RECQ1-depletion leads to aphidicolin sensitivity, aberrant replication stress response and chromosomal fragility in HeLa cells. A. Cells transfected with control or RECQ1 siRNA were exposed in quadruplicate to increasing doses of aphidicolin (μM) or grown in regular complete medium and their survival was measured 72 h later by MTS assay. Percentage of control growth was plotted for each data point, representing the mean ± SD of three independent experiments. B. Surviving fraction was determined for control or RECQ1 siRNA transfected cells growing in regular medium or medium supplemented with aphidicolin (μM) by cell count after 72 h and presented as the mean ± SD of three independent experiments. C. Detection of the activated forms of Chk1 kinase and the phosphorylated forms of RPA32 and H2AX in untreated or aphidicolin (0.5 μM) treated HeLa cells transfected with control or RECQ1 siRNA. Depletion of RECQ1 by siRNA is also shown. GAPDH serves as loading control. D and E. Chromosomal breaks/gaps in RECQ1-depleted cells. Metaphase spreads of control or RECQ1 siRNA transected cells (72 h after siRNA) grown in the absence or presence of aphidicolin (0.5 μM, 24 h) were scored for chromosome gaps/breaks. Ten metaphases of each cell condition were analyzed. In untreated condition, RECQ1-depleted cells showed significantly more breaks than control cells (p < 0.05); aphidicolin treatment induced chromosome breaks and gaps in control or RECQ1 siRNA transfected cells (D). Representative partial metaphase spreads for RECQ1-depletion is shown (E); bottom panel shows an enlarged section. APH, aphidicolin; HU, hydroxyurea.