pi3kp110α  (Echelon Biosciences)

Journal: Molecular Cancer

Article Title: PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells

doi: 10.1186/1476-4598-9-85

Figure Lengend Snippet: Differential expression of PI3K catalytic isoforms and DOCK2 by PCa cell lines . (A) Total cell lysates (60 μg) from RWPE-1, LNCaP, and PC3 cells were resolved by SDS-PAGE and subjected to immunoblotting using antibodies against PI3Kp110α, p110β, p110γ, p110δ, and DOCK2 (Santa Cruz). GAPDH detection served as a loading control. (B) DOCK2 silencing conditions were optimized by transfecting PC3 cells with 1 μg of DOCK2 siRNA duplex incubated cells for 0, 24, 48, and 72 hours. The efficacy of DOCK2 silencing was determined by Western blot analysis.

Article Snippet: All cell lines were serum starved overnight prior to treatment with 0 or 100 ng/ml of CXCL13 (PeproTech) in the presence or absence of: isotype control antibody or anti-human CXCR5 antibody (1 μg/ml, R&D systems), pertussis toxin (100 ng/ml, List Biological Laboratories), G protein β and γ inhibitor (U-73122, 5 μM, Sigma), wortmannin (1 μM, Sigma), small molecule inhibitors of PI3Kp110α (PI-103, 3 μM, Echelon), PI3Kp110β (TGX221, 1 μM, Cayman Chemicals), and PI3Kp110γ (AS605240, 3 μM, Echelon), Src (SU6656, 5 μM, Sigma), FAK (PF-573228, 5 μM, Pfizer), or DOCK2 siRNA (1 μg, Santa Cruz).

Techniques: Expressing, SDS Page, Western Blot, Incubation

Journal: Molecular Cancer

Article Title: PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells

doi: 10.1186/1476-4598-9-85

Figure Lengend Snippet: CXCR5 activate PI3K isoforms through Gα-Src-FAK pathway or via Gβ/γ pathway leading to subsequent activation of ERK1/2 . LNCaP cells express PI3Kp110α, PI3Kp110β and PI3Kp110δ, while PC3 cells express PI3Kp110α, PI3Kp110β and PI3Kp110γ. Perhaps owing to their more aggressive phenotype, PC3 cells are capable of exploiting both Gα-Src-FAK and Gβ/γ-mediated events leading to enhanced migration and invasion, while LNCaP cells are only able to take advantage of the Gα-Src-FAK-mediated pathway following CXCL13-CXCR5 interaction.

Article Snippet: All cell lines were serum starved overnight prior to treatment with 0 or 100 ng/ml of CXCL13 (PeproTech) in the presence or absence of: isotype control antibody or anti-human CXCR5 antibody (1 μg/ml, R&D systems), pertussis toxin (100 ng/ml, List Biological Laboratories), G protein β and γ inhibitor (U-73122, 5 μM, Sigma), wortmannin (1 μM, Sigma), small molecule inhibitors of PI3Kp110α (PI-103, 3 μM, Echelon), PI3Kp110β (TGX221, 1 μM, Cayman Chemicals), and PI3Kp110γ (AS605240, 3 μM, Echelon), Src (SU6656, 5 μM, Sigma), FAK (PF-573228, 5 μM, Pfizer), or DOCK2 siRNA (1 μg, Santa Cruz).

Techniques: Activation Assay, Migration

Journal: BMC Biology

Article Title: BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex

doi: 10.1186/1741-7007-12-43

Figure Lengend Snippet: BMP2 induces chemotaxis of multipotent mesenchymal C2C12 mouse myoblasts. (A) Trajectories of multipotent mouse mesenchymal C2C12 cells migrating in a 2D chemotaxis chamber over period of 16 hours exposed to a linear BMP2 gradient compared to non-stimulated control or in the presence of the PI3K p110α selective inhibitor PI103 (8 nM). The gradient was produced by application of BMP2 to the upper reservoir. It was allowed to generate a linear concentration profile with a maximum concentration of approximately 10 nM reaching the cells on the edge of the observation area as described by the manufacturer. (B) Syntaxin 6 and DAPI stainings of C2C12 cells after BMP2-induced chemotaxis compared to non-stimulated control or PI103 [8 nM] pre-treatment. The location of the depicted cells within the chemotaxis chamber is indicated. Scale bar represents 20 μm. (2D) two dimensional; (BMP2) Bone Morphogenetic Protein 2; (PI3K) Phosphoinositide 3-kinase; (p110α) Class I PI3K catalytic subunit alpha; (DAPI) 4',6-diamidino-2-phenylindole.

Article Snippet: LY294002 was purchased from Cell Signaling Technology (Cell Signaling Technology Inc., Danvers, MA, USA) and PI103 was purchased from Echelon Bioscience (Echelon Bioscience Inc., Salt Lake City, USA).

Techniques: Chemotaxis Assay, Produced, Concentration Assay

Journal: BMC Biology

Article Title: BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex

doi: 10.1186/1741-7007-12-43

Figure Lengend Snippet: The PIP3-binding protein LL5β localises to BMP2-induced cortical actin-rich lamellipodia. (A) Upper panel shows colloidal Coomassie Blue staining of protein precipitates gained by precipitation of PIP2-, PIP3-coated and control beads from C2C12 cell lysates. Lower panel shows LL5β detection (approximately 160 kDa) by western blot. LL5β binds to PIP3 (lane 3) but not PIP2 or control beads (lanes 1 and 2). (B) LL5β-specific peptides (marked in yellow) as identified by mass spectrometry upon precipitation of PIP3-coated beads from C2C12 cell lysates. (C) Immunocytochemical stainings of endogenous LL5β and actin in C2C12 cells upon 45 minutes’ stimulation with 10 nM BMP2. Arrow indicates co-localisation of LL5β with cortical actin in BMP2-induced cell protrusions at the C2C12 cell leading edge (magnified region of interest). (D) PI103 pre-treatment blocks BMP2-induced co-localisation of LL5β with cortical actin. C2C12 cells were stimulated with 10 nM BMP2 for 45 minutes in the presence of DMSO or 8 nM PI103 respectively. The magnified region of interest depicts the co-localisation of LL5β with cortical actin. Scale bars represent 20 μm.

Article Snippet: LY294002 was purchased from Cell Signaling Technology (Cell Signaling Technology Inc., Danvers, MA, USA) and PI103 was purchased from Echelon Bioscience (Echelon Bioscience Inc., Salt Lake City, USA).

Techniques: Binding Assay, Staining, Western Blot, Mass Spectrometry

Journal: Oncotarget

Article Title: Amalgamation of PI3K and EZH2 blockade synergistically regulates invasion and angiogenesis: combination therapy for glioblastoma multiforme

doi: 10.18632/oncotarget.27842

Figure Lengend Snippet: ( A ) Boyden chamber analysis was performed for cell migration properties. Combination of drugs shows that a smaller number of migrated cells compared to control. ( B ) Wound healing assay shows the similar pattern of migration inhibition during combination of PI-103 & EPZ-6438. ( C ) Cytoskeleton analysis depicted the deregulation of actin and tubulin after treatment. It accounts for the morphological changes in the cellular architecture after the treatment EPZ and PI-103. ( D ) Adhesion properties of GBM U-87 cells was altered during treatment of combination of drugs and is one of the reasons of reduced cell migration during treatment.

Article Snippet: Inhibitor PI-103 was procured from Echelon Biosciences and EPZ-6438 from APExBIO.

Techniques: Migration, Wound Healing Assay, Inhibition

Journal: Oncotarget

Article Title: Amalgamation of PI3K and EZH2 blockade synergistically regulates invasion and angiogenesis: combination therapy for glioblastoma multiforme

doi: 10.18632/oncotarget.27842

Figure Lengend Snippet: ( A ) Cell cycle analysis was conducted to confirm the effect of EPZ and PI-103 on cell migration. Cells were arrested in G1 phase (85.7%) after the treatment with combination of drugs. ( B ) Cell apoptotic analysis was performed to get the insight into the effect of drug. DCFDA analysis showed the reduction of ROS in GBM U-87 cells. GBM U-87 cells have the high frequency of ROS at basal level. This data suggests that GBM U-87 cell progression is reduced after reduction in ROS. This data showed that cells are stuck in G1 phase but not going under apoptosis at this concentration.

Article Snippet: Inhibitor PI-103 was procured from Echelon Biosciences and EPZ-6438 from APExBIO.

Techniques: Cell Cycle Assay, Migration, Concentration Assay

Journal: Oncotarget

Article Title: Amalgamation of PI3K and EZH2 blockade synergistically regulates invasion and angiogenesis: combination therapy for glioblastoma multiforme

doi: 10.18632/oncotarget.27842

Figure Lengend Snippet: ( A ) Invasion assay was performed to analyze the effect of drug molecules on GBM U-87 cells. We found that invaded cells were less compared to the control cells as confirmed with Gelatin degradation assay. ( B ) Small patches showed the gelatin degradation in control and treated cells. We found that the number of invaded patches were less in combination of EPZ and PI-103 treated cells. ( C ) Invaded cells activity was also analyzed with MMP2 assay. We found that MMP -2 expression reduced while treating the cells. MMP-2 reduction suggests the inability of GBM (U-87) cells to invade the surrounding tissue. ( D ) CD24 expression represents the aggression of GBM U-87 cells. We found that combination of EPZ-6438 and PI-103 reduced the expression of CD24. Less expression of CD24 indicates higher reduction in metastasis. ( E ) Western blot analysis performed and showed effective reduction in EMT specific markers. Inhibition of β-catenin along with SMAD1 and SNAI3 showed the reduction of metastatic potential of GBM U-87 cells.

Article Snippet: Inhibitor PI-103 was procured from Echelon Biosciences and EPZ-6438 from APExBIO.

Techniques: Invasion Assay, Degradation Assay, Activity Assay, Expressing, Western Blot, Inhibition

Journal: Oncotarget

Article Title: Amalgamation of PI3K and EZH2 blockade synergistically regulates invasion and angiogenesis: combination therapy for glioblastoma multiforme

doi: 10.18632/oncotarget.27842

Figure Lengend Snippet: ( A ) Mammosphere assay was conducted for stemness properties. Two weeks analysis showed a high reduction in spheroid formation. We found that combination of EPZ and PI-103 reduces the effective stemness properties which promotes spheroid formation and sphere cells in-vitro . ( B ) To get the further role of treatment in GBM U-87 cells we performed colony forming assay. Clusters of cells during treatment highly reduced and cells were segregated. Treated cells were not able to make colonies for 2 weeks. Scale bar represents 100 μM.

Article Snippet: Inhibitor PI-103 was procured from Echelon Biosciences and EPZ-6438 from APExBIO.

Techniques: In Vitro

Journal: Oncotarget

Article Title: Amalgamation of PI3K and EZH2 blockade synergistically regulates invasion and angiogenesis: combination therapy for glioblastoma multiforme

doi: 10.18632/oncotarget.27842

Figure Lengend Snippet: ( A and B ) IFA and WB data showed that metastatic markers are altered during combination treatment. Which strongly support that metastasis inhibition during treatment of combination of drugs. ( C ) High level of Ca+2 helps in GBM (U-87) proliferation and stemness potential. We treated the cells with EPZ and PI-103 and found a significant reduction of Ca+2 levels. ( D ) Tube formation assay was performed for GBM (U-87) induced angiogenesis. We found that TCM of treated GBM U-87 cells showed a smaller number of branches sand tubes compare to alone and control cells. This data suggests the role of combined treatment of EPZ and PI-103 in tumor microenvironment. Graphical representation shows the nodes and branches details.

Article Snippet: Inhibitor PI-103 was procured from Echelon Biosciences and EPZ-6438 from APExBIO.

Techniques: Inhibition, Tube Formation Assay

Journal: Oncotarget

Article Title: Amalgamation of PI3K and EZH2 blockade synergistically regulates invasion and angiogenesis: combination therapy for glioblastoma multiforme

doi: 10.18632/oncotarget.27842

Figure Lengend Snippet: ( A ) Cytokine array analysis showed the various proteins of angiogenesis and metastasis are effectively changed during the course of combination treatment. ( B ) Heat map data depicted the intensity of upregulation and down regulation of all the affected proteins. ( C ) Schematic representation of effect of EPZ-6438 and PI-103 in GBM U-87 cells.

Article Snippet: Inhibitor PI-103 was procured from Echelon Biosciences and EPZ-6438 from APExBIO.

Techniques:

Journal: Translational Oncology

Article Title: Collaboration Between RSK-EphA2 and Gas6-Axl RTK Signaling in Arginine Starvation Response That Confers Resistance to EGFR Inhibitors

doi: 10.1016/j.tranon.2019.12.003

Figure Lengend Snippet: Activation of EphA2 by ADI is mediated by RSK1. (A) Time-dependent activation of p-RSK1 by ADI; (B) knockdown of RSK1 using two siRNAs shows suppression of EphA2 induction by ADI; (C) inhibition of EphA2 activation by RSK1 inhibitor BI-D1870; (D) and (E) inhibitions of ADI-induced activation of RSK1 and EphA2 by PI3K inhibitors PI-103 and Ly294002, respectively; (F) effects of AKT inhibitor (perifosine) on RSKs and EphA2 treated with ADI. (B–F) A2058 cells were treated with the indicated drugs for 24 hours. ADI, arginine deiminase.

Article Snippet: Reagents were obtained from the following sources: ADI-PEG20 (specific activity, 5∼10 IU/mg) from Polaris Pharmaceuticals Inc. (San Diego, CA); sulforhodamine B (SRB), Ly294002 from Sigma–Aldrich (St. Louis, MO); perifosine, PLX4720 and foretinib (XL880 or GSK1363089) from Selleck Lab (Houston, TX); PI-103 from Echelon Biosciences (Salt Lake City, UT); gefitinib and lapatinib from LC laboratories (Woburn.

Techniques: Activation Assay, Inhibition

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: PI3K inhibition is required and sufficient for intracellular δR retention. (A) Representative confocal image from a fixed primary cultured TG neuron expressing a FLAG-tagged δR. Internal δR (red in merge) colocalizes with the Golgi marker GPP130 (green in merge). (B) Example image (of three independent experiments) of a PC12 cell showing surface δR. NGF treatment (60 min at 100 ng/μl) causes internal δR accumulation (arrow), colocalizing with TGN-38 (Golgi, green). (C) NGF-treated PC12 cells, chased with cycloheximide (CHX) for 1 h to prevent additional δR delivery, in the presence of inhibitors of MEK (10 µM U0126, denoted as U01), ROCK (5 µM Y-27632, denoted as Y-2), and PLC (10 µM U73122, denoted as U73). Percentage of cells with Golgi-localized δR >100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. ctrl). None of the inhibitors prevented NGF-mediated δR retention. (D) Representative images (of three independent experiments) showing that inhibition of PI3K with 10 μM Wtm or LY is sufficient to cause δR retention. (E) Quantitation of percentage of cells with δR Golgi localization showing that PI3K inhibition with Wtm or LY is sufficient for δR Golgi localization (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. NT). (F) Quantitation of the percentage of δR localization within the Golgi normalized to the total cell δR fluorescence, showing that PI3K inhibition with Wtm or LY is sufficient for δR retention (>100 cells each; mean ± SEM; *** p < 0.001). (G) Example images (of three independent experiments) for PC12 cells expressing FLAG-δR, untreated (left two columns) or treated with NGF (100 ng/ml) for 1 h. NGF treatment induces intracellular retention of δR (red), which colocalizes with the Golgi (green). Activation of PI3K by the p85 subunit–binding peptide 740Y PDGFR (50 µg/ml) decreased NGF-induced Golgi localization of δR. Images without and with 740Y PDGFR . (H) Quantitation of percentage of cells with δR Golgi localization, showing significant reduction in percentage of cells with Golgi-localized δR in the NGF condition after addition of the PI3K-activating peptide 740Y PDGFR (>100 cells each; mean ± SEM; ** p < 0.01 by one-way ANOVA with Dunn’s multiple comparison test). (I) Image analysis and quantification shows a significant reduction in percentage of total δR fluorescence that overlaps with the Golgi in the NGF condition after addition of PI3K-activating peptide 740Y PDGFR . 740Y PDGFR had no effect on Golgi localization of δR on its own (>100 cells each; mean ± SEM; *** p < 0.001 by one-way ANOVA with Dunn’s multiple comparison test).

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Inhibition, Cell Culture, Expressing, Marker, Quantitation Assay, Fluorescence, Activation Assay, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: PI3K inhibition does not induce intracellular retention of μ R. (A) Representative images (of three independent experiments) of PC12 cells expressing FLAG- μ R as a control do not show retention after PI3K inhibition. (B) Quantitation of percentage of cells showing μ R retention after PI3K inhibition (>100 cells each; mean ± SEM; not significant [ p > 0.05] by two-sided t test vs. control). (C) Quantitation of percentage of total μ R fluorescence that overlaps with the Golgi after PI3K inhibition (>100 cells each; mean ± SEM; not significant [ p > 0.05] by two-sided t test vs. control). (D) Representative images (of three independent experiments) for δR endocytosis estimated by selectively labeling the surface vs. total pool of δR as described in Materials and Methods . PI3K inhibition did not cause endocytosis. The δR agonist DADLE was used as a control for endocytosis. (E) Pearson’s r of colocalization of the primary and secondary antibodies. High correlation denotes minimal endocytosis. DADLE significantly reduced the correlation, consistent with endocytosis (three representative fields; mean ± SEM; **** p < 0.0001 by two-sided t test vs. control).

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Inhibition, Expressing, Quantitation Assay, Fluorescence, Labeling

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: PI3K inhibition delays export of δR from the Golgi in HEK 293 cells. (A) Example images of HEK 293 cells expressing FLAG-δR (δR). In control conditions (ctrl), δR is present on the cell surface; however, in HEK 293 cells, PI3K inhibition by Wtm (10 µM) increases the Golgi localization of δR. (B) Image analysis and quantification show a significant increase in percentage of cells with the Golgi-localized δR after PI3K inhibition by 10 µM Wtm (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. ctrl) (C) Image analysis and quantification reveal a significant increase in percentage of total δR fluorescence that overlaps with the Golgi after PI3K inhibition by 10 µM Wtm (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. ctrl). (D) Example images of HEK 293 cells expressing FLAG- μ R. In control conditions (ctrl), μ R is present on the cell surface; however, unlike for δR, PI3K inhibition by 10 µM Wtm or LY does not increase the Golgi localization of μ R. (E) Image analysis and quantification resulted in a nonsignificant change in percentage of cells with the Golgi-localized μ R after PI3K inhibition by Wtm or LY (ctrl, n = 45; Wtm, n = 80; LY, n = 88; mean ± SEM; not significant by two-sided t test vs. ctrl). (F) Image analysis and quantification show a nonsignificant change in percentage of total μ R fluorescence that overlaps with the Golgi after PI3K inhibition by Wtm or LY (ctrl, n = 45; Wtm, n = 80; LY, n = 88; mean ± SEM; not significant by two-sided t test vs. ctrl). (G) Example time-lapse images (of four independent experiments) for HEK 293 cells expressing VSVG-δRtail-GFP imaged at the permissive temperature of 32°C. PI3K inhibition by 10 µM Wtm and LY resulted in sustained intracellular Golgi signal compared with control. (H) Quantification of the Golgi-associated fluorescence signal revealed a kinetic slowing of VSVG-δRtail-GFP trafficking from the Golgi after treatment with PI3K inhibitors Wtm and LY (control, 12 cells; Wtm, 14 cells; LY, 12 cells; mean ± SEM). (I) Quantification of the Golgi-associated VSVG fluorescence signal after 30 min, normalized to total, reveals that VSVG-δRtail-GFP requires the δRtail to show PI3K-dependent trafficking from the Golgi (control VSVG, 9 cells; VSVG LY294002, 9 cells; VSVG-δRtail control, 11 cells; VSVG-δRtail LY294002, 11 cells; mean ± SEM; *** p < 0.001).

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Inhibition, Expressing, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Class I PI3K is not required for Golgi retention of δR. (A) Example images (of three independent experiments) for PC12 cells expressing FLAG-δR, untreated (ctrl), or treated with NGF (100 ng/ml) for 1 h. NGF treatment induces intracellular retention of δR (red), which colocalizes with the Golgi (green). Inhibition of PI3K via 1 µM Wtm leads to partial accumulation of δR in the Golgi. Inhibition of PI3K via 10 µM Wtm leads to large accumulation of δR in the Golgi. (B) Image analysis and quantification show significant increase in percentage of cells with Golgi-localized δR after NGF treatment and PI3K inhibition by 1 and 10 µM Wtm (ctrl, 105 cells; NGF, 64 cells; 1 µM Wtm, 56 cells; 10 µM Wtm, 67 cells; mean ± SEM; ** p < 0.01, **** p < 0.0001 by two-sided t test vs. ctrl). (C) Further quantification demonstrated a significant increase in percentage of total δR fluorescence that overlaps with the Golgi in the NGF condition and a dose-dependent effect on percentage of δR-Golgi localization for 1 and 10 µM Wtm (ctrl, 105 cells; NGF, 64 cells; 1 µM Wtm, 56 cells; 10 µM Wtm, 67 cells; mean ± SEM; * p < 0.05, **** p < 0.0001 by two-sided t test vs. ctrl). (D) Example images (of three independent experiments) for PC12 cells expressing FLAG-δR, untreated (ctrl), or treated with NGF (100 ng/ml), LY (10 µM), or Wtm (10 µM) for 1 h induces intracellular retention of δR (red), which colocalizes with the Golgi (green). Class I–specific PI3K inhibitors PI-103 (PI3K C1 α, 50 nM), IC87114 (PI3K C1 δ, 5 µM), and AS605240 (PI3K C1 γ, 25 nM) were not sufficient to cause Golgi retention of δR. (E) Quantification demonstrated a significant increase in percentage of cells with Golgi-localized δR after NGF treatment and PI3K inhibition by LY (10 µM) or Wtm (10 µM) but not after class I–specific PI3K inhibition by PI3K inhibitors PI-103 (PI3K C1 α, 50 nM), IC87114 (PI3K C1 δ, 5 µM), and AS605240 (PI3K C1 γ, 25 nM; ctrl, 42 cells; NGF, 73 cells; LY, 77 cells; Wtm, 45 cells; α, 82 cells; δ, 99 cells; γ, 28 cells; mean ± SEM; *** p < 0.001 by one-way ANOVA with Dunn’s multiple comparison test). (F) Image analysis and quantification demonstrated a significant increase in percentage of cells with the Golgi-localized δR after NGF treatment and PI3K inhibition by 10 µM LY or Wtm but not after class I–specific PI3K inhibition by PI3K inhibitors PI-103 (PI3K C1 α, 50 nM), IC87114 (PI3K C1 δ, 5 µM), and AS605240 (PI3K C1 γ, 25 nM; ctrl, 42 cells; NGF, 73 cells; LY, 77 cells; Wtm, 45 cells; α, 82 cells; δ, 99 cells; γ, 28 cells; mean ± SEM; ** p < 0.01, **** p < 0.0001 by one-way ANOVA with Dunn’s multiple comparison test).

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Expressing, Inhibition, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Class II PI3K C2A is required for surface trafficking of δR. (A) Immunoblotting for PI3K C2A (C2A) confirmed knockdown of C2A in PC12 cells stably expressing C2A lentiviral shRNA. Three different shRNA sequences were used to determine the most efficient knockdown. Representative immunoblot, with actin as a loading control. Blot densitometry was performed to quantitate the percentage of knockdown for C2A shRNA sequences 1–3 compared with control (Ctl) cells. (B) Densitometry (of three independent experiments) revealed C2A shRNA sequences 2 and 3 as providing significant reduction in C2A protein expression, with shRNA 3 providing the best knockdown at 49% (three independent experiments; mean ± SEM; ** p < 0.01 by two-sided t test vs. Ctl). (C) Example images (of three independent experiments) for PC12 cells expressing FAP-δR (δR) with and without lentiviral PI3K C2A shRNA 3 (C2A shRNA) with a GFP reporter. Cells stably expressing the PI3K C2A shRNA were identified via GFP expression and had increased intracellular δR. (D) Image analysis and quantification revealed a significant increase in percentage of total δR fluorescence that overlaps with the Golgi in cells stably expressing the PI3K C2A shRNA compared with δR-only cells (δR, 102 cells; δR + C2A shRNA, 75 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR). (E) Further quantification confirmed a significant increase in percentage of cells with Golgi-localized δR in cells stably expressing the PI3K C2A shRNA compared with δR-only cells (δR, 102 cells; δR + C2A shRNA, 75 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR). (F) Example images from PC12 FAP- δR cells expressing EPAC cAMP biosensor and control nontargeted siRNA. Images reveal an increase in cAMP EPAC FRET ratio over time after 5 µM forskolin addition and subsequent decrease in cAMP and receptor internalization after δR agonist DADLE (10µM) addition. (G) Quantitative analysis of the EPAC FRET ratio, showing an increase in cAMP after forskolin addition and decrease after δR agonist DADLE addition in control (Ctl) siRNA cells. Similar experiments performed in cells transfected with PI3K C2A siRNA exhibited no decrease in cAMP after δR agonist DADLE (Ctl siRNA, 12 cells; C2A siRNA, 10 cells; mean ± SEM). (H) Analysis of percentage of cAMP inhibition after δR agonist addition demonstrates PI3K C2A siRNA ability to significantly reduce cAMP inhibition compared with control siRNA cells (Ctl siRNA, 12 cells; C2A siRNA, 10 cells; mean ± SEM; **** p < 0.0001 by two-sided t test Ctl vs. C2A siRNA).

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Fluorescence, Transfection, Inhibition

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Expression of class II PI3K C2A is sufficient to induce surface trafficking of δR. (A) Example images (of three independent experiments) for PC12 cells expressing GFP-PI3K C2A (green) demonstrate a pool of GFP-PI3K C2A enriched around the Golgi (TGN-38, red) in control conditions (Ctl) that is reduced after 1 h of treatment with NGF (100 ng/ml). (B) Quantification of GFP-PI3K C2A mean fluorescence intensity revealed a significant decrease in Golgi localization after NGF treatment (control, 36 cells; NGF, 45 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. control). (C) Additional quantification of the observed shift in GFP-PI3K C2A distribution shows a shift from perinuclear localization to diffuse cytoplasmic localization after NGF treatment (control, 28 of 36 perinuclear; NGF, 12 of 33 perinuclear; mean ± SEM; **** p < 0.0001 by two-sided Fisher’s exact test vs. control). (D) Example images (of three independent experiments) for PC12 cells expressing FAP-δR (δR, red) alone and in combination with overexpression of GFP-PI3K C2A (PI3K C2A, green) untreated (Ctl) or treated with NGF (100 ng/ml) for 1 h. Overexpression of GFP-PI3K C2A was sufficient to prevent the NGF-induced Golgi retention of δR. (E) Quantification and image analysis show a significant decrease in percentage of total δR fluorescence that overlaps with the Golgi in cells treated with NGF when overexpressing GFP-PI3K C2A compared with δR-only cells (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR Ctl). (F) Further quantification demonstrated a significant decrease in percentage of cells with Golgi-localized δR in cells treated with NGF when overexpressing GFP-PI3K C2A compared with δR-only cells (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR Ctl). (G) Example image showing GFP-PI3K C2A (green) expression in primary TG neuron counterstained live with Hoechst DNA stain (blue). TG neurons expressing GFP-PI3K C2A have ∼3.27-fold higher deltorphin II–Cy3 binding compared with nonexpressing (without PI3K-C2A) TG neurons (without PI3K-C2A, 33 cells; expresses PI3K-C2A, 35 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. without PI3K-C2A).

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Expressing, Fluorescence, Over Expression, Staining, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Optogenetic recruitment of the PI3K C2A kinase domain to the TGN is sufficient to induce δR surface trafficking. (A) Schematic showing the optogenetic recruitment strategy for targeting the PI3K C2A kinase domain to the TGN upon stimulation with blue light. The CRY2-CIBN optical recruitment system was adapted by cloning the PI3K C2A kinase domain (amino acids 863–1405, KD) onto the C-terminus of an mCherry-CRY2 chimeric construct to make mCherry-CRY2-PI3K-C2A-KD. In addition, the GFP-CIBN was adapted for TGN-specific recruitment by cloning the N-term of 2,6-sialyltransferase onto the C-terminus of GFP-CIBN to make GFP-CIBN-SialyT. The GFP-CIBN-SialyT has a basal TGN localization and the mCherry-CRY2-PI3K-C2A-KD is mostly cytoplasmic before recruitment. Optorecruitment is activated by blue light stimulation at 488 nm driving the mCherry-CRY2-PI3K-C2A-KD to the TGN. (B) PC12 cells were transiently transfected with the mCherry-CRY2-PI3K-C2A-KD (C2A KD, red), GFP-CIBN-SialyT (CIBN-TGN, green), and the TGN-localized PI(4)P sensor PH-FAPP1-iRFP (PH-FAPP1, blue) for live imaging of the optogenetic recruitment. During the live stimulation with 488-nm light, recruitment of the C2A KD to the CIBN-TGN and PH-FAPP1 can be observed within 5 s. (C) Analysis of the recruitment and colocalization of the C2A KD to the CIBN-TGN and PH-FAPP1 region was performed via a line-scan plot (example line in yellow) to identify relative spatiotemporal fluorescence intensity over a pixel area. Over time, the peak fluorescence intensity and the fluorescence intensity distribution for C2A KD (red), CIBN-TGN (green), and PH-FAPP1 (blue) are all highly overlapping after 5 s. (D) PC12 cells were transiently transfected with the mCherry-CRY2-PI3K-C2A-KD (C2A KD, blue), GFP-CIBN-SialyT (CIBN-TGN, green), and FAP-δR (δR, red) for live imaging of the optogenetic recruitment and induced surface trafficking after NGF-induced retention of δR. NGF (100 ng/ml) was added for 1 h to cause Golgi retention of δR (yellow arrow). Optical recruitment of the mCherry-CRY2-PI3K-C2A-KD to the TGN was performed to stimulate PI3K C2A and induce surface trafficking of the retained pool of δR. After recruitment, the Golgi pool of δR decreases and appears to vesiculate. (E) Analysis of live-imaging frames reveals the formation of vesicles containing δR beginning in the TGN (yellow arrow), bud off, and appear to be trafficking to the cell surface. (F) Image analysis and quantification of the complete recruitment movie demonstrate a decrease in the δR fluorescence intensity within the TGN over time after active and sustained recruitment of the mCherry-CRY2-PI3K-C2A-KD to the TGN. (G) Quantification and analysis (of eight independent experiments) show the fold change in δR membrane and TGN-localized fluorescence normalized to their starting values (dotted blue line) after recruitment of mCherry-CRY2-PI3K-C2A-KD to the TGN. There is a significant decrease in the fluorescence intensity of δR within the TGN after recruitment (TGN δR, 37%), as well as a significant increase in total δR membrane fluorescence (membrane δR, 7%; membrane δR, eight cells; TGN δR, eight cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. respective Ctl).

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Clone Assay, Construct, Transfection, Imaging, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Pharmacological compounds used throughout the article.

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Concentration Assay

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Pharmacological compounds used throughout the article.

Article Snippet: PI-103 , PI3K C1 α inhibitor , 50 nM , Echelon Biosciences , B0303.

Techniques: Concentration Assay