Figure 4.
VAMP2 depletion in PC12 cells and striatal neurons decreased MOR recycling. (A) Schematic of the treatment and labeling conditions for the ratiometric recycling assay, as described in the text. (B) Representative confocal images of PC12 cells co-expressing FLAG-MOR and shVAMP2 either treated with (top panel, +Dox) or without (bottom panel, −Dox) 200 ng/ml Dox for 48 h prior to the ratiometric recycling assay. Scale bar = 15 µm. (C) Quantification of ratios between 488 nm fluorescence and 647 nm fluorescence (488/647) calculated using integrated density (ID) for PC12 cells of indicated conditions (NT, T, WO: −Dox: n = 20, 20, 33 cells; +Dox: n = 20, 21, 29 cells, from two independent coverslips). +Dox cells showed significantly lower MOR recycling compared with −Dox cells (two-way ANOVA, post-hoc Šidák test, +Dox vs. −Dox: NT: P = 0.9929; T: P = 0.9173; WO: P = 0.0073). (D and E) Representative confocal images of E18 rat striatal neurons expressing shScramble (D) or shVAMP2 (E) after 48 h of Dox treatment, fixed and immunostained for endogenous VAMP2 (anti-VAMP2, magenta) and tagBFP (anti-tagBFP, blue). Scale bar = 20 µm. (F) Scatter plots of mean fluorescence intensity of VAMP2 immunostaining in the cell bodies of shScramble or shVAMP2 expressing neurons (median ± 95% CI, n = 12 or 15 cells, respectively, from one experiment), showing depletion of VAMP2 in the latter (P < 0.0001, unpaired two-tailed t test). (G) Representative confocal images of rat striatal neurons co-expressing FLAG-MOR and shVAMP2 either treated with (top panel, +Dox) or without (bottom panel, −Dox) 48 h Dox prior to the ratiometric recycling assay. Scale bar = 15 µm. (H) Similar quantification of ratios as in C for rat striatal neurons of indicated conditions (NT, T, WO: −Dox: n = 27, 33, 41 neurons; +Dox: n = 22, 39, 46 neurons, from two independent neuronal cultures). +Dox neurons showed significantly lower MOR recycling compared to −Dox neurons (two-way ANOVA, post-hoc Šidák test, +Dox vs. −Dox: NT: P = 0.9624; T: P = 0.9949; WO: P < 0.0001).

VAMP2 depletion in PC12 cells and striatal neurons decreased MOR recycling. (A) Schematic of the treatment and labeling conditions for the ratiometric recycling assay, as described in the text. (B) Representative confocal images of PC12 cells co-expressing FLAG-MOR and shVAMP2 either treated with (top panel, +Dox) or without (bottom panel, −Dox) 200 ng/ml Dox for 48 h prior to the ratiometric recycling assay. Scale bar = 15 µm. (C) Quantification of ratios between 488 nm fluorescence and 647 nm fluorescence (488/647) calculated using integrated density (ID) for PC12 cells of indicated conditions (NT, T, WO: −Dox: n = 20, 20, 33 cells; +Dox: n = 20, 21, 29 cells, from two independent coverslips). +Dox cells showed significantly lower MOR recycling compared with −Dox cells (two-way ANOVA, post-hoc Šidák test, +Dox vs. −Dox: NT: P = 0.9929; T: P = 0.9173; WO: P = 0.0073). (D and E) Representative confocal images of E18 rat striatal neurons expressing shScramble (D) or shVAMP2 (E) after 48 h of Dox treatment, fixed and immunostained for endogenous VAMP2 (anti-VAMP2, magenta) and tagBFP (anti-tagBFP, blue). Scale bar = 20 µm. (F) Scatter plots of mean fluorescence intensity of VAMP2 immunostaining in the cell bodies of shScramble or shVAMP2 expressing neurons (median ± 95% CI, n = 12 or 15 cells, respectively, from one experiment), showing depletion of VAMP2 in the latter (P < 0.0001, unpaired two-tailed t test). (G) Representative confocal images of rat striatal neurons co-expressing FLAG-MOR and shVAMP2 either treated with (top panel, +Dox) or without (bottom panel, −Dox) 48 h Dox prior to the ratiometric recycling assay. Scale bar = 15 µm. (H) Similar quantification of ratios as in C for rat striatal neurons of indicated conditions (NT, T, WO: −Dox: n = 27, 33, 41 neurons; +Dox: n = 22, 39, 46 neurons, from two independent neuronal cultures). +Dox neurons showed significantly lower MOR recycling compared to −Dox neurons (two-way ANOVA, post-hoc Šidák test, +Dox vs. −Dox: NT: P = 0.9624; T: P = 0.9949; WO: P < 0.0001).

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