Supplementary Figure 3: Analyses of human T-cell acute lymphoblastic leukemia (T-ALL) cells.

(a) Human T-ALL MOLT-16 cells were transduced with viruses expressing anti-cyclin C shRNA or control shRNA (CTR). Shown are mean percentages of cells in the indicated cell cycle phases, error bars denote standard deviation of triplicate samples. G₁ CTR to G₁ cyclin C shRNA P = 0.052, S CTR to S cyclin C shRNA P = 0.993, G₂/M CTR to G₂/M cyclin C shRNA P = 0.151 (t-test). (b) MOLT-16 cells were transduced with anti-cyclin C or control shRNAs (CTR). Cells were harvested and lysates probed with the indicated antibodies. (c) MOLT-16 cells were transduced with viruses expressing anti-cyclin C shRNAs (1, 2 or 3) or control shRNA (CTR). Cycloheximide (20 μg/ml) was added to the media and cells were harvested at the indicated time-points. Lysates were immunoblotted with the indicated antibodies. Note that knockdown of cyclin C strongly increased the half-life of ICN1, but it had no major effect on half-lives of other Fbw7 targets (c-Myc, cyclin E, mTOR, Mcl1; see also Supplementary Fig. 1f). The half-life of ICN1 in cells depleted of cyclin C and in control cells was determined by densitometric analysis (ImageJ, NIH). (d) Combined knock-down of cyclin C and Fbw7. MOLT-16 cells were transduced with lentiviral vectors expressing shRNA against Fbw7 or cyclin C, or both. Lysates were immunoblotted using the indicated antibodies. Note that knockdown of cyclin C led to upregulation of ICN1 (compare ICN1, lanes 1 versus 2). Knockdown of Fbw7 also increased ICN1 levels, as expected (compare ICN1, lanes 1 versus 3). Importantly, cyclin C-knockdown did not further increase ICN1 levels in cells depleted of Fbw7 (ICN1, lanes 3 versus 4), indicating that the two proteins operate in the same pathway. Fbw7-knockdown increased the levels of an Fbw7 target, cyclin E (compare lanes 1 versus 3). (e) MOLT-16 cells were transduced with viruses encoding cyclin C, CDK8, or with empty vectors (EV). The levels of the indicated proteins were determined by immunoblotting. (f) MOLT-16 cells were transduced with viruses encoding HA-tagged cyclin C, CDK8, CDK3, CDK19 or empty vectors (EV). The levels of the indicated proteins were determined by immunoblotting. For detection of HA-tagged proteins, an anti-HA antibody was used. This antibody detects a background band that co-migrates with HA-CDK19 (star).