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Fig. S5 In situ hybridization of Cre in transgenic Tg(hsp70-Cre) embryos with chemical treatment. (A,B) Tg(hsp70-Cre) embryos with or without heat shock were fixed and subjected to ISH for Cre as controls. (C,D) Tg(hsp70-Cre) embryos were incubated in chemicals (Triptolide and TSA) from 22 hpf, heat shocked from 24-25 hpf, fixed at 30 hpf, and analyzed by ISH for Cre. The Cre expression levels were suppressed in comparison to B, indicating that these chemicals interfere with heat shock promoter activation or transcription. (E-H) Tg(hsp70-Cre) embryos were incubated in chemicals (TPCK, PD98059, LY171883 and 12-methoxydodecanoate) from 22 hpf, heat shocked from 24-25 hpf, fixed at 30 hpf, and analyzed by ISH for Cre. The Cre expression levels were not suppressed in comparison to B, indicating that these chemicals do not interfere with heat shock promoter activation or transcription; therefore, these chemicals were confirmed as suppressors of the RAS pathway.