Fig 2: Analysis of cell lines with overexpression and knockdown of RBP2. (a) Stable βlox5 cells overexpressing RBP2, mRBP2 or empty vector were assessed for proliferation every day for 4 days. Similar proliferation assays were performed every other day for days for the H727 and QGP-1 cells lines that were transiently transfected with scrambled si or RBP2si, with the assay beginning 24 h after transfection. (b) mRNA levels of p21, p27 and p57 in stable overexpressing βlox5 cells, and H727 and QGP-1 transiently transfected with RBP2 siRNAs. (c) Representative western blots for the same targets in cell lines as above in (b) and protein quantitation via densitometry of p21, p27 and p57 resulting from three separate experiments using actin for normalization. Significance was measured relative to the respective control (*P<0.05 **P<0.02 #P<0.005).

Fig 3: CITED1 silencing restrains cell cycle progression and reduces cell viability.(A) A bar chart showing the % change in cell cycle distribution in #1 siCITED1 treated HT144 cells relative to siNEG treated HT144 cells. The reduction in the total S-phase is shown at 33 h, 48 h and 72 h post-transfection in addition to the corresponding increase in the diploid G1 fraction. (B) Western blots showing upregulation of CDKN1A/P21 following CITED1 silencing in HT144 cells and suppression of CDKN1C/P57 following CITED1 overexpression in A2058 cells. (C) An Alamar Blue based metabolic assay shows a reduction in cell viability over 5 days in HT144 cells treated with siCITED1 relative to those treated with siNEG. Stars indicate significance for siNEG vs. #1 siCITED1 where ∗∗∗ p < = 0.0005, ∗∗ p < = 0.005 and ∗ p < = 0.05. In the case of siNEG vs. #1 siCITED1, the difference is significant (*) at 96 and 120 h.

Fig 4: Imatinib and Dasatinib increase quiescence in primary CML stem cells and reduce the p18INK4c and p57Kip2 Cyclin-Dependent Kinase Inhibitors. LSC derived from CML patients were culture in the absence (Control) or in the presence of Dasatinib (50 nM) or Imatinib (2.5, uM) for 48 hours and their cell cycle status and CDKI content was then analyzed. Results correspond to a representative plot for LSC enrichment and each analysis. Corresponding mean ± SD (from 4 different experiments) for cell cycle status and MFI for each protein are indicated in squares below the each cythometric plot. ***p < 0.05 as compared to the corresponding control.

Fig 5: Cell cycle analysis.HeLa and HEK293T cells were transfected with plasmids expressing wild-type or one of four mutant CDKN1C proteins, p.Ile272Ser, p.Asp274Asn, p.Phe276Val, and p.Phe276fs*10. Mock transfections were performed by using pCMV-3Tag-1B vector alone. At 48 h after transfection, cells were fixed in 70% ethanol, stained by propidium iodide, and then subjected to cell cycle analysis by FACSCanto II. Percentages of the cells in G1 phase are presented by bar graphs: blue bars, HeLa cells; red bars, HEK293T cells.