Fig 2: Basal levels of SSTR2 protein expression. (A) Basal expression level of SSTR2 in human fibroblast cell lines (917 and WI38), aggressive thyroid cancer cell lines (TPC, FTC236, and Hth7) and in neuroendocrine cancer cell lines (medullary thyroid cancer: TT and MZ; pancreatic NE cancer: BON; pulmonary carcinoid: H727). H727 cell line has the lowest basal expression of SSTR2 among all NET cells (Supplementary Figure S1). (B) A comparison of basal SSTR2 expression at the protein level between two pulmonary carcinoid cell lines: UMC-11 and H727 (Supplementary Figure S2). Error bars show standard deviation.

Fig 3: Identification and cell-based validation of peptide ligand–GPCR interactions. a Commercially available Dynorphin A was Cy5-labeled and probed to a VirD-GPCR array. Quantitative analysis showed that it bound to VirD-OPRD1 with the highest Z-score followed by ADCYAP1R1 and P2RY2. b A commercially available peptide SRIF-14 was Cy3-labeled and probed to a VirD-GPCR array. Quantitative analysis revealed that it bound to several unexpected off-targets in addition to its canonical receptor, SSTR2. c Vero cells were separately infected with SSTR2, GABBR2, NTSR1, KISS1R, or K082 virus. Infected cells were then incubated with Cy5-labeled SRIF-14 at 8 µM in the absence (upper panel) or presence of cold SRIF-14 (middle panel) or cyclosomatostatin (cycloSST, lower panel). d Quantitative analysis of binding signals. Each binding assay was performed in triplicate and the obtained binding signals were normalized to those of the K082 controls. Data were analyzed by two-way ANOVA with repeated measures followed by Bonferroni post-test. *P < 0.05, comparison between VirD-GPCRs and K082 in the absence of competitor ligands; #P < 0.001, comparison between binding signals obtained in the absence and presence of the competitor ligands. n = 3, biologically independent samples

Fig 4: 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 5: Increase in surface SSTR2 incidence. (A) The pulmonary carcinoid cell line H727 showed a significant increase in the percentage of cells expressing SSTR2 after being administered 6 nM of TDP-A (B) UMC-11, another pulmonary carcinoid cell line, showed significant increases in the percentage of SSTR2-positive cells after receiving either 2 nM or 6 nM of TDP-A (C) A papillary thyroid carcinoma cell line, TPC, did not show any change in SSTR2 expression after TDP-A administration. ns: not significant.