Fig. 3
RNAi mediated knockdown of LSF reduced expression of mitotic regulators. a Schematic of experimental protocol. siRNAs targeting LSF or a non-expressed target were transfected into HeLa cells at the indicated concentrations, followed by synchronization of cells and release from the G1/S block. Cells were harvested between 0 h and when control cells visually reached mitosis. b Representative immunoblot of LSF and β-actin from lysates collected following release from the final thymidine block for 0, 4 and 8 h (as in panel a). The images were cropped to indicate the proteins of interest; full images are in Additional file 5. c Cells synchronized during treatment with 20 nM of LSF (+) or control siRNA (−) were harvested for RNA at 0 or when control cells reached mitosis (~ 8 h after release from the final G1/S block). TFCP2 (which encodes LSF), AURKB, and CDC20 RNA levels were measured and normalized to those of GAPDH from the same time point. The relative gene expression levels are reported as the fraction of the RNA levels in the control siRNA-treated cells at 0 h. Data points and means are from 3 to 4 independent experiments. *p = 0.042; **p = 0.0010 (AURKB), p = 0.0019 (CDC20). d Representative immunoblots of the indicated proteins or protein modification are shown for lysates harvested when control cells visually reached mitosis (~ 8 h post release from the final G1/S block). Molecular weight markers are indicated on the right side. Relative intensities can only be compared within each separate immunoblot of each protein showing levels at increasing FQI1 concentrations, not between separate immunoblots. The images were cropped to indicate the proteins of interest; full images are in Additional file 6. e Independent quantification of AURKB and CDC20 protein levels from cell lysates harvested for mitotic expression (as in panel d). Each target protein was normalized to the level of β-actin in the same lysate. Data points and means are from 2 to 3 independent experiments. *p = 0.027 (AURKB), p = 0.021 (CDC20); **p = 0.0087; ***p = 0.0003