Effect of LCP1 I232F on cellular stress responses. (A) Flow cytometric analysis of Annexin V and propidium iodide (PI)-based apoptosis assay was performed on isogenic 32D cell lines as indicated. Camptothecin (CPT; 1 μM) was used as positive control. (B) 32D cells (wtLCP1 and LCP1 I232F) grown under conditions of IL-3 withdrawal for 48 hours were analyzed for apoptosis by flow cytometry–based Annexin V/PI staining (n = 3). Annexin V⁺ and Annexin V⁺/PI⁺ cells were identified as early and late apoptotic cells, respectively. (C) Analysis apoptotic markers, PARP1 and Caspase-3, for their cleavage by immunoblotting was performed on 32D cells expressing wtLCP1 and LCP1 I232F as indicated. β-actin was used as loading control. (D) Real-time quantitative polymerase chain reaction analysis investigating makers of unfolded protein response Bip, Chop, Xbp1/Xbp1s, and Atf4 was performed at indicated time points with Actin as the housekeeping gene (n = 4). Data are shown as mean ± standard deviation. Statistical analysis was assessed by using the Student t test. dox, doxycycline; DMSO, dimethyl sulfoxide.