CCND3 knockdown modulates gene expression outside of the CDK4/6 complex

We compared gene expression profiles of B-ALL cell lines of different genotypes after CCND3 knockdown or inhibition of its associated kinases CDK4/6 by palbociclib and found that a distinct subset of genes is exclusively regulated after CCND3 knockdown, likely responsible for induction of apoptosis after CCND3 knockdown but not treatment with palbociclib. Overall design: B-ALL cell lines NALM-6, RS4;11 and BV-173 were either transduced with shCCND3 or scrambled control in the pRSI12-U6-shUbiC-TagRFP lentiviral vector (BioCat, Heidelberg, Germany) or treated with 1 µM palbociclib or equivalent amounts of DMSO for 3 days. Transduced cells were isolated on day 4 post transduction, palbociclib treated cells were collected after three days. RNA was isolated with the RNeasy mini kit (QIAGEN). RNA-sequencing and data processing was done by BGI (Hong-Kong). RNA libraries were prepared for sequencing using standard BGI protocols. SOAPnuke was used for data filtering and Bowtie2 was used to map clean reads to reference. RSEM was used to calculate gene expression levels and DEseq2 was used to detect DEGs. hg38 was used as reference genome.