(A) Schematic diagram of reporter cassette HRF measuring gene conversion, crossing over, and SSA. The cassette contains two copies of defective GFP-Pem1. The first copy has two inverted I-SceI recognition sites, and a 22-nt deletion on the first exon of GFP, and the second copy of GFP lacks the start codon. I-SceI induced DSBs can be repaired by gene conversion, crossing over or SSA, resulting in functional GFP. SD, splice donor; SA, splice acceptor; the yellow line stands for the homologous arm. (B) Schematic diagram of reporter cassette HRIF measuring gene conversion and crossing over. The positions of the two copies of defective GFP is different in HRF and HRIF. In HRF reporter, both copies are downstream CMV promoter while in HRIF reporter the CMV promoter is located between the two copies of GFP. In response to I-SceI digestion, only gene conversion or crossing over can reconstitute functional GFP. SD, splice donor; SA, splice acceptor; the yellow line stands for the homologous arm. (C) Effect of farrerol and RS-1 treatment on SSA efficiency. The two reporters were integrated into HCA2-hTERT cells, and a pool of colonies containing chromosomally integrated reporters were mixed for further analysis. HRF minus HRIF was employed as the measure of SSA efficiency. 5 μg I-SceI vector and 15 ng pDsRed2-N1 were transfected into reporter cells pre-treated with different small molecules. Small molecules were still added to the cells until FACS analysis three days after transfection. The ratio of GFP positive cells versus DsRed positive cells was used as the measure of repair efficiency. Error bars represent the s.d. *p<0.05, **p<0.01, ***p<0.001, n.s., not significant, t-test. All experiments were repeated at least three times.