Skip to main content
. Author manuscript; available in PMC: 2012 Oct 1.
Published in final edited form as: Nat Cell Biol. 2012 Mar 4;14(4):375–385. doi: 10.1038/ncb2463

Figure 5.

Figure 5

p100 degradation promotes multiple myeloma cell survival. (a) In B-cell lines, p100 is cytoplasmic, whereas Fbxw7α is largely nuclear. Subcellular fractionation was carried out on HMMCLs (ARP-1, KMS11, MM1.S and U266B1), DLBCL (Ly10, SudHL6 and Farage) and Burkitt’s lymphoma (Raji) cell lines. Lysates were immunoblotted as indicated. IκBα and α-tubulin, cytoplasmic controls. c-Myc, nuclear control. (b) Stable p100(Ser707/711Ala) and p100(4×NES) are correctly processed in HMMCLs. HMMCLs were infected with retroviruses encoding HA-tagged p100, p100(Ser707/711Ala), p100(4×NES) or empty vector (EV). Cell extracts were immunoblotted as indicated. Short exposure (s.e.) and long exposure (l.e.) are shown. (c) Expression of stable p100 mutant impairs HMMCL growth. HMMCLs were infected with retroviruses expressing p100, p100(Ser707/711Ala) or empty vector. Cell proliferation was monitored by MTS assay and normalized on empty vector at day 1, arbitrarily set as 100%. Error bars represent s.d., n = 4. (d) Stable p100 expression impairs NFκB-dependent transcription in HMMCLs. HMMCLs were infected as in c. Steady-state levels of the indicated mRNAs were analysed by quantitative PCR (±s.d., n = 3). PCR product amount in empty vector was set as 1. (e) Expression of stable p100 promotes apoptosis of HMMCLs. HMMCLs were infected as in c. At 72 h post-infection, cells were stained with Annexin V/7-AAD and analysed by flow cytometry. Apoptosis and cell viability were calculated as the percentage of Annexin V/7-AAD double-positive and double-negative cells, respectively. (f) Forced nuclear localization of p100 inhibits HMMCLs proliferation. HMMCLs were infected with retroviruses encoding p100, p100(4×NES) mutant or empty vector. Cell viability was monitored as in c. Values were normalized on the empty vector at time 0. Error bars represent s.d., n = 4. (g) Fbxw7 depletion impairs HMMCL growth. HMMCLs were infected with the indicated shRNA-encoding lentiviruses. Cell viability was monitored as in c (left panels). Values were normalized on Luc shRNA at time 0. Error bars represent s.d., n = 4. Fbxw7 protein levels were analysed by immunoblotting (right panel). (h) Fbxw7 depletion stabilizes nuclear p100. HMMCLs were infected as in g. Total (T), cytoplasmic (C) and nuclear (N) fractions were immunoblotted as indicated. IκBα and Lamin A/C are cytoplasmic and nuclear markers, respectively. Uncropped images of blots are shown in Supplementary Fig. S8.