Stellar chemically competent cells

Two sets of primers were designed to amplify the full-length cDNA (Supplementary Material Table S1), keeping in mind that the vector would be linearized with SspI restriction enzyme (New England Biolabs, Ipswich, USA). The online Expasy translate tool was used to double-check the continuity of the ORF-His tag fusion protein. A nested polymerase chain reaction (PCR) was performed to amplify three predicted full length TPS cDNAs with the first set of primers according to CloneAmp^TM HiFi PCR premix (Takara Bio, Kusatsu, Japan) protocol with a 55 °C annealing temperature. PCR products were diluted 10-fold in water and used for PCR using a second set of primers and 60 °C annealing temperature. Resulting single-band products were cloned into SspI-digested pETHis6GST expression vector (Addgene) by the Sequence and Ligation Independent Cloning method (SLIC; [95 (link)]). The mix was transformed into Stellar chemically competent cells (Takara Bio, Kusatsu, Japan). Colonies with recombined plasmid were identified by colony PCR, used for plasmid purification. Inserted fragments were fully sequenced by Sanger, confirming expected nucleotide sequences. All resulting recombinant proteins contained the open reading frame with an N-terminal His tag.

Stellar chemically competent cells (Takara, Saint-Germain-en-Laye, France, #636763) were used for transformation of pHDAC5^WT, pSNAI-2, pTCF4 and pAPCDD1^WT, whereas pHDAC5^MUT and pAPCDD1^MUT were transformed into XL10-Gold Ultracompetent cells (Agilent, #200314) after site-directed mutagenesis. Plasmid extraction was performed using PureLink™ HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific, #K210004).