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A selective role for CDCA7 in lymphomagenesis
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Figure 1. The expression profiles between BL and LCL are different. A. Experimental design. The expression profile of 5 BL cell lines (BL2, Ramos, DG-75, Akata, and Mutu-I) was compared with that of a pool of LCL lines (X50-7, IB4 and Dana). Each BL cell line was considered as a biological replica for data analysis. B. Venn diagram showing overlapping of genes significantly regulated relative to the pool of LCL. The total number of overlapping up- and down-regulated genes is shown. C. Genes up- (59 genes) or down-regulated (425 genes) >3-fold were selected and the most statistically significant genes (TOP 100) were further selected. The only genes among the TOP 100 that were up-regulated in BL cell lines are shown indicating their relative expression (Fold), adjusted P value, Ensemble gene identification number, and the biological process in which these genes might be involved.
was pooled and used as a universal control for the whole experi- ment. The RNA from DG-75, Ramos, BL2, Mutu-I, and Akata BL cell lines was mixed with an equal amount of universal control pool and processed for microarray analysis using 22K-oligo microarrays (CapitalBio Corporation). Genes with similar expres- sion within BL cell lines were ranked according to their P-value for differential expression (adj. P value <0.05). Gene expression data are available at http://www.ncbi.nlm.nih.gov/projects/geo/ (accession number GSE41865).
Northern blotting and quantitative PCR analysis
Northern blotting was performed as described27 using 32P-labeled 5’ CDCA7-2 and ACTG DNA fragments. Real-time quantitative RT-PCR (q-PCR) was performed using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for both iso- forms of human CDCA7 (Hs00230589_m1), CDCA7-1 (Hs00912235_m1), CDCA7-2 (Hs00914361_m1), or TBP (Hs00427621_m1).
Cell Transfection, lentivirus production and cell transduction
HEK-293T cells were transfected using the calcium phosphate method.28 Lentiviral particles were produced as previously described.29 MISSION pLKO.1-puro-based vectors encoded either a non-targeting shRNA (SHC002) or the following CDCA7 target- ing shRNAs: sh-25 (TRCN0000140725), sh-40 (TRCN0000139240), sh-56 (TRCN0000139556) sh-83 (TRCN0000145183). DG-75, Ramos, BL2, Toledo, Molt-4 and S1F cell lines were transduced as described30 and selected in 1mg/ml puromycin >96h.
Antibodies
The anti-CDCA7 S99 polyclonal rabbit serum was raised against the CRGRHPLPGSDSQSRRPR KLH-conjugated peptide as described.31
Cell proliferation and cell cycle analysis
Cell proliferation was assessed by EdU incorporation as described.32 Cell cycle analysis was performed as previously described.30
Transformation assays
Transformation assays were performed as described.30 All mice were inoculated with control cells in one flank and CDCA7- silenced cells in the opposite flank.
Results
Transcriptomic analysis of immortalized and tumoral cells
To identify genes participating in stages of malignant transformation beyond or independent of replicative immortalization, we compared the transcriptome of immortalized B cells (LCLs) and BL cell lines. Both cell types display similar growth, replication and viability rates when cultured in liquid media on plastic.30 LCLs cul- tured in liquid media grow mostly in clumps (Online Supplementary Figure S1). Single cells can also be found in these cultures, and most of them are anchored to the cul- ture vessel surface, as indicated by their marked spreading
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