Expression and mutational status of USP8 in tumors causing ectopic ACTH secretion syndrome

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   Figure 1

   Immunohistochemical stainings of ACTH, EGFR and USP8 in a representative typical bronchial carcinoid, an atypical bronchial carcinoid and a small-cell lung carcinoma. Counterstaining was done with toluidine blue (ACTH) and hematoxylin (EGFR and USP8).
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   Figure 2

   Effect of USP8 on POMC transcription in neuroendocrine DMS79 and NCI-H727 cells. (A) Endogenous POMC transcription in cells overexpressing human USP8 as determined by real-time RT-PCR vs pituitary ACTH-secreting AtT-20 cells. The empty pME-Flag vector was used as control (mock). (B) Endogenous POMC transcription after knocking down human (DMS79, NCI-H727) or mouse (AtT-20) USP8 with a specific shRNA for USP8. A scramble shRNA was used as control. Both plasmids also coded for GFP. After transfection cells were left in medium supplemented with 2% fetal calf serum for four days and then green fluorescent cells were sorted. Both RNA and proteins were extracted for qPCR (B) and Western blot (G). Data are means ± standard deviation of 3 measurements, calculated as POMC/TFIIB and presented as % of each control. nd, not-detected. (C, D, E and F) Effect of USP8 overexpression or knock-down on rat Pomc (C and D) or human POMC promoter activity (E and F). Data are represented as means ± standard deviation of 3 measurements (error bars), calculated as luciferase-to-β-galactosidase ratio and presented as percentage of each control. RLA, relative luciferase activity. Measurements were compared to the control condition using t-test. *P < 0.05. (G) Knock-down efficacy was monitored by Western blot for USP8. β actin was used as control.

• © 2017 Society for Endocrinology