Fig. 4

MGD-28 exhibited a significant degradation effect on IKZF1/2/3 and CK1α via a Cullin-CRBN dependent pathway. A-B. Flow cytometry plot showing wild type and CRBN^–/– NCI-H929 cells treated with indicated doses of MGD-4, MGD-28 or pomalidomide (Pom) for 3 days, and cells were stained with Annexin V-PE and DAPI (A). Apoptosis was measured by flow cytometry using Annexin V as a marker (B). Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), **p < 0.01. C. Viability of wild type and CRBN^−/− NCI-H929 cells treated with MGD-28 for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. D. MV-4-11 cells viability in the absence and presence of pomalidomide (Pom, 10 µM) for 96 h. Data shown are a representative graph of three independent experiments; mean ± SD of triplicates. E. Western blot analysis of CK1α, IKZF1, IKZF2 and IKZF3 degradation in NCI-H929 cells CRBN knockout, pre-treated with MLN4924, MG132 or pomalidomide for 1 h and treated with MGD-28 for 24 h. F. Western blot analysis of p53, p21 and MDM2 in NCI-H929 cells treated with different doses of MGD-28 for 24 h. G-H. PCR analysis of CDKN1A/p21 (G) and MDM2 (H) in MV-4-11 cells treated with different doses of MGD-28, lenalidomide (Len, 10 µM) or pomalidomide (Pom, 10 µM) for 24 h. Error bars denote standard deviations (independent experiments, n = 3). Student’s t test, ns (no significance), **p < 0.01. I. Western blot analysis of CK1α, p53, and p21 in NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector control treated with lenalidomide (Len, 10 µM) or MGD-28 (1 µM). J. NCI-H929 cells overexpressing CK1α WT, CK1α G40N, or vector were treated with MGD-28. Data shown are a representative result of three independent experiments; mean ± SD of triplicates.