,5)P 2 [metabolically stable PI(4,5)P 2 P-F4516; Echelon Biosciences] was combined with unlabeled Shuttle PIP Carrier 3 (P-9C3; Echelon Biosciences) per the manufacturer's instructions, and the complex was diluted to a final concentration of 10 μM in growth medium.

Techniques: Staining, Expressing, Construct, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: INPP5E Preserves Genomic Stability through Regulation of Mitosis

doi: 10.1128/MCB.00500-16

Figure Lengend Snippet: INPP5E regulates nucleation of spindle microtubules at mitotic centrosomes. (A) Assay schematic. (B) Cold spindle destabilization in control and INPP5E knockdown prometaphase cells. (C) Impaired microtubule spindle reassembly in a representative INPP5E knockdown cell compared to a control. (D and E) Quantification of the number of microtubules per centrosome (D) and the microtubule length (E). P values were calculated by two-tailed t tests ( n = 14 centrosomes/170 microtubules for controls and 20 centrosomes/76 microtubules for INPP5E knockdown cells). (F) Representative image of prometaphase microtubule repolymerization in cold-treated cells following treatment with carrier only (top panel) or 20 μM PI(4,5)P 2 (bottom panel). (G) Quantification of microtubule length. the P value was calculated with an unpaired t test. For control cells, n = 175; for PI(4,5)P 2 -treated cells, n = 76.

Article Snippet: For paclitaxel-PI(4,5)P 2 treatment, PI(4,5)P 2 [metabolically stable PI(4,5)P 2 P-F4516; Echelon Biosciences] was combined with unlabeled Shuttle PIP Carrier 3 (P-9C3; Echelon Biosciences) per the manufacturer's instructions, and the complex was diluted to a final concentration of 10 μM in growth medium.

Techniques: Two Tailed Test

Journal: The Journal of General Physiology

Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

doi: 10.1085/jgp.200609576

Figure Lengend Snippet: Mechanism of NGF-mediated sensitization. Simplified cartoon representation of the TRPV1-PI3K-trkA signal transduction complex (above) and two models of NGF-mediated sensitization (below). The PIP2 headgroups are shown in green and the PIP3 headgroups are shown in pink.

Article Snippet: DiC16-PIP2 (Echelon Biosciences) was solubilized in tetrahydrofuran:water (4:1), dried under N 2 , and resuspended to a stock concentration of 0.5 mM in water by bath sonication for 45 min. Stocks were divided into aliquots and frozen at −80°C.

Techniques: Transduction

Journal: The Journal of General Physiology

Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

doi: 10.1085/jgp.200609576

Figure Lengend Snippet: PIP2 is a potentiating molecule for TRPV1, not an inhibitory molecule. (A) Excised inside-out membrane patches were pulled from F-11 cells transfected with TRPV1. Points are steady-state currents recorded during a 100-ms pulse to +80 mV (positive inside relative to outside) from a holding potential of 0 mV. Zero current is indicated by the dotted line. Capsaicin and polylysine (>300 kD) were applied for the duration of the bars. We chose 30 μg/ml polylysine because it has recently been shown to be effective in sequestering PIP2 from TRPM4 channels . Inhibition by polylysine did not spontaneously reverse over the time scale of our experiments, suggesting that its unbinding from the PIP2 is quite slow. (B) Currents and cells as in A. PIP2 (10 μM) and capsaicin were applied during the time of the bar. The delay observed between PIP2 application and the increase in current arose from the special system we devised to apply very small amounts of the expensive phosphoinositide to our cell chamber (see Materials and methods). (C) Application of PIP2 to an untransfected F-11 cell. (D) Water-soluble DiC8-PIP2 (red bar) reversibly potentiated capsaicin-activated current. Inside-out excised patch from F-11 cell transfected with TRPV1. (E) Two representative inside-out patches from mouse DRG neurons. The open and filled black bars as above. The red bar represents DiC8-PIP2. The time scale bar applies to both panels, but each has its own scale bar for current.

Article Snippet: DiC16-PIP2 (Echelon Biosciences) was solubilized in tetrahydrofuran:water (4:1), dried under N 2 , and resuspended to a stock concentration of 0.5 mM in water by bath sonication for 45 min. Stocks were divided into aliquots and frozen at −80°C.

Techniques: Transfection, Inhibition

Journal: The Journal of General Physiology

Article Title: Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

doi: 10.1085/jgp.200609576

Figure Lengend Snippet: Ruthenium red (RR) and capsazepine (CPZ) reduce the amplitude of PIP2-potentiated currents. (A) Currents from inside-out excised patches in which the membrane was held and 0 mV and the potential was jumped to +80 mV, to −80 mV, and back to 0 mV. Note that RR is believed to be a voltage-dependent blocker of the pore, and thus is less effective at hyperpolarized potentials. In contrast, CPZ may allosterically inhibit activation, giving similar reductions in current at depolarized and hyperpolarized potentials. RR (blue, top) and CPZ (red, bottom) were applied to different patches. (B) Box plot showing the percent reduction in current at +80 mV for RR and CPZ. For comparison, the percent inhibition of capsaicin-activated currents by RR in the absence of PIP2 is shown (first box). Boxes enclose the 25th to 75th percentile of the data, lines within the boxes represent the median, and whiskers extend to the 10th and 90th percentiles.

Article Snippet: DiC16-PIP2 (Echelon Biosciences) was solubilized in tetrahydrofuran:water (4:1), dried under N 2 , and resuspended to a stock concentration of 0.5 mM in water by bath sonication for 45 min. Stocks were divided into aliquots and frozen at −80°C.

Techniques: Activation Assay, Inhibition