Fig. 5: CDK8 downregulation contributes to the cytotoxicity induced by CCND3-knockdown.

A Heatmap generated from RNA-Seq data performed for the three B-ALL cell lines NALM-6, RS4;11 & BV-173, which were either treated with 1 µM palbociclib for 3 days or sorted after 4 days post lentiviral transduction with either a shRNA targeting CCND3 (shC3-1) or control vector (scr). The heatmap represents the result of unsupervised hierarchical clustering of the 12 genes that were exclusively regulated only by CCND3-knockdown in all cell lines. (Supplementary Fig. 13). q = 0.1, p < 0.05. B CDK8 protein levels are downregulated by CCND3-knockdown, not by palbociclib treatment. CDK8 levels were analyzed by immunoblot either after three days of 1 µM palbociclib treatment or four days post transduction with a CCND3-targeting shRNA. n = 3, image representative. C ShRNA-dependent CDK8 knockdown was controlled by immunoblot, image representative of n = 3. D Both CDK8-targeting shRNAs reduce cell fitness in a competitive growth assay (see legend to Fig. 3B). Data shown as mean ± SD, n = 3. E CDK8-knockdown by shRNA induces significant apoptosis in B-ALL cell lines. Cells were transduced as described in the legend of Fig. 5D and sorted four days after transduction for 100% RFP⁺ cells, then cultured for two more days. Specific apoptosis was determined by staining with PI and for Annexin-V, measured via flow cytometry. Data shown as mean ± SD, n = 3 Statistical analysis was performed by Student’s t-test.