Fig. 5. Golgi-restricted DOR activation does not promote a transcriptional response.

(A) Confocal images of HEK293 cells stably expressing FLAG-DOR (HEK293-DOR). Cells were fixed, permeabilized, and immunolabeled with anti-FLAG (cyan) and anti-giantin (magenta) antibodies. Scale bar, 10 μm. (B and C) Volcano plots of differentially expressed genes in HEK293-DOR cells treated with DPDPE (100 nM) for 1.5 hours (B) or 6 hours (C) versus mock-treated control cells. Results are presented as the mean differential gene expression from three replicates and expressed as log₂FC. Genes significantly up-regulated are shown in red [quasi-likelihood F-test (QL F-test), FC > 1.5, adjusted P < 0.05]. Genes significantly down-regulated are shown in blue (QL F-test, FC < –1.5, adjusted P < 0.05). (D) Confocal images of HEK293-DOR cells expressing EGFP-Nb33 (gray) and GalT-DsRed (magenta). Control cells and cells treated for 6 hours with ICI (100 μM, 5 min preincubation) and SNC80 (100 nM). Scale bar, 10 μm. (E and F) Volcano plots of differentially expressed genes in HEK293-DOR cells treated with ICI-SNC80 (ICI = 100 μM, SNC80 = 100 nM) for 1.5 hours (E) or 6 hours (F) versus control cells (100 μM ICI treated). Results are presented as the mean differential gene expression from three replicates and expressed as log₂FC. No genes are significantly changed in their expression [criteria as in (B) and (C)]. (G) Immunoblot of ERK1/2 activation upon treatment of HEK293-DOR cells with ICI-SNC80 (100 μM and 100 nM) or SNC80 (100 nM) for indicated times or untreated control (CTR). (H) Quantification of pERK1/2 protein levels normalized to total ERK1/2 in HEK293-DOR cells treated as in (G), for 5 min and 25 min. N = 3, mean ± SEM.