Fig. 4

SAV1 is necessary for MERTK inhibition-mediated suppression of Akt activation in RCC cells. a, b IB analysis of WCL derived from HEK293 cells transfected with Flag-SAV1 and HA-Akt1. Where indicated, cells were treated with indicated doses of MK2206 (a) or GDC0068 (b) for 10 h before cell collection. c, d Cell viability assays were performed with WT- and SAV1-depleted RCC4 cells treated with either indicated doses of MK2206 (c) or GDC0068 (d) for 3 days (mean ± SD, n = 3). 1000 cells were plated in each well on 96-well plates. *P < 0.05 (Student’s t test). e IB analysis of WCL derived from indicated RCC4 cells treated with MERTK inhibitor UNC2025 (400 nM) for 2 h before cell collection. f, g Cell viability assays were performed with WT- and SAV1-depleted RCC4 (f) or UMRC6 (g) cells treated with indicated doses of MERTK inhibitor UNC2025 for 3 days (mean ± SD, n = 3). *P < 0.05 (Student’s t test). h IB analysis of WCL derived from 786-O cells transfected with indicated SAV1 constructs and treated with indicated doses of MERTK inhibitor UNC2025 (400 nM) for 2 h before cell collection. i Cell viability assays were performed with 786-O cells transfected with indicated SAV1 constructs and treated with indicated doses of MERTK inhibitor UNC2025 for 3 days (mean ± SD, n = 3). *P < 0.05 (Student’s t test). j A schematic proposed model to demonstrate that SAV1 is necessary for MERTK-inhibition governed inactivation of Akt. Specifically, when SAV1 expression is high, MERTK inhibitors reduce Akt1-pY26 signals, subsequently enhancing SAV1 binding to Akt to block Akt plasma membrane attachment and binding to Akt upstream activating kinases, thus leading to suppression of Akt. On the other hand, when SAV1 expression is low, MERTK inhibition, although still reduces Akt1-pY26 signals, would achieve limited effects due to the inability to recruit SAV1 for suppressing Akt