Figure 3. WBSCR16 knockout leads to markedly decreased cell growth, increased apoptosis, decreased cyclin E levels and blockage at the G₁-S transition.

(A) MTT assay growth curves for untransfected HeLa cells, empty vector-transfected HeLa cells, or CRISPR-Cas9 WBSCR16 knockout (KO) HeLa cells. Ordinate axis: 1 is OD at time 0, with increasing numbers denoting fold increases thereafter. (B) FACS analysis shows increased apoptotic cells (sub-G₁ DNA content) upon WBSCR16 CRISPR/Cas9 knockout. ***, P<0.005. Immunoblots show (C) increased cleaved caspase 3 and (D) reduced cyclin E in WBSCR16 CRISPR/Cas9 KO cells, compared with untransfected cells and cells transfected with empty vector. GAPDH, loading control. Cyclin A, additional loading control in D, showing cyclin E effects to be specific. (E) G₁-phase HeLa cells, transfected with empty vector (WT) entered S-phase immediately. In contrast, G₁-phase HeLa cells subjected to CRISPR-Cas9 WBSCR16 KO took at least 9 h to enter S-phase, with some entering after 12 h (blocked 9–12 h at the G₁-S transition). Once in S, both WT and KO cells took 6 h to enter G₂/M. (F) Both WT and WBSCR16-KO G₂/M-phase cells entered G₁-phase immediately. WT cells then transitioned quickly to S after G₁, but KO cells were blocked in G₁ for ~11 h, entering G₁ only at the 15-h time point. Thus, experiments with G₁-phase (E) and G₂/M-phase (F) cells both indicate a block at G₁-S transition in consequence of WBSCR16 ablation. See also Table S1.