Figure 1: CDK4/6 inhibition inhibits cancer metastasis.

(a) MDA-MB-231 cells were treated with PD0332991 as indicated. The migratory potential of cells was analysed by wound-healing assay and results were quantified (right panel). Scale bars, 100 μm. The results represent the means±s.d. of three independent experiments. ^##P<0.01. (b–h) Passage 3 tumours from HCI001 were used to test the effect of PD0332991 on metastasis. When primary tumours reached 100–150 mm³, mice were randomized and treated either with saline or PD0332991 for 5 weeks (n=8). Tumour weights (b) were measured after mice were killed. Data are expressed as mean±s.d. Statistical analyses were performed with the Student’s t-test. Liver (c–e) and lung (f–h) metastatic nodules were examined macroscopically or detected in paraffin-embedded sections stained with H&E. Scale bars, 50 μm. Arrowheads indicate metastases. Fisher’s exact test was calculated and statistical significance is represented (^#P<0.05). (i) MDA-MB-231 cells were treated with PD0332991 and western blot was performed with indicated antibodies. (j,k) MDA-MB-231 cells were treated with PD0332991 for 24 h. Then, cells were treated with either vehicle or MG-132 for an additional 6 h. SNAIL1 level was detected by western blotting. Three independent experiments were performed and results are quantified in (k). ^##P<0.01. (l,m) MDA-MB-231 cells were treated with PD0332991 for 24 h. Cycloheximide pulse-chase assay was performed in cells. Three independent experiments were performed and results are quantified in m. (n) MDA-MB-231 cells were transfected with indicated constructs and treated with vehicle or PD0332991 for 24 h in the presence of MG-132. Ni-NTA beads were used to pull down His-tagged ubiquitin, and the polyubiquitylated SNAIL1 protein was examined. (o) MDA-MB-231 cells were treated vehicle or PD0332991 for 24 h in the presence of MG-132 and cell lysates were subjected to immunoprecipitation with control IgG, anti-SNAIL1 and the ubiquitination of SNAIL1 protein was examined.