12xcsl d1egfp

Wnt/β-catenin, Notch, Hedgehog, mTOR, NF-kB, P38, JNK, ERK and PKA reporter plasmids were overexpressed liver cancer cells, and treated with lactic acid or DM-αKG^{59 (link)}. The activity levels of each signaling pathway were detected by FACS. For example, Wnt activity level = (TOP-GFP intensity)/(FOP-GFP intensity). The plasmids used in this assay are: TOP-GFP (addgene no. 35489), FOP-GFP (addgene no. 35490), 12XCSL-d1EGFP (addgene no. 47684), 7Gli:GFP (addgene no.110494), TORCAR (addgene no. 64927), NF-kB-eGFP (addgene no. 118093), TORCAR(T/A) (addgene no. 64928), p38KTRmCerulean3 (addgene no. 59155), JNKKTRmRuby2 (addgene no. 59154), PKAKTRClover (addgene no.59153), ERKKTRClover (addgene no. 59150).

Template DNAs for EGFP and BFP mRNAs were prepared via PCR using KOD Dash (TOYOBO, Japan) with 12XCSL-d1EGFP (Addgene, Watertown, MA, USA) and pTagBFP-N (Evrogen, Russia) vectors as templates. The template DNAs for EGFP and BFP mRNAs were amplified using the following primers (sequence of the T7 promoter is underlined): 5′-CCG GGT AAT ACG ACT CAC TAT AGG GAC ACA ACT GTG TTT ACT TGC-3′ and 5′-GAT GCT ATT GCT TTA TTT GTA AC-3′ for EGFP, and 5′-CCG GGT AAT ACG ACT CAC TAT AGG TCT ATA TAA GCA GAG CTG G-3′ and 5′-GTT AAC AAC AAC AAT TGC ATT C-3′ for BFP. Capped mRNAs were prepared via in vitro transcription using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen) and a polyA addition with the Poly (A) Tailing Kit (Invitrogen) according to the manufacturer's protocols.