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. 2021 Jun 28;28(12):3357–3370. doi: 10.1038/s41418-021-00823-x

Fig. 6. DREAM cooperates with RB for repression of MKI67 gene expression.

Fig. 6

Expression of Mki67 mRNA or expression from MKI67 reporter constructs in knockout cell models. Wild-type mouse NIH3T3 and knockout cells deficient for Lin37, Rb, or Lin37 and RB (DKO) were employed for the assays. A Mki67 mRNA expression in serum-starved and restimulated cells. Wild-type (wt, blue), Lin37-deficient (Lin37−/−, yellow), Rb-deficient (Rb−/−, gray), and DKO (black) cells were arrested by serum starvation (0 h) followed by addition of serum to the medium, to stimulate cells to re-enter the cell cycle. Hours after serum addition are given. Relative Mki67 mRNA levels were quantified using real-time RT-qPCR. All values were normalized to the highest measurement from the wild-type cells. Mean values ± SD are given of two technical replicates for each time point. The fold-change of Mki67 mRNA levels was calculated for each cell line by dividing maximal expression by levels at 0 h. Gray bars at 0 and 18 h time points were introduced to emphasize comparison of expression between serum-starved and restimulated cells, respectively. B Expression from MKI67 promoter luciferase reporter constructs and phenotype rescue in cells lacking Lin37 and Rb. Serum-starved NIH3T3 wild-type cells (wt) and NIH3T3 cells deficient for both Lin37 and Rb (DKO) were transfected before starvation with different expression plasmids: empty pcDNA vector (vector control, gray), carrying a cDNA coding for Rb (Rb, green), containing a Lin37 cDNA (Lin37, yellow) or expressing both Lin37 and RB (Lin37 + RB, blue). In addition, cells were transfected with reporter plasmids containing wild-type (wt) or mutated in CHRprox, CDE, and CHRdist elements (3mut) MKI67 promoter constructs. Luciferase relative light units (RLUs) of one representative out of three biological replicates were measured. Mean values ± SD of three technical replicates are given and significances were calculated using the Student’s t-test (n.s., not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).