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. Author manuscript; available in PMC: 2021 Oct 15.
Published in final edited form as: Cancer Res. 2021 Feb 16;81(8):2086–2100. doi: 10.1158/0008-5472.CAN-20-2218

Figure 3. Enhanced macropinocytosis in Tsc2-deficient cells is mediated via DGKA.

Figure 3.

(A) Macropinocytosis is enhanced (3-fold) in Tsc2−/− MEFs compared to Tsc2+/+ MEFs. Ritanserin (10uM; 16 hours) inhibited the macropinocytic uptake of dextran (0.5mg/ml, FITC-Dextran) selectively in Tsc2−/− MEFs. PA (100uM) restored macropinocytosis of ritanserin-treated Tsc2−/− MEFs to levels compared to that of untreated cells. (B) Exogenous protein uptake (0.5mg/ml, BSA-TMR) was increased in ritanserin (10uM; 16 hours) treated Tsc2−/− MEFs compared to Tsc2+/+ MEFs. PA (100uM) partially rescued macropinocytosis in Tsc2−/− MEFs. As expected, EIPA (25uM;16 hours) inhibited macropinocytic dextran and BSA uptake. (C) Confocal microscopy shows that ritanserin inhibits macropinocytic uptake of dextran (10uM; 16 hours). (D) Genetic downregulation of DGKA inhibits macropinocytic dextran uptake (0.5mg/ml, FITC-Dextran). (E) Lysotracker staining revealed that ritanserin (10uM; 16 hours) reduces lysosome numbers in Tsc2−/− MEFs but not in Tsc2+/+ MEFs. Combination treatment with CQ and ritanserin further inhibited lysosomal numbers. mTORC1 inhibitor rapamycin (20nM; 16 hours) strongly decreased lysosomes in Tsc2−/− MEFs but not in Tsc2+/+ MEFs. (F-G) Lysosomal activity is enhanced in Tsc2−/− MEFs compared to Tsc2+/+ MEFs. Ritanserin treatment (10uM; 16 hours) decreased lysosomal activity by 50% (0.2mg/ml; DQ-BSA). Data represented as mean +/− standard deviation from three biological replicates. Statistical significance was assessed using two-way ANOVA with Bonferroni correction with ***p<0.001, ****p < 0.0001.