puma oe plasmid  (Addgene inc)

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Paradoxical role of BBC3 in the methamphetamine-induced decrease in microglial survival. (A) The effect of methamphetamine on the survival of microglia in the hippocampus of WT and Bbc3 KO mice. WT and Bbc3 KO mice were treated with methamphetamine (intraperitoneal, 30 mg/kg) for 48 h, followed by AIF1 immunostaining. Representative image of AIF1 immunostaining in the hippocampus of mice injected with saline/methamphetamine. Scale bars: 200 μm (upper panel), 100 μm (middle panel), and 50 μm (lower panel). (B) Stereology of AIF1-positive microglia in the hippocampus. N = 6 animals/group. *, p < 0.05, **, p < 0.01 vs. the saline-treated group; #, p < 0.05 vs. the methamphetamine-treated group using one-way ANOVA followed by the Holm-Sidak test. (C) Bbc3 siRNA lentivirus was transduced to decrease BBC3 expression (upper panel), and the cells were transduced with BBC3 OE lentivirus to increase BBC3 expression (lower panel). (D and E) Transduction of cells with Bbc3 siRNA lentivirus failed to ameliorate the decreased microglial survival as assessed by CCK8 assay in both BV-2 cells (D) and primary mouse microglia (E). (F and G) Transduction of cells with a BBC3 OE lentivirus ameliorated the decreased microglial survival as assessed by CCK8 in both BV-2 cells (F) and primary mouse microglia (G). All data are given as the mean ± SD of 3 independent experiments. **, p < 0.01 and ***, p < 0.001 vs. the siRNA-control or vector group; #, p < 0.05, ##, p < 0.01 and ###, p < 0.001 vs. the methamphetamine-treated group using one-way ANOVA followed by the Holm-Sidak test. Meth, methamphetamine.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: Immunostaining, Injection, Saline, Expressing, Transduction, CCK-8 Assay, Control, Plasmid Preparation

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Methamphetamine mediated upregulation of BBC3 in microglia. (A and B) Methamphetamine increased Bbc3 expression at the mRNA level in BV-2 cells (A) and in primary mouse microglia (B) as determined by real-time PCR. (C and D) Methamphetamine induced a biphasic response of BBC3 expression in BV-2 cells (C) and in primary mouse microglia (D) as determined by Western blot analysis. Cells were treated with methamphetamine (1.5 mM) for 6, 12 and 24 h, followed by collection of RNA and protein for assessment of BBC3 expression. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05, **, p < 0.01 and ***, p < 0.001 vs. the control group using one-way ANOVA followed by the Holm-Sidak test. (E) Representative image of BBC3 immunofluorescence staining in primary mouse microglia. Cells were treated with methamphetamine (1.5 mM) for 6 h. Red, AIF1; green, BBC3; blue, DAPI. Scale bar: 50 μm. (F) Methamphetamine increased TRP53 expression and induced TRP53 translocation into the nuclei of BV-2 cells. Cells were treated with methamphetamine for 5, 15, 30, 60 and 180 min followed by isolation of nuclear and cytosolic fractions as well as collection of the total cell lysates for the detection of TRP53 expression using Western blot analysis. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05, **, p < 0.01 and ***, p < 0.001 vs. the control group using one-way ANOVA followed by the Holm-Sidak test. (G and H) Pretreatment of primary mouse microglia with the TRP53 inhibitor pifithrin-α (PFT-α; 10 μM) for 1 h (G) or Trp53 siRNA (H), followed by treatment with methamphetamine for 6 h. Inhibition of the TRP53 pathway significantly decreased the BBC3 expression induced by methamphetamine. All data are presented as the mean ± SD of 3 independent experiments. **, p < 0.01 vs. the control group; ##, p < 0.01 vs. the methamphetamine-treated group using one-way ANOVA followed by the Holm-Sidak test. Meth, methamphetamine.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Immunofluorescence, Staining, Translocation Assay, Isolation, Inhibition

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Mir143 regulates BBC3 expression at the post-transcriptional level in microglia. (A) Putative Mir143 binding sites in the Bbc3 gene. The potential complementary residues are shown in red. (B) Relative luciferase activity of wild type 3′-UTR mutant constructs of Bbc3 cotransfected with the Mir143 OE vector and the pmiR-GLO plasmid. All the data are indicated as the mean ± SD of 3 individual experiments. *, p < 0.05 vs. the Mircontrol co-transfected with WT group using one-way ANOVA followed by the Holm-Sidak test. (C) Effect of methamphetamine on Mir143 expression at the mRNA level in primary mouse microglia as determined by real-time PCR. Cells were incubated with methamphetamine (1.5 mM) for 6, 12 and 24 h, followed by collection of RNA for assay of Mir143 expression. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs. the control group using one-way ANOVA followed by the Holm-Sidak test. (D) Fluorescence in situ hybridization of mature Mir143 in primary mouse microglia combined with immunostaining for the microglial marker AIF1. Cells were incubated with methamphetamine (1.5 mM) for 24 h. Red, Mir143; green, AIF1; blue, DAPI. Scale bar: 20 µm. (E and F) Primary mouse microglia infected with Mircontrol + Mir143 and anti-Mircontrol + anti-Mir143 lentivirus for 48 h were assessed for BBC3 expression at the mRNA (E) and protein (F) levels. All data are presented as the mean ± SD of 3 individual experiments. **, p < 0 .01 vs. the Mircontrol or anti-Mircontrol group using Student ttest. Meth, methamphetamine.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Mutagenesis, Construct, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Incubation, Control, Fluorescence, In Situ Hybridization, Immunostaining, Marker, Infection

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Role of BBC3 in methamphetamine-induced cell apoptosis of BV-2 cells. (A to C) Exposure of BV-2 cells to methamphetamine (1.5 mM) for different time periods (3, 6, 12 and 24 h) increased the expression of BAX-BCL2L1 (A), cleaved CASP9:CASP9 (B) and cleaved CASP3:CASP3 (C). All data are presented as the mean ± SD of 3 independent experiments. **, p < 0.01 and ***, p < 0.001 vs. the control group using one-way ANOVA followed by the Holm-Sidak test. (D to F) Methamphetamine-induced expression of BAX-BCL2L1 (D), cleaved CASP9:CASP9 (E) and cleaved CASP3:CASP3 (F) was attenuated by BBC3 knockdown in BV-2 cells using Bbc3 siRNA in BV-2 cells. BV-2 cells were transfected with Bbc3 siRNA for 24 h followed by methamphetamine treatment (1.5 mM) for another 6 h, and the cells were homogenized for detection of cleaved CASP9 and CASP3 expression using Western blotting. (G and H) TUNEL staining (green) in BV-2 cells transfected with Bbc3 siRNA for 24 h and treated with methamphetamine for another 24 h. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05 and **, p < 0.01 vs. the control group; #, p < 0.05 and ##, p < 0.01 vs. the methamphetamine-treated group using one-way ANOVA followed by the Holm-Sidak test.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: Expressing, Control, Knockdown, Transfection, Western Blot, TUNEL Assay, Staining

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Role of BBC3 in methamphetamine-induced microglial apoptosis. (A and B) Exposure of primary mouse microglia to methamphetamine (1.5 mM) for different time periods (6, 12 and 24 h) increased the expression of cleaved CASP9 and CASP3. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05 and **, p < 0.01 vs. the control group using one-way ANOVA followed by the Holm-Sidak test. (C) Bbc3 siRNA inhibited the methamphetamine-induced increase in cleaved CASP3 expression. Primary mouse microglia were transduced with Bbc3 siRNA for 24 h followed by methamphetamine treatment (1.5 mM) for another 6 h, and the cells were homogenized for detection of cleaved CASP3 expression using Western blotting. All data are presented as the mean ± SD of 3 independent experiments. **, p < 0.01 vs. the control group; #, p < 0.05 vs. the methamphetamine-treated group using one-way ANOVA followed by the Holm-Sidak test. (D and E) Expression of BAX-BCL2L1 (D) and cleaved CASP3:CASP3 (E) in the hippocampus of WT and Bbc3 KO mice as determined by Western blotting. WT and Bbc3 KO mice were treated with methamphetamine (intraperitoneal, 30 mg/kg) for 48 h followed by examination for apoptosis. N = 6 animals/group. *, p < 0.05 and **, p < 0.01 vs. the saline-treated WT group; ##, p < 0 .01 vs. the methamphetamine-treated WT group using one-way ANOVA followed by the Holm-Sidak test. Meth: methamphetamine.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: Expressing, Control, Transduction, Western Blot, Saline

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Role of BBC3 in methamphetamine-induced autophagy of microglia. (A) Pretreatment of BV-2 cells (left panel) and primary mouse microglia (right panel) with the autophagy inducer rapamycin (0.1 μM) for 1 h enhanced the methamphetamine-induced increase in MAP1LC3-II expression. (B) Effect of methamphetamine on the formation of MAP1LC3-II puncta. Cells were pretreated with rapamycin for 1 h and then treated with methamphetamine (1.5 mM) for another 24 h. MAP1LC3-II puncta were then analyzed by confocal microscopy. Scale bar: 20 μm. (C) Knockdown of Bbc3 expression by siRNA decreased the methamphetamine-induced expression of MAP1LC3-II in primary mouse microglia. Primary mouse microglia were transfected with Bbc3 siRNA for 24 h followed by methamphetamine treatment (1.5 mM) for another 24 h, and the cells were processed for detection of MAP1LC3-II expression using Western blotting. (D) Transduction of primary mouse microglia with BBC3 OE lentivirus further enhanced the methamphetamine-induced increase in MAP1LC3-II expression in primary mouse microglia. Primary mouse microglia were transduced with BBC3 OE lentivirus for 24 h followed by methamphetamine treatment (1.5 mM) for another 24 h, and the cells were processed for detection of MAP1LC3-II expression using Western blotting. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05, **, p < 0.01 and ***, p < 0.001 vs. the control group; #, p < 0.05 and ##, p < 0.01 vs. the methamphetamine-treated group using one-way ANOVA followed by the Holm-Sidak test. (E) Expression of MAP1LC3-II in the hippocampus of WT and Bbc3 KO mice as determined by Western blotting. WT and Bbc3 KO mice were treated with methamphetamine (intraperitoneal, 30 mg/kg) for 48 h, followed by examination for autophagy. N = 6 animals/group. *, p < 0.05 and **, p < 0.01 vs. the saline-treated WT group; ##, p < 0.01 vs. the methamphetamine-treated WT group using one-way ANOVA followed by the Holm-Sidak test. Meth, methamphetamine; Rap, rapamycin.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: Expressing, Confocal Microscopy, Knockdown, Transfection, Western Blot, Transduction, Control, Saline

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Role of Mir143-BBC3 in methamphetamine-induced apoptosis and autophagy in vitro. (A to C) Transduction of cells with anti-Mir143 attenuated the methamphetamine-induced decrease in cell viability as determined by CCK8 assay (A) and enhanced the level of cleaved CASP3 (B) and MAP1LC3-II (C) as determined by Western blotting. Cells were transduced with anti-Mir143 lentivirus for 24 h and then treated with methamphetamine (1.5 mM) for 24 h to examine the cell viability and MAP1LC3-II expression and for 6 h to evaluate the level of cleaved CASP3. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05, **, p < 0.01 and ***, p < 0.001 vs. the anti-Mircontrol group; #, p < 0.05 and ###, p < 0.001 vs. the anti-Mircontrol treated with methamphetamine group using one-way ANOVA followed by the Holm-Sidak test. (D to F) Transduction of cells with the Bbc3 siRNA lentivirus significantly inhibited the anti-Mir143-induced increase in cell viability as determined by CCK8 assay (D) and decreased the levels of cleaved CASP3 (E) and MAP1LC3-II (F) as determined by Western blotting. Cells were transduced with Bbc3 siRNA lentivirus for 24 h, and then were treated with methamphetamine (1.5 mM) for another 24 h to examine the cell viability and MAP1LC3-II expression, and for another 6 h to evaluate the level of cleaved CASP3. All data are presented as the mean ± SD of 3 independent experiments. *, p < 0.05 and **, p < 0.01 vs. the anti-Mircontrol group; #, p < 0.05 and ##, p < 0.01 vs. the anti-Mir143 cotransduced with siRNA-control group using one-way ANOVA followed by the Holm-Sidak test. Meth: methamphetamine.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: In Vitro, Transduction, CCK-8 Assay, Western Blot, Expressing, Control

Journal: Autophagy

Article Title: Mir143 -BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy: Implications for methamphetamine-mediated neurotoxicity

doi: 10.1080/15548627.2016.1191723

Figure Lengend Snippet: Role of Mir143 in methamphetamine-induced microglial death in vivo. (A) Representative images of C57BL/6J mice microinjected with either anti-Mir control-RFP lentivirus or anti-Mir143-RFP lentivirus (LV) in microglia of the hippocampus. Scale bar: 100 μm. Mice were microinjected bilaterally with anti-Mir-RFP lentivirus (2 μl of 109 viral genomes µl−1) into the hippocampus, and 2 wk after microinjection, mice were sacrificed and examined for RFP and AIF1 expression. (B) Anti-Mir143 lentivirus injection successfully increased BBC3 expression as determined by Western blotting. Two wk after microinjection, mice were sacrificed and examined for BBC3 expression. (C) Effect of methamphetamine on the survival of microglia in the hippocampus of C57BL/6J mice, which were microinjected with either anti-Mircontrol-RFP lentivirus or anti-Mir143-RFP lentivirus. Two wk after microinjection, mice were treated with methamphetamine (intraperitoneal, 30 mg/kg) for another 48 h, followed by AIF1 immunostaining. Representative images of AIF1 immunostaining in the hippocampus of mice injected with saline or methamphetamine. Scale bars: 200 μm (upper panel), 100 μm (middle panel), and 50 μm (lower panel). (D) Stereology of AIF1-positive microglia in the hippocampus. N = 5 animals/group. *, p < 0.05 vs. the anti-Mircontrol mice treated with saline group; #, p < 0.05 vs. the anti-Mircontrol mice treated with methamphetamine-treated group using one-way ANOVA followed by the Holm-Sidak test. Meth, methamphetamine.

Article Snippet: BBC3 OE plasmid (pHA-BBC3; 16588 deposited by Bert Vogelstein) was obtained from Addgene.

Techniques: In Vivo, Control, Microinjection, Expressing, Injection, Western Blot, Immunostaining, Saline