Journal: British journal of pharmacology

Article Title: Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

doi: 10.1111/bph.16503

Figure Lengend Snippet: FIGURE 8 Effects of inhibitors of AMPK and PP2A on decreased 5-HT receptor expression on astrocytes induced by exposure to clozapine and L-BAIBA. Cultured astrocytes were incubated in fDMEM containing 3 μM clozapine (CLZ), 3 μM clozapine plus 10 μM dorsomorphin (CLZ + Dor), 3 μM clozapine plus 3 μM LB-100 (CLZ + LB) or 30 μM L-BAIBA (BAIBA), 30 μM L-BAIBA plus 10 μM dorsomorphin (BAIBA + Dor) or 30 μM L-BAIBA plus 3 μM LB-100 (BAIBA + LB) for 14 days. In the upper-side histograms, ordinate: mean ± SD (n = 6) of the relative levels of 5-HT1A (a and d), 5-HT2A (b and e) and 5-HT7 receptors (c and f) relative to control in the plasma membrane fraction of cultured astrocytes. *P <0.05 relative to 3 μM clozapine alone or 30 μM L-BAIBA alone using one-way ANOVA with Scheffe's post hoc test. The lower panel indicates pseudo-gel images of capillary immunoblotting. F values of the effects of clozapine, dorsomorphin and LB-100 on 5-HT1A, 5-HT2A and 5-HT7 receptors in plasma membrane fraction were [F(2,15) = 23.9 (P <0.05)], [F(2,15) = 19.4 (P <0.05)] and [F(2,15) = 65.6 (P <0.05)], respectively. F values of effects of L-BAIBA, dorsomorphin and LB-100 on 5-HT1A, 5-HT2A and 5-HT7 receptors in the plasma membrane fraction were [F(2,15) = 15.9 (P <0.05)], [F(2,15) = 28.7 (P <0.05)] and [F(2,15) = 12.1 (P <0.05)], respectively.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_ 10001915, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (8475, RRID:AB_3102029, 1:50, Cell Signalling Technology, Danvers, MA, USA), γ-actin (GTX101794, RRID:AB_2036153, 1:100, GeneTex, Irvine, CA, USA), connexin43 (C6219, RRID:AB_476857, 1:100, Sigma-Aldrich), PP2A (AF1653, RRID:AB_354917, 1 μg ml 1, R&D systems, Minneapolis, MN, USA), phosphorylated-PP2A (pPP2A: AF3989, RRID:AB_2169636, 10 μg ml 1, R&D Systems), Akt (AF1775, RRID:AB_354982, 1 μg ml 1, R&D Systems), phosphorylated-Akt (pAkt: AF887, RRID:AB_355685, 5 μg ml 1, R&D Systems), AMPKα (2603, RRID:AB_490795, 1:50, Cell Signalling Technology, Danvers, MA, USA), phosphorylated-AMPKα (pAMPK: 2535, RRID:AB_331250, 1:50, Cell Signalling Technology), 5-HT1A receptor (5-HT1A-R: NBP2–21590, RRID:AB_3102030, 1:100, Novus Biologicals, Littleton, CO, USA), 5-HT2A receptor (5-HT2A-R: LS-C172270, RRID:AB_3102031, 1:50, Lifespan Biosciences, WA, USA), 5-HT7 receptor (5-HT7-R, NB100-56352, RRID:AB_2264237, 1:50, Novus Biologicals) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio).

Techniques: Expressing, Cell Culture, Incubation, Control, Clinical Proteomics, Membrane, Western Blot

Journal: British journal of pharmacology

Article Title: Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

doi: 10.1111/bph.16503

Figure Lengend Snippet: FIGURE 9 Interactions among clozapine, L-BAIBA and AMPK inhibitors on PP2A without clozapine, with 3 μM clozapine alone (CLZ), 3 μM clozapine plus 10 μM dorsomorphin (CLZ + Dor) (a), or without 30 μM L-BAIBA, with 30 μM L-BAIBA alone (BAIBA), or 30 μM L-BAIBA plus 10 μM dorsomorphin (BAIBA + Dor) (b) for 14 days. In the upper histograms, ordinate: mean ± SD (n = 6) of the relative levels of pPP2A relative to control. *P <0.05 relative to control (clozapine or L-BAIBA free) by one-way ANOVA with Scheffe's post hoc test. The lower panel indicates pseudo-gel images of capillary immunoblotting. The F values for the effects of clozapine and dorsomorphin on pPP2A were F(2,15) = 22.9 (P <0.05). The F values for the effects of L-BAIBA and dorsomorphin on pPP2A were [F(2,15) = 20.9 (P <0.05)].

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_ 10001915, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (8475, RRID:AB_3102029, 1:50, Cell Signalling Technology, Danvers, MA, USA), γ-actin (GTX101794, RRID:AB_2036153, 1:100, GeneTex, Irvine, CA, USA), connexin43 (C6219, RRID:AB_476857, 1:100, Sigma-Aldrich), PP2A (AF1653, RRID:AB_354917, 1 μg ml 1, R&D systems, Minneapolis, MN, USA), phosphorylated-PP2A (pPP2A: AF3989, RRID:AB_2169636, 10 μg ml 1, R&D Systems), Akt (AF1775, RRID:AB_354982, 1 μg ml 1, R&D Systems), phosphorylated-Akt (pAkt: AF887, RRID:AB_355685, 5 μg ml 1, R&D Systems), AMPKα (2603, RRID:AB_490795, 1:50, Cell Signalling Technology, Danvers, MA, USA), phosphorylated-AMPKα (pAMPK: 2535, RRID:AB_331250, 1:50, Cell Signalling Technology), 5-HT1A receptor (5-HT1A-R: NBP2–21590, RRID:AB_3102030, 1:100, Novus Biologicals, Littleton, CO, USA), 5-HT2A receptor (5-HT2A-R: LS-C172270, RRID:AB_3102031, 1:50, Lifespan Biosciences, WA, USA), 5-HT7 receptor (5-HT7-R, NB100-56352, RRID:AB_2264237, 1:50, Novus Biologicals) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio).

Techniques: Control, Western Blot

Journal: British journal of pharmacology

Article Title: Impacts of exposure to and subsequent discontinuation of clozapine on tripartite synaptic transmission.

doi: 10.1111/bph.16503

Figure Lengend Snippet: FIGURE 11 Proposed hypothesis of pathophysiology of clozapine discontinuation syndrome. During clozapine exposure, clozapine increases L-BAIBA and Akt signalling, but inhibits 5-HT receptors (and histamine H1 receptors) (Carli et al., 2021; Fukuyama et al., 2023a, 2023b, 2023c). Inhibition of 5-HT2 receptors decreases IP3 synthesis, leading to attenuation of IP3 receptors (Okada et al., 2005). Increased intracellular calcium levels activate ATP synthase activity (Carli et al., 2021; de Brito & Scorrano, 2010). Clozapine also increases L-BAIBA synthesis, which activates AMPK signalling via possibly MRGPRD activation (Fukuyama et al., 2023a, 2023c; Kitase et al., 2018; Minato et al., 2022). Therefore, clozapine exposure activates AMPK signalling through 5-HT2 receptor inhibition and increases L-BAIBA. Clozapine-induced activation of hypothalamic AMPK signalling plays important roles in weight-gain and metabolic complication of clozapine (Carli et al., 2021). Activation of AMPK signalling suppresses PP2A signalling, resulting in decreasing 5-HT receptors in the plasma membrane via suppression of receptor recycling (Raote et al., 2013; Tsao et al., 2001). On the contrary, connexin43 trafficking is enhanced via activation of Akt signalling, resulting in increased hemichannels containing connexin43 in the astroglial plasma membrane (Fukuyama et al., 2023a; Okada, 2022; Smith et al., 2014). Astroglial D-serine release through hemichannels increases during clozapine exposure. Clozapine discontinuation, intracellular L-BAIBA level and signallings AMPK, Akt and PP2A immediately recovered. PP2A signalling immediately recovered during subsequent clozapine discontinuation, in comparison clozapine exposure transiently increased 5-HT receptor expression (until 7 days of discontinuation), resulting in transiently increased intracellular IP3 levels. PP2A also activates phosphatidylinositol turnover (Zhong et al., 2008). Connexin43 expression also recovered after clozapine discontinuation, but increased astroglial D-serine release persisted until 7 days after clozapine discontinuation due to transiently activation of IP3 receptors via transiently increased IP3 level.

Article Snippet: Primary antibodies against GAPDH (NB300-327, RRID:AB_ 10001915, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (8475, RRID:AB_3102029, 1:50, Cell Signalling Technology, Danvers, MA, USA), γ-actin (GTX101794, RRID:AB_2036153, 1:100, GeneTex, Irvine, CA, USA), connexin43 (C6219, RRID:AB_476857, 1:100, Sigma-Aldrich), PP2A (AF1653, RRID:AB_354917, 1 μg ml 1, R&D systems, Minneapolis, MN, USA), phosphorylated-PP2A (pPP2A: AF3989, RRID:AB_2169636, 10 μg ml 1, R&D Systems), Akt (AF1775, RRID:AB_354982, 1 μg ml 1, R&D Systems), phosphorylated-Akt (pAkt: AF887, RRID:AB_355685, 5 μg ml 1, R&D Systems), AMPKα (2603, RRID:AB_490795, 1:50, Cell Signalling Technology, Danvers, MA, USA), phosphorylated-AMPKα (pAMPK: 2535, RRID:AB_331250, 1:50, Cell Signalling Technology), 5-HT1A receptor (5-HT1A-R: NBP2–21590, RRID:AB_3102030, 1:100, Novus Biologicals, Littleton, CO, USA), 5-HT2A receptor (5-HT2A-R: LS-C172270, RRID:AB_3102031, 1:50, Lifespan Biosciences, WA, USA), 5-HT7 receptor (5-HT7-R, NB100-56352, RRID:AB_2264237, 1:50, Novus Biologicals) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio).

Techniques: Inhibition, Activity Assay, Activation Assay, Clinical Proteomics, Membrane, Comparison, Expressing

Journal: Oncotarget

Article Title: A novel anti-melanoma SRC-family kinase inhibitor

doi: 10.18632/oncotarget.26787

Figure Lengend Snippet: ( A ) SAB298 increases the levels of inactivated PP2A (pPP2A Y307) in YUSIK and YUSIV melanoma cells in a fashion similar to okadaic acid (Okadaic a). Active (unphosphorylated) PP2A is not dependent on the presence of pY416 SFK. Panels B and C show SHP2 inactivation by SAB298 in YUSIK, YUROB and YUSOC, but not YUGASP melanoma cells (pSHP2 Y542). Suppression of SHP2 is associated with SAB298 induced inactivation of ERBB2 (pERBB2 Y1196) ( C , D ).

Article Snippet: We probed with the following primary antibodies: phospho-SRC Tyr416 (CST #2101S), pSRC Tyr527 (#2105), SRC (#2108), pPI3K p85 Y458/p55 Y199 (#4228), LYN (C13F9, #2796), FYN (#4023), phospho-MEK1/2 pSer217/221 (#9121), MEK1/2, phospho-ERK2 pThr202/Tyr204 (#9101), ERK1/2 (#9107), phospho-p38 kinase T180/Y182 (#9216), p38 kinase (#9212), PP2A (#2259), pSHP2 Y542 (#3751), SHP2 (D50F2, #3397), ERBB2 (D8F12 XP(R), #4290), phospho-ERBB2 Y1196 (D66B7 #6942), MYC (D84C12, #5605); GAPDH (14C10, #2118), all from Cell Signaling Technology, Beverly, MA; phospho-PP2A Y307 (AF3989), and MITF (clone D5, #AF5769) from R&D Systems, Inc., Minneapolis, MN; p27KIP1 (#10241) and YES (#610375) from BD Biosciences, San Jose, CA; Cyclin D1 (#04-221) and β-actin (mouse mAb A5316) from MilliporeSigma, St. Louis, MO; PI3K Antibody (p85 alpha, NSJ R30480) from Bioreagents, 9921 Carmel Mountain Rd #352, San Diego, CA.

Techniques:

Journal: PLoS ONE

Article Title: Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

doi: 10.1371/journal.pone.0027627

Figure Lengend Snippet: Effect of okadaic acid (OA; 10 −9 M) on corticosteroid sensitivity (A), GR nuclear translocation (B), phosphorylation levels of GR-Ser 226 (C) and JNK1 (D) in U937 cells (n = 3–4). E: Effect of PP2A siRNA on IC 50 of dexamethasone on TNFα-induced IL-8 (n = 7). Values represent means ± SEM. # P <0.05, ## P <0.01 (vs. non-treatment control; NT), * P <0.05.

Article Snippet: 2 µg of DNA/plasmids (pCMV6 Entry, OriGene Technologies, Rockville, MD) containing the human PP2A catalytic subunit, alpha isoform (PP2A Cα ) gene were transfected to U937 cells pretreated with 50 ng/ml of PMA for 4 h. 20 h after the transfection, the medium was changed to the appropriate treatment in 1% FBS medium.

Techniques: Translocation Assay

Journal: PLoS ONE

Article Title: Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

doi: 10.1371/journal.pone.0027627

Figure Lengend Snippet: Effects of IL-2/IL-4 co-treatment for 48 h on IC 50 of dexamethasone on TNFα-induced IL-8 release (A), PP2A C protein expression (B), immunoprecipitated PP2A (IP-P2A) activity (C), PP2A C -Tyr 307 phosphorylation(D), GR-Ser 226 phosphorylation (E) and JNK1 phosphorylation (F). Values represent means ± SEM (n = 3–4). # P <0.05, ## P <0.01 (vs. non-treatment control; NT).

Article Snippet: 2 µg of DNA/plasmids (pCMV6 Entry, OriGene Technologies, Rockville, MD) containing the human PP2A catalytic subunit, alpha isoform (PP2A Cα ) gene were transfected to U937 cells pretreated with 50 ng/ml of PMA for 4 h. 20 h after the transfection, the medium was changed to the appropriate treatment in 1% FBS medium.

Techniques: Expressing, Immunoprecipitation, Activity Assay

Journal: PLoS ONE

Article Title: Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

doi: 10.1371/journal.pone.0027627

Figure Lengend Snippet: PP2A C protein expression (A), PP1 protein expression (B), immunoprecipitated PP2A (IP-PP2A) activity (C), phosophorylation levels of GR-Ser 226 (D) and PP2A C -Tyr 307 (F) in PBMCs from severe asthmatics (SA) and healthy volunteers (HV). E. Correlation between PP2A C expression and GR-Ser 226 phosphorylation. The dotted lines show 95% confidence interval. # P <0.05, ## P <0.01 (vs. HV).

Article Snippet: 2 µg of DNA/plasmids (pCMV6 Entry, OriGene Technologies, Rockville, MD) containing the human PP2A catalytic subunit, alpha isoform (PP2A Cα ) gene were transfected to U937 cells pretreated with 50 ng/ml of PMA for 4 h. 20 h after the transfection, the medium was changed to the appropriate treatment in 1% FBS medium.

Techniques: Expressing, Immunoprecipitation, Activity Assay

Journal: PLoS ONE

Article Title: Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

doi: 10.1371/journal.pone.0027627

Figure Lengend Snippet: (A) PP2A C and JNK1 expression in GR-immunoprecipitates. Expression levels of PP2A C in GR (B)- or JNK1 (D)-immunoprecipitates. PP2A activity in GR (C)- and JNK1 (E) immunoprecipitates were also determined. Values represent means of four experiments ± SEM. # P <0.05, ## P <0.01 (vs. non-treatment control; NT).

Article Snippet: 2 µg of DNA/plasmids (pCMV6 Entry, OriGene Technologies, Rockville, MD) containing the human PP2A catalytic subunit, alpha isoform (PP2A Cα ) gene were transfected to U937 cells pretreated with 50 ng/ml of PMA for 4 h. 20 h after the transfection, the medium was changed to the appropriate treatment in 1% FBS medium.

Techniques: Expressing, Activity Assay

Journal: EBioMedicine

Article Title: PP2AC Level Determines Differential Programming of p38-TSC-mTOR Signaling and Therapeutic Response to p38-Targeted Therapy in Colorectal Cancer

doi: 10.1016/j.ebiom.2015.11.031

Figure Lengend Snippet: PP2AC determines differential programming of p38-to-mTORC1 signaling. A. The protein level of PP2A-A, PP2A-B and PP2A-C in different CRC cells as analyzed by immunoblot. B. HCT116 cells were transfected with four different PP2AC siRNAs (1, 2, 3 and 4), and then treated with 10 μM SB202190 for 2 h. Phosphorylation of TSC2, S6K1, MK2, ERK and RSK was analyzed by immunoblot. C. SW620 cells were transfected with four different PP2AC siRNAs (1, 2, 3 and 4), and then treated with 10 μM SB202190 for 2 h. Phosphorylation of TSC2, S6K1, S6, MK2, ERK and RSK was analyzed by immunoblot. D. RKO and SW480 cells were transfected with PP2AC plasmid and then treated with 10 μM SB202190 for 2 h. Phosphorylation of TSC2, S6K1, S6, MK2, ERK and RSK was analyzed by immunoblot. E. Effect of p38i on ERK-PP2AC interaction in sensitive and resistant CRC cells. HCT116, SW620, SW480 and RKO cells were treated without or with 10 μM SB202190 for 2 h. Endogenous PP2AC was immunoprecipitated with a PP2AC-specific antibody and analyzed for binding to ERK by Western blot. F. Immunohistochemistry (IHC) staining of primary human CRC tissue microarray. Shown are staining of tumor tissue sections (400 ×) representative of high PP2AC, medium PP2AC and low PP2AC staining. G. PP2AC is differentially expressed in CRC. Scatter plot shows PP2AC staining level in individual tumor as H-score.

Article Snippet: siRNA for human PP2AC, TSC1 and TSC2 were designed and synthesized by OriGene Technologies (MD, USA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Immunohistochemistry, Microarray, Staining

Journal: EBioMedicine

Article Title: PP2AC Level Determines Differential Programming of p38-TSC-mTOR Signaling and Therapeutic Response to p38-Targeted Therapy in Colorectal Cancer

doi: 10.1016/j.ebiom.2015.11.031

Figure Lengend Snippet: PP2AC predicts therapeutic outcome of p38i in patient-derived xenograft (PDX) CRC models. A. H&E staining, and IHC and IF staining of PP2AC in three representative PDX tumors (14, 25, 33) with low, and three representative PDX tumors (30, 31, 40) with high PP2AC expression. B. PDX tumors with low PP2AC expression are sensitive to SB202190. Mice bearing PDX tumors 14, 25 and 33 were treated with SB202190 or drug vehicle (VC) and measured for tumor volume (mean ± s.d., n = 6). *P < 0.01 versus VC. C. Representative p38i-sensitive PDX tumors at the end of the experiment. D. SB202190 enhances growth of PDX tumors with high PP2AC expression. Mice bearing PDX tumors 30, 31 and 40 were treated with SB202190 or drug vehicle (VC) and measured for tumor volume (mean ± s.d., n = 6). *P < 0.05, **P < 0.01 versus VC. E. Representative p38i-resistant PDX tumors at the end of the experiment. F. T/C value was calculated for the subgroup of 9 PDX tumors with low PP2AC expression and the subgroup of 9 PDX tumors with high PP2AC expression treated with SB202190 (t test, t = 9.903, df = 16, p < 0.0001). G. IHC staining of P-MK2, P-S6 and Ki67 in SB202190-treated PDX tumors 14, 25, 33 (upper panel), and 30, 31 and 40 (lower panel).

Article Snippet: siRNA for human PP2AC, TSC1 and TSC2 were designed and synthesized by OriGene Technologies (MD, USA).

Techniques: Derivative Assay, Staining, Expressing, Immunohistochemistry

Journal: EBioMedicine

Article Title: PP2AC Level Determines Differential Programming of p38-TSC-mTOR Signaling and Therapeutic Response to p38-Targeted Therapy in Colorectal Cancer

doi: 10.1016/j.ebiom.2015.11.031

Figure Lengend Snippet: A model showing differential programming of p38-to-mTORC1 signaling determines the outcome of p38-targeted therapies. It shows the primary signal transduction flow and effect of p38 inhibition on downstream effectors when PP2AC expression is low (left) or high (right) in CRCs.

Article Snippet: siRNA for human PP2AC, TSC1 and TSC2 were designed and synthesized by OriGene Technologies (MD, USA).

Techniques: Transduction, Inhibition, Expressing