Panel B, Western blot analysis of autophagy markers in the striatum. Panel A, Motor coordination (RotaRod test, time to fall relative to pre-treatment n = 7 animals per group). ( A–C) D 1R-CB 1R KO mice and CB 1R-floxed control littermates were treated with THC (10 mg/kg as a single i.p.injection) or its vehicle for 4 hr. Raw numerical data and further statistical details are shown in Figure 4-source data 1. *p<0.05, **p<0.01 from vehicle-treated group, or #p<0.05, #p<0.01 from THC-treated group, by one-way ANOVA with Tukey’s multiple comparisons test. Representative images with encircled examples of a high-intensity cell ( panel D) or double-positive cells ( panel E) are shown. ( D, E) Immunofluorescence analysis of p62 (p62 fluorescence intensity per DARPP32-positive cell panel D) and phosphorylated ribosomal protein S6 (phospho-S6/DARPP32 double-positive cells relative to total cells panel E) in the dorsal striatum ( n = 4 animals per group). Representative blots of each condition, together with optical density values relative to those of loading controls, are shown ( n = 4 animals per group). ( B, C) Western blot analysis of autophagy markers ( panel B) and mTORC1 signaling pathway markers ( panel C) in the striatum. Wild-type C57BL/6N mice were given trehalose (10 g/L) or plain water ad libitum for 24 hr, and, subsequently, were treated with THC (10 mg/kg as a single i.p.injection) or its vehicle for 4 hr. ( A) Motor coordination (RotaRod test, time to fall relative to pre-treatment n = 11–14 animals per group). Raw numerical data and further statistical details are shown in Figure 3-source data 1. Electrophoretic migration of molecular weight markers is depicted on the right-hand side of each blot. ( A) Motor coordination (RotaRod test, time to fall relative to pre-treatment n = 8–9 animals per group). Wild- type C57BL/6N mice were treated with temsirolimus (1 mg/kg as a single i.p. injection) or its vehicle for 20 min, and, subsequently, with THC (10 mg/kg as a single i.p. Raw numerical data and further statistical details are shown in Figure 2-source data 1. *p<0.05, **p<0.01 from vehicle-treated group by two-way ANOVA with Tukey’s multiple comparisons test. Representative images of selected experimental conditions with encircled examples of high-intensity cells are shown ( n = 3–6 independent cell preparations per condition). ( B) p62 immunoreactivity (p62 fluorescence intensity relative to total cells). Representative images with encircled examples of double-positive cells are shown ( n = 3–6 independent cell preparations per condition). Primary striatal neurons from C57BL/6N mice were exposed for 24 hr to THC (0.75 µM) or HU-210 (10 nM), alone or in combination with hydroxychloroquine (0.1 mM), E64d (0.1 µM) and/or pepstatin A (10 ng/ml), or their vehicles. ( A) LC3-II immunoreactivity (number of cells with three or more LC3-positive dots relative to total cells upper panel) and LC3-II/LAMP1 immunoreactivity (number of LAMP1-positive cells with three or more LC3 dots relative to total cells lower panel).
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