Figure 4 from GDF-15 Predicts Epithelioid Hemangioendothelioma Aggressiveness and Is Downregulated by Sirolimus through ATF4/ATF5 Suppression

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Sirolimus downmodulated GDF-15 release in patient-derived models of EHE through the inhibition of ATF4 and ATF5. A, GDF-15 released in culture medium of control cells and cells exposed for 3 days to doxorubicin or different sirolimus concentrations as detected by ELISA. Data were normalized as amount (pg) of released GDF-15 to total released (mg) proteins and reported as mean ± SD of three independent experiments. B, GDF-15 released in the blood collected from untreated mice and after the end of treatment with doxorubicin or different sirolimus doses. Data were normalized as amount (pg) of released GDF-15 to tumor weight (g) and reported as mean ± SD of three mice/experimental group. C, Western blot analysis of GDF-15, ATF4, and ATF5 expression in untreated cells (−) and cells treated with different sirolimus concentrations for 3 days (left) and in tumors removed from untreated (−) and sirolimus-treated mice after the first round of treatment with different drug doses (right). Western blot data of GDF-15 expression obtained on tumors from three untreated mice (−) and three mice exposed to 2.5 mg/kg sirolimus. Cropped images of selected proteins are shown. D, siRNA-mediated downregulation of ATF4 (left) and ATF5 (right) in the EHE cell line as detected at the mRNA level by RT-qPCR. E, Effects of ATF4 and ATF5 downregulation on GDF-15 protein expression as detected by level by Western blotting (left) and on cytokine release in cell culture medium as measured by ELISA (right). ELISA data were normalized as amount (pg) of released GDF-15 to total released (mg) proteins and reported as mean ± SD of three independent experiments. Cropped images of selected proteins are shown. F, The working model of sirolimus-induced GDF-15 downregulation. Sirolimus-induced mTORC1 inhibition led to reduced 4E-BP1 phosphorylation/activation followed by decreased ATF4 and ATF5 expression, thus resulting in GDF-15 downregulation. (F, Created with BioRender.com.)

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