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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Leukemia. 2015 Aug 12;30(1):219–228. doi: 10.1038/leu.2015.222

Figure 6. AUY922 upregulates the expression of BIM and BAD proteins, which are sequestered by overexpressed BCL2 in JURKAT cells.

Figure 6

(a) BCL2-overexpressing JURKAT cells were treated with the indicated concentrations of AUY922 or DMSO (AUY922 0 nM) for 48 h. Anti-BCL2 immunoprecipitate from 320 μg of each total cell lysate (IP BCL2) was immunoblotted for BCL2, BIM and BAD (lanes 4-6). Whole-cell lysate (20 μg; WCL) was also loaded and immunoblotted for BCL2, BIM, BAD, and α-tubulin (lanes 1-3). Three isoforms of BIM (BIMEL, BIML and BIMS) are shown. Band intensities of BIMEL and BAD on the short-exposure film were measured by ImageJ, and relative intensities are shown. (b) BIM and BAD mRNA expression in T-ALL cell lines treated with 30 nM of AUY922, 5 μM of ABT-199 or DMSO for 48 h. LOUCY cells were treated with 1 nM of ABT-199 instead of 5 μM of ABT-199, based on the high sensitivity of the cells to ABT-199. BIM and BAD mRNA expression was measured by quantitative RTPCR and normalized by GAPDH expression. Expression levels relative to DMSO-treated cells are shown as mean ± s.d. of triplicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-sample, two-tailed t test. (c) Western blot analysis to assess the effect of AUY922 on BIM and BAD protein expression levels in T-ALL cell lines. KOPT-K1, HPBALL, JURKAT, MOLT-4 and LOUCY cells were treated with 30 nM of AUY922 or DMSO (AUY922 0 nM) for 48 h, and subjected to immunoblot analysis with antibodies specific for BIM, BAD and α-tubulin. (d) JURKAT and KOPT-K1 cells were lentivirally transduced with each of TYK2-targeting shRNAs (shTYK2 #2 and #3) or a control shRNA targeting luciferase (shLuc). Whole-cell extracts were analyzed by immunoblotting with antibodies specific for TYK2, BIM, BAD and α-tubulin.