Supplementary Material for: Cytoskeleton Protein Filamin A Is Required for Efficient Somatostatin Receptor Type 2 Internalization and Recycling through Rab5 and Rab4 Sorting Endosomes in Tumor Somatotroph Cells D.Treppiedi F.Mangili E.Giardino R.Catalano M.Locatelli A.G.Lania A.Spada M.Arosio D.Calebiro G.Mantovani E.Peverelli 2020 The high expression of somatostatin receptor 2 (SST<sub>2</sub>) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST<sub>2</sub> signaling and expression. The aim of this study was to explore FLNA involvement in SST<sub>2</sub> intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST<sub>2</sub> internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST<sub>2</sub> internalization, control versus FLNA small interfering RNAs (siRNA) cells, respectively, <i>p</i> < 0.001) and human GH-secreting primary cultured cells (70.3 ± 21.1 vs. 24 ± 19.2% SST<sub>2</sub> internalization, control versus FLNA siRNA cells, respectively, <i>p</i> < 0.05). In addition, confocal imaging revealed impaired SST<sub>2</sub> recycling to the plasma membrane in FLNA silenced GH3 cells. Coimmunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely dependent recruited to octreotide-stimulated SST<sub>2</sub> receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST<sub>2</sub> complex in GH3 cells, it significantly impaired efficient SST<sub>2</sub> loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and 4, respectively (33.7 ± 8.9% down to 25.9 ± 6.9%, <i>p</i> < 0.05, and 28.4 ± 7.4% down to 17.6 ± 5.7%, <i>p</i> < 0.01, for SST<sub>2</sub>-Rab5 and SST<sub>2</sub>-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST<sub>2</sub> trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes.