Pharmacologic inhibition of GSK3, Cullin–RING ligases or the proteasome impairs the growth of multiple myeloma cells. (a) GSK3 inhibition induces nuclear accumulation of p100 in HMMCLs. KMS11 and ARP-1 cells were treated with GSK3i IX (2 μM for 8 h). Total (T), cytoplasmic (C) and nuclear (N) fractions were isolated and analysed by immunoblotting as indicated. Cytoplasmic and nuclear markers, IκBα and c-Myc, respectively, are also shown. (b) Proteasome or Cullin–RING ligase inhibitors induce nuclear accumulation of p100 in HMMCLs. KMS11 cells were treated with either bortezomib or MLN4924 for 4 h. Total, cytoplasmic and nuclear fractions were analysed by immunoblotting as indicated. Cytoplasmic and nuclear markers, IκBα and c-Myc, respectively, are also shown. (c) NF-κB-dependent transcription is impaired in HMMCLs treated with a GSK3 inhibitor. KMS11 cells were treated with GSK3i IX and the steady-state levels of the indicated mRNAs were analysed by real-time PCR (±s.d., n = 3). The value for the amount of PCR product present in DMSO-treated cells was set as 1. (d) Inhibition of GSK3, Cullin–RING ligases or the proteasome decreases the viability of HMMCLs. KMS11, MM1.R and ARP-1 cells were treated with increasing concentrations of GSK3i IX, MLN4924 or bortezomib. Cell viability was measured by MTS assay at 72 h after treatment and individually normalized to untreated cells (0 nM), arbitrarily set as 100%. Error bars represent s.d., n = 4. (e) Next-generation GSK3 inhibitors induce accumulation of nuclear p100 in HMMCLs. KMS11 cells were treated with GSK3i XVI or GSK3i XXII (5 μM and 1 μM for 8 h, respectively). Total, cytoplasmic and nuclear fractions were isolated and analysed by immunoblotting as indicated. Cytoplasmic and nuclear markers, IκBα and Lamin A/C, respectively, are also shown. (f) Next-generation GSK3 inhibitors decrease the viability of HMMCLs. KMS11, MM1.R and ARP-1 cells were treated with increasing concentrations of GSK3i XVI or GSK3i XXII. Cell viability was measured by MTS assay at 72 h after treatment. Each value was individually normalized on the DMSO-treated cells, arbitrarily set as 100%. Error bars represent s.d., n = 4. Uncropped images of blots are shown in Supplementary Fig. S8.