Fig. 3. Sin1–4A mutant inhibits AKT-pSer⁴⁷³ in response to DNA damage.

(A) MCF7-Sin1^KO cells were reconstituted with Sin1-WT or Sin1–4A. EV was a negative control. Cells were treated with 10 μM etoposide (Eto) for 30 min before performing immunofluorescent staining. Representative images of AKT-pSer⁴⁷³ and DAPI are shown; n = 3 independent experiments. Scale bar, 10 μm. (B) IB analysis of WCL derived from MCF7-Sin1^KO cells stably expressing HA-Sin1-WT or Sin1–4A. EV was a negative control. Cells were treated with 10 μM etoposide for 60 min before harvesting. n = 3 independent experiments. (C) In vitro kinase assays analysis of mTORC2 kinase activity. Flag-Sin1 IP was derived from MCF7-Sin1^KO cells reconstituted with Flag-Sin1-WT or Sin1–4A. EV was a negative control. Cells were treated with 10 μM etoposide for 60 min before harvesting. n = 3 biological replicates. (D) MCF7-Sin1^KO cells reconstituted with Flag-Sin1-WT or Sin1–4A were serum-starved for 12 hours and then stimulated with 100 nM insulin for 30 and 60 min before harvesting for IB analysis. n = 3 independent experiments.