FIG 2.
Calpain activity is essential for mediating resistance to 17AAG. (A) Immunoblot analysis of MDA-MB-231 cells infected with a lentivirus expressing the vector alone (control), CAPN1, CAPN2, or protease-inactive mutant CAPN2 (CAPN2-C105S). (B) Assays of the death of the MDA-MB-231 cells described in panel A treated for 48 h with the concentrations of 17AAG indicated. Data are mean ± SD. LD50s: control, 16.7 μM; capn1, 43.9 μM; capn2, 58.4 μM; capn2-C105S, 27.2 μM (P = 0.0063). (C) Immunoblot analysis of CAPN1 in MDA-MB-231 cells treated with 50 μM SNJ1945 for 24 h. RasGAP blot assays were used as a loading control. An autoproteolytic fragment of CAPN1 and a nonspecific (ns) band are indicated. (D) Assays of the death of MDA-MB-231 control, calpain knockdown (shRNA-Capns1), or control cells treated with 50 μM SNJ1945 and challenged with increasing concentrations of 17AAG for 48 h. Data are mean ± SD. LD50s: control, 166 μM; shRNA-Capns1, 13.3 μM; control plus SNJ1945, 16.9 μM. Student's t tests were performed. *, P ≤ 0.01 relative to the control. One-way ANOVA was used to detect significant differences between LD50s.