P3xflag myc cmv 24

The complete MBD3 sequence was amplified by RT–PCR using primers MBD3-all-F (5′-CGGAATTCCGATGGAGCGGAAGAGCCCGAGCG-3′) and MBD3-all-R (5′-GGGGTACCCCCTAGACGTGCTCCATCTCCGGGT-3′) from a cDNA library of PANC1 cells, then inserted into the expression Vector p3xFLAG-Myc-CMV-24 (Sigma, St. Louis, MO, USA). The sh-EGFP and sh-MBD3 plasmids were previously constructed in our laboratory (Sigma) and kept at the School of Basic Medical Sciences, Jiangsu University. Plasmids were transfected into colon cancer cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The specific sequences are shown in Table S2. Methods for generating retroviruses encoding reprogramming factors and further infecting NPCs were referred to a previous paper (Pontes et al., 2014).

The entire Lin28B sequence was amplified with RT‐PCR using primers Lin28B‐all‐F, 5′‐CGGGGTACCAATGGCCGAAGGCGGGGCTA‐3′ and Lin28B‐all‐R, 5′‐CGCGGATCCTTATGTCTTTTTCCTTTTT‐3′ and then cloned into the expression vector p3xFLAG‐Myc‐CMV™‐24 (Sigma‐Aldrich, San Francisco, CA, USA). The Lin28B, TET3, PKCβ and KRAS short hairpin (sh)RNA oligos (Lin28B‐shRNA‐F, 5′‐CCGGGCAGGCATAATAAGCAAGTTACTCGAGTAACTTGCTTATTATGCCTGCTTTTTG‐3′; Lin28B‐shRNA‐R, 5′‐AATTCAAAAAGCAGGCATAATAAGCAAGTTACTCGAGTAACTTGCTTATTATGCCTGC‐3′; TET3‐shRNA‐F, 5′‐CCGGGAACCTTCTCTTGCGCTATTTCTCGAGAAATAGCGCAAGAGAAGGTTCTTTTTG‐3′; TET3‐shRNA‐R, 5′‐AATTCAAAAAGAACCTTCTCTTGCGCTATTTCTCGAGAAATAGCGCAAGAGAAGGTTC‐3′; sh‐PKCβ‐F, 5′‐CCGGCAAGTTTAAGATCCACACGTACTCGAGTACGTGTGGATCTTAAACTTGTTTTT‐3′; sh‐PKCβ‐R, 5′‐AATTCAAAAACAAGTTTAAGATCCACACGTACTCGAGTACGTGTGGATCTTAAACTTG‐3′; sh‐KRAS‐F, 5′‐CCGGCTATACATTAGTCCGAGAAATCTCGAGATTTCTCGGACTAATGTATAGTTTTTG‐3′; sh‐KRAS‐R, 5′‐AATTCAAAAACTATACATTAGTCCGAGAAATCTCGAGATTTCTCGGACTAATGTATAG‐3′) produced by Sangon Biotech Co. Ltd. (Shanghai, China), were first annealed into double strands and then cloned into pLKO.1‐puro or Tet‐on‐pLKO‐puro vector.