Figure 1. siORC1 impairs recovery of U2OS but not 1BR3hTERT cells from HU.

1BR3hTERT (A) or U2OS (B) cells were transfected with siControl or siORC1 oligonucleotides (0.1-20 nM), and viability assessed 7 days later. Results represent mean +/− SD from triplicate samples. Black arrows indicate the oligonucleotide concentration subsequently utilised. (C) 1BR3hTERT and U2OS cells were transfected with 5 or 0.6 nM siORC1, respectively. ORC1 protein levels were assessed by immunoblotting 48 hours later. β-actin was a loading control. (D-E) 1BR3hTERT and U2OS cells were transfected with siRNA for 48 hours and treated with HU (0.03-40 mM) for 24 hours. Viability was assessed 4 days following HU removal. (F-G) 1BR3hTERT and U2OS cells transfected with siRNA as described were treated with 2 mM HU for indicated times and HU-induced S-phase damage was assessed by immunofluorescence labelling of γH2AX. Nuclei containing bright γH2AX pan-nuclear staining were scored as γH2AX⁺. Black arrows indicate the time of HU treatment required to obtain 100% γH2AX⁺ cells. (H) Viability was assessed after transfection with siRNA and treatment with HU for 24 or 48 hours as in (D-E). HU IC₅₀ values were estimated from the viability graphs. (I) U2OS cells were treated with siRNA as described above and then with HU (0.05-2 mM) for 24 hours. Clonogenic survival was estimated at 10 days following HU removal. (J) As in (I) except 1BR3hTERT cells were treated with HU for 48 hours and clonogenic survival was estimated 21 days following HU removal. Additional controls are shown in Supplementary Fig. S1. (K) ORC1 protein levels were assessed in chromatin-bound and unbound fractions in 1BR3hTERT and ORC1-P4hTERT by immunoblotting. (L) 1BR3hTERT or ORC1-P4hTERT were treated with HU (0.03-40 mM) for 24 hours. Viability was assessed 4 days post HU removal. (M) 1BR3hTERT or ORC1-P4hTERT cells were treated with 0.5 mM HU for 48 or 72 hours, respectively. Viability was assessed as above.