The cells were cultured in 100-mm diameter dishes or six-well plates, and total RNA was extracted from cells using ISOGEN (NIPPON GENE CO., Tokyo, Japan) or Nucleospin RNA® (Macherey-Nagel GmbH & Co. KG, Düren, Germany). cDNA was synthesized by ReverTra Ace® (TOYOBO). The RT-qPCR was done using THUNDERBIRD® qPCR Mix (TOYOBO) and Step One Plus™ (Thermo Fisher Scientific). Gene expression was quantified and then normalized against the RPL30 housekeeping gene expression10 (link). The primer sequences were as follows: endogenous DKK3, CAG GCT TCA CAG TCT GGT GCT TG (forward) and ACA TTG TTT CCA TCT CCT CCC CTC (reverse)11 (link); DKK3-HA-Tag, AGG AAC TGA TGG AGG ACA CG (forward) and CTT CTG CCT TCT TCG TCT CC (reverse); RPL30, ACA GCA TGC GGA AAA TAC TAC (forward) and AAA GGA AAA TTT TGC AGG TTT (reverse); cyclin D1, CTT CCT CTC CAA AAT GCC AG (forward) and AGC GTG TGA GGC GGT AGT AG (reverse); and c-myc, TCC TCG GAT TCT CTG CTC TC (forward) and GTT GTG CTG ATG TGT GGA GA (reverse). The PCR conditions were 95°C for 1 min followed by 40 cycles of 95°C for 15 s and 60°C for 45 s. Absolute quantification was used for analysis as previously described12 (link).