Icr 191

2-aminoanthracene (2-AA), Mitomycin C (MMC), ICR-191, Benzo(a)pyrene (BP), Sodium azide (SA) and 2-nitrofluorene (2-NF) were from Sigma-Aldrich, St Louis Missouri United States.

Negative, including blank sterility controls, and positive controls were included in the testing of microPEA, both in the presence and absence of S9 to verify spontaneous mutation frequencies and strain‐specific responses to known mutagens. Positive control compounds without S9 mix were as follows: sodium azide (Sigma Aldrich St. Louis, MO, USA) for TA1535, ICR 191 (Sigma Aldrich) for TA97a; 3‐methylmethane sulfonate (Sigma Aldrich) for TA100 and TA102; and, 4‐nitroquinoline‐N‐oxide (Acros Organics, Geel, Belgium) for TA98. Positive controls with S9 mix were as follows: 2‐aminoanthracene (Sigma Aldrich) for TA1535; 2‐aminofluorene (Sigma Aldrich) for TA97a, TA98, and TA100; and, danthron (Sigma Aldrich) for TA102.

To isolate chemically mutagenized CDT-resistant clones, ten pools of CHO-pgs A745 cells (A745, provided by Jeff Esko, UCSD) were treated with ICR191 (Sigma Aldrich) at a concentration high enough to kill 90% of the cells [58] (link). The resulting cells were counted, seeded at 1×10⁶ cells per 10 cm plate and selected with 20 nM Aa-CDT. Resulting resistant cells were subjected to limiting dilutions to obtain single cell clones, expanded and reselected with Aa-CDT.
Selection of retrovirally mutagenized CDT-resistant clones was performed similar to a previously reported protocol [44] (link). Briefly, an Hd-CDT-sensitive clonal A745 cell line expressing tetR-KRAB (A745TKR) was established. Ten pools of 1×10⁶ A745TKR parental cells were mutagenized by transduction with murine leukemia virus encoding the transcription response element TetO₇ in the long terminal repeat (pCMMP.GFP-NEO-TRE) at a multiplicity of infection of 0.1. These pools were transcriptionally repressed at proviral integration sites for 96 hours in the absence of doxycycline then selected with 5 nM Hd-CDT for 24 hours. After selection, two of the ten pools yielded colonies; these colonies were picked, expanded and reselected with Hd-CDT. None of the CDT-resistant clones displayed doxycycline dependant sensitivity to CDT, so they were further maintained in the presence of doxycycline.