Figure 3.
VAMP2 depletion inhibits the recycling of SpH-MOR but not that of SpH-B2AR and TfR-SpH. (A) Representative confocal image of PC12 cells expressing shScramble-tagBFP (blue) immunostained for endogenous VAMP2 (magenta) after 48 h of Dox treatment. (B) Representative confocal image of PC12 cells expressing shVAMP2-tagBFP (blue) immunostained for endogenous VAMP2 (magenta) after 48 h of Dox treatment, showing depletion of VAMP2. Scale bar = 20 µm. (C) Scatter plots of normalized IDoT of VAMP2 immunostaining in shScramble-tagBFP (shScramble) or shVAMP2-tagBFP (shVAMP2) expressing cells (median ± 95% CI, n = 45 or 46 cells, respectively, from two independent experiments), showing depletion of VAMP2 in the latter (P < 0.0001, unpaired two-tailed t test). (D) The number of TfR-SpH puffs per cell per minute (/cell • min) in cells expressing either TfR-SpH alone (Dox alone) or in cells co-expressing shScramble or shVAMP2, treated with 48 h of Dox, show no significant difference (Dox alone: n = 11; shScramble: n = 10; shVAMP2: n = 10, from two independent experiments; one-way ANOVA, post-hoc Tukey test). (E) A similar comparison shows no difference in the numbers of SpH-B2AR puffs across the three conditions (Dox alone: n = 27; shScramble + Dox: n = 24; shVamp2 + Dox: n = 36, from three independent experiments, one-way ANOVA, post-hoc Tukey test). (F) A similar comparison shows significant reduction in the number of SpH-MOR puffs in cells co-expressing shVAMP2 compared with the other two conditions (Dox alone: n = 27; shScramble: n = 23; shVAMP2: n = 28, from three independent experiments, one-way ANOVA, post-hoc Tukey test). (G) A similar comparison in cells co-expressing SpH-MOR and shScramble or VAMP4 shRNA (shVAMP4), shows no difference in the numbers of SpH-MOR puffs (shScramble, n = 15; shVAMP2, n = 13, from one experiment, unpaired two-tailed t test, checked by power analysis). Cells from E–G were treated with agonists for 5 min at 37°C before imaging. Filled dots in red from E–G represented outliers identified by the Tukey plot that were included in the statistical analysis. “+” in the Tukey plots from D–G represented mean. (H) Schematic of the treatment and labeling conditions for the flow cytometry assay. Cells were labeled with M1-Alexa-647 (M1-647) antibody at the endpoint of treatment conditions to measure surface MOR levels: at baseline (NT), after DAMGO for 20 min at 37°C (T), or after washing out DAMGO and incubating in Naltrexone for 20 min at 37°C (WO). (I) Representative histograms of PC12 cells co-expressing FLAG-MOR and shVAMP2, either without (shVAMP2 −Dox) or with (shVAMP2 +Dox) pretreatment of Dox for 48 h. (J) Quantification of mean (left panel) and geometric mean (geomean, right panel) of surface MOR levels normalized to unlabeled baseline (±SEM, three samples each condition from one experiment).

VAMP2 depletion inhibits the recycling of SpH-MOR but not that of SpH-B2AR and TfR-SpH. (A) Representative confocal image of PC12 cells expressing shScramble-tagBFP (blue) immunostained for endogenous VAMP2 (magenta) after 48 h of Dox treatment. (B) Representative confocal image of PC12 cells expressing shVAMP2-tagBFP (blue) immunostained for endogenous VAMP2 (magenta) after 48 h of Dox treatment, showing depletion of VAMP2. Scale bar = 20 µm. (C) Scatter plots of normalized IDoT of VAMP2 immunostaining in shScramble-tagBFP (shScramble) or shVAMP2-tagBFP (shVAMP2) expressing cells (median ± 95% CI, n = 45 or 46 cells, respectively, from two independent experiments), showing depletion of VAMP2 in the latter (P < 0.0001, unpaired two-tailed t test). (D) The number of TfR-SpH puffs per cell per minute (/cell • min) in cells expressing either TfR-SpH alone (Dox alone) or in cells co-expressing shScramble or shVAMP2, treated with 48 h of Dox, show no significant difference (Dox alone: n = 11; shScramble: n = 10; shVAMP2: n = 10, from two independent experiments; one-way ANOVA, post-hoc Tukey test). (E) A similar comparison shows no difference in the numbers of SpH-B2AR puffs across the three conditions (Dox alone: n = 27; shScramble + Dox: n = 24; shVamp2 + Dox: n = 36, from three independent experiments, one-way ANOVA, post-hoc Tukey test). (F) A similar comparison shows significant reduction in the number of SpH-MOR puffs in cells co-expressing shVAMP2 compared with the other two conditions (Dox alone: n = 27; shScramble: n = 23; shVAMP2: n = 28, from three independent experiments, one-way ANOVA, post-hoc Tukey test). (G) A similar comparison in cells co-expressing SpH-MOR and shScramble or VAMP4 shRNA (shVAMP4), shows no difference in the numbers of SpH-MOR puffs (shScramble, n = 15; shVAMP2, n = 13, from one experiment, unpaired two-tailed t test, checked by power analysis). Cells from E–G were treated with agonists for 5 min at 37°C before imaging. Filled dots in red from E–G represented outliers identified by the Tukey plot that were included in the statistical analysis. “+” in the Tukey plots from D–G represented mean. (H) Schematic of the treatment and labeling conditions for the flow cytometry assay. Cells were labeled with M1-Alexa-647 (M1-647) antibody at the endpoint of treatment conditions to measure surface MOR levels: at baseline (NT), after DAMGO for 20 min at 37°C (T), or after washing out DAMGO and incubating in Naltrexone for 20 min at 37°C (WO). (I) Representative histograms of PC12 cells co-expressing FLAG-MOR and shVAMP2, either without (shVAMP2 −Dox) or with (shVAMP2 +Dox) pretreatment of Dox for 48 h. (J) Quantification of mean (left panel) and geometric mean (geomean, right panel) of surface MOR levels normalized to unlabeled baseline (±SEM, three samples each condition from one experiment).

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