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. 2018 Sep 21;8:14161. doi: 10.1038/s41598-018-32256-x

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Macropinocytosis is increased in Tsc2-deficient cells. (A) Uptake of macropinocytotic cargo dextran (FITC-Dextran, 70 kDa; 0.5 mg/ml) was significantly increased in Tsc2^−/− MEFs compared to Tsc2^+/+ MEFs. Rapamycin (20 nM) had no impact on macropinocytosis in Tsc2^−/− MEFs, while Torin1 (250 nM) reduced dextran uptake to levels comparable to Tsc2^+/+ MEFs. (B) Increased dextran uptake in mouse inner medullary collecting duct epithelial cells (mIMCD3) with Tsc2 knockout (sgTsc2) compared to sgCtl cells. (C) Immunoblot of serum starved mIMCD3 cells showing knockout of Tsc2 and increased phosphorylation of S6 ribosomal protein. The blot was cropped to highlight the relevant bands. The full-length blot is presented in Supplementary Figure 2B. (D) Increased dextran uptake in Tsc2-deficient embryonic fibroblasts (MEFs) derived from Tsc2^fl/fl Rosa26-CreERT2 mice (Tsc2ko) compared to Tsc2-expressing MEFs (Tsc2wt). (E) Immunoblot of MEFs treated with ethanol or 4-hydroxytamoxifen to knockout Tsc2. Cells were either grown in 10% FBS DMEM or serum starved for 16 hours. (F) Representative images of Tsc2^+/+ and Tsc2^−/− MEFs treated for 24 hours with vehicle, rapamycin (20 nM) or Torin1 (250 nM). The macropinocytic cargo dextran was internalized at higher levels in Tsc2^−/− MEFs, compared to Tsc2^+/+ MEFs. Rapamycin did not impact macropinocytosis in Tsc2^−/− MEFs. FITC-Dextran uptake decreased with Torin1. (G) Increased DQ Red BSA processing in Tsc2^−/− MEFs, compared to Tsc2^+/+ MEFs. Data represented as mean +/− standard deviation of three biological replicates. Statistical significance was assessed using two-way and one-way ANOVAs with Bonferroni correction with *p < 0.05, ***p < 0.001, ****p < 0.0001.