Fast taqman mix

Cells were dissolved in the TRIzol reagent (Invitrogen, 12044977) for total RNA extraction. RNA quality was confirmed by optical density measurement. cDNA was synthesized from 1 μg of total RNA using random primers and Moloney murine leukemia virus reverse transcriptase (MultiScribe, Applied Biosystems, 10117254). The cDNA was used for qRTPCR in an Mx3005P Real-Time PCR System (ThermoFisher Scientific). Reactions were carried out in duplicate for all conditions using a Sybr Green Master mix (ThermoFisher Scientific, 4344463) or Fast Taqman mix (ThermoFisher Scientific, 4444557) and expression of mouse Gapdh mRNA (Life Technology, 433764T) was used as endogenous control in the comparative cycle threshold method (2⁻ΔΔ^Ct). Primer sequences used are listed in Supplementary Table S2.

Total RNA was extracted from frozen tumors, lungs, and cultured cells with TRIzol (Invitrogen, 12044977). cDNAs (synthesized using random primers and Moloney murine leukemia virus reverse transcriptase (MultiScribe, Applied Biosystems, 10117254)) were used for qRT–PCR in an Mx3005P Real‐Time PCR System (Thermo Fisher Scientific) with Sybr Green Master mix (Thermo Fisher Scientific, 4344463) or Fast Taqman mix (Thermo Fisher Scientific, 4444557). Expression of mouse Gapdh mRNA (Life Technology, 433764T) was used as endogenous control in the comparative cycle threshold method (2‐ΔΔC_t) with the listed primers (Appendix Table S7).