Fig 2: Lysosomal targeting of endogenous Sstr2 by Dvl1.A, IMR32 cells were treated with vehicle or 20 mM NH4Cl for 2 h and then fixed and stained for Sstr2 (green), Lamp1 (magenta), and DNA (blue). Shown are representative z-plane images. The scale bar represents 10 µm. B, quantification of colocalization of Sstr2 and Lamp1 using Pearson’s coefficient, from three separate experiments. Bars are median values. C, IMR32 cells were transfected with the siRNAs shown and then fixed and stained for cell surface Sstr2 (red) and DNA (blue). Shown are representative micrographs. The scale bar represents 20 µm. D, quantification of Sstr2 surface expression divided by cell area in IMR32 cells. Each dot represents one cell. Results are combined from three separate experiments. E, representative Western blots for control, Dvl1, or pan-Dvl siRNA transfected IMR32 cells. F, quantification of cell surface Sstr2 in IMR32 cells by whole cell ELISA is shown. Three experiments with six replicates per condition are combined. G, representative Western blots for Dvl1 expression in siRNA transfected IMR32 cells used for cell surface ELISAs. Significance in (B) and (F) was determined with unpaired Student’s t test. Significance in (D) was determined with one-way ANOVA with multiple comparisons, Tukey’s post hoc test. ***p < 0.001; ****p < 0.0001; n.s. = not significant.

Fig 3: Effects of Dvl1 knockdown on octreotide-stimulated Sstr2 internalization, recycling, and signaling.A, HEK293 cells were transfected with the siRNAs shown and then retransfected with HA-Sstr2. The cells were incubated with HA-epitope antibody and then treated with octreotide for 30 min. Surface fraction of HA-Sstr2 was determined by ELISA of nonpermeabilized cells. Shown are the results from four separate experiments, each done in triplicate. Significance was assessed by ANOVA with multiple comparisons, Tukey’s post hoc test. B, representative Western blot of HEK293 cells transfected with the siRNAs shown. C, HEK293 cells were transfected with the siRNAs shown and then retransfected with HA-Sstr2. Octreotide (100 nM) was added for 30 min, after which the Sstr2 antagonist PRL1392 (100 nM) was added for the times shown. Cells were fixed and surface Sstr2 was measured by ELISA. Shown are the results from three separate experiments combined, each performed in triplicate. D, representative Western blot of siRNA transfected cells. E and F, HEK293 cells stably expressing HA-Sstr2 were transfected with the siRNAs shown and then infected with a BacMam expressing a cADDis cAMP reporter. Cells were treated with the forskolin analog NHK477 (10 μM), minus or plus octreotide (100 nM), and reporter fluorescence was measured in real time. Shown are the average of three separate experiments. G, representative Western blot of HEK293 cells transfected with the siRNAs shown. H, HEK293 stably expressing HA-Sstr2 were transfected with control or Dvl1-specific siRNAs and then stimulated with the agonist octreotide (100 nM) for the times shown. ERK1/2 activation was assessed by Western blot analysis. Shown is a representative experiment from three independent experiments. I, quantification of ERK1/2 activation in control or Dvl1 knockdown cells. Shown is the average of three independent experiments. Errors are SEM. Significance in (C), (E), and (F) was determined by one-way ANOVA. n.s. = not significant. Significance in (I) was determined by Student’s t test. ∗p < 0.05.

Fig 4: Wnt signaling inhibits Sstr2 expression.A, HEK293 cells were transfected with HA-Sstr2 plus the Wnts shown. Whole cells lysates were examined for HA-Sstr2 expression by Western blotting. A representative experiment from five separate experiments is shown. B, quantification of HA-Sstr2 expression in Wnt transfected cells. Bars are median values. C, Western blot for endogenous Sstr2 expression in IMR32 cells stably expressing empty vector (control) or Wnt7a. Shown are representative Western blots. D, quantification of endogenous Sstr2 expression in control or Wnt7a expressing IMR32 cells. E, effects of overnight treatment with a pan-Dvl PDZ domain inhibitor (NSC668036, 10 µM), a CK1d/e inhibitor (PF670462, 10 µM), or a porcupine inhibitor (LGK974, 10 µM) on endogenous Sstr2 expression in IMR32 cells. F, quantification of Sstr2 expression in IMR32 cells after treatment with Wnt pathway inhibitors. Bars are the medians from 4 to 5 separate experiments. G, growth hormone (GH) secretion from GH3 cells was assessed following Dvl PDZ inhibitor treatment (10 µM, 4 h), in the presence and absence of octreotide (100 nM, 15 min). Shown is the average of three replicates performed in duplicate. H, model for regulation of Sstr2 trafficking to the lysosome by Dvl1. In (B) and (F), significance was assessed by ANOVA with multiple comparisons, Dunnett’s post hoc test. For (G), significance was assessed by ANOVA with Tukey’s multiple comparisons test. Significance in (D) was assessed by unpaired student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. = not significant.

Fig 5: Dvl1 inhibits Sstr2 expression.A, HEK293 cells were transfected with HA-Sstr2 and the Flag-tagged PDZ domain-containing proteins shown. Cells were lysed in an SDS- and urea-containing buffer (whole cell lysate) and HA-Sstr2 expression was assessed by Western blotting. Shown is a representative experiment from five separate experiments. B, quantification of HA-Sstr2 expression after cotransfection of PDZ domain-containing proteins. Bars denote median values. C, HEK293 cells were transfected with HA-Sstr2 and Flag-tagged Dvl1, Dvl2, or Dvl3. HA-Sstr2 expression in whole cells lysates was assessed by Western blotting. Shown is a representative experiment from four separate experiments. D, quantification of HA-Sstr2 expression after cotransfection of Flag-tagged Dvl1, 2, or 3. Each point represents the average number of puncta per cell in a field, with a minimum of three separate fields quantified per experiment. Bars denote median values. E, HEK293 cells were transfected with control, Dvl1, or pan-Dvl-specific siRNAs. One day later the cells were retransfected with HA-Sstr2. HA-Sstr2 expression was examined by Western blotting. Shown is a representative experiment from nine separate experiments. F, quantification of HA-Sstr2 expression after control, Dvl1, or pan-Dvl knockdown. Bars denote median values. G, IMR32 cells were transfected with control, Dvl1, or pan-Dvl siRNAs and tested for effects on endogenous Sstr2 expression. Shown is a representative experiment from six separate experiments. H, quantification of Sstr2 expression after Dvl1 or pan-Dvl knockdown. Each point represents the average number of puncta per cell in a field, with a minimum of three separate fields quantified per experiment. Bars denote median values. Significance in (B), (D), and (H) was assessed by one-way ANOVA with multiple comparisons, Dunnett’s post hoc test. Significance in (F) was assessed by unpaired Student’s t test. *p < 0.05; ***p < 0.001. n.s. = not significant.