Pcmv 3tag 1c

Human c‐Myc cDNA (1365 bp) and MYCN construct were kind gifts from Drs. William P. Tansey and Arturo Sala. The LMNA cDNA (1995 bp) was introduced into lentiviral construct from GeneChem Co., Ltd. Their truncated fragments were prepared by PCR assay with primer sets (Table S5), and introduced into pCMV‐HA, pCMV‐3Tag‐1C, pET28‐A or pGEX‐6P‐1 (Addgene). Oligonucleotides specific for shRNAs or small guide RNAs (Table S6) were introduced into GV298 (GeneChem Co., Ltd.) or dCas9‐BFP‐KRAB (Addgene).

Human SPI1 cDNA (816 bp, Shanghai GeneChem Co., Ltd, China) were inserted into CV186 lentivirus vector (Genechem Co., Ltd). Human SPIB cDNA (789 bp) construct was provided by Dr. Zhe Liu.⁴⁷ Their truncated fragments obtained using primer sets (Table S5) were inserted into pCMV‐N‐Myc, pCMV‐3Tag‐1C, pGEX‐6P‐1 or pET‐28a (Addgene, Watertown, MA). The shRNAs for SPI1 or SPIB were established by inserting oligonucleotides (Table S6) into GV298 vector (Shanghai GeneChem Co., Ltd), while small interfering RNAs (siRNAs) were synthesized (Table S6).

Human MZF1 coding sequence (CDS, 2205 bp), MZF1 cDNA (2920 bp), YY1 cDNA (1245 bp) and corresponding truncations were obtained from NB tissues by PCR primers (Table S2), and inserted into pcDNA3.1 (Invitrogen), pEGFP-N1, pCMV-3Tag-1C, pCMV-C-Flag, pCMV-N-MYC (Addgene, Cambridge, MA), or lentiviral expression vector CV186 (Genechem Co., Ltd, Shanghai, China), respectively. Human HK2 and PGK1 expression vectors were obtained from Genechem Co., Ltd. Mutation and frame-shift deletion of GFP or MZF1-uORF was prepared with GeneTailor^TM Site-Directed Mutagenesis System (Invitrogen) and primers (Table S2). Oligonucleotides encoding short hairpin RNAs (shRNAs) specific for MZF1, HK2, PGK1, MZF1-uORF, or YY1 (Table S3) were subcloned into GV298 (Genechem Co., Ltd). Single guide RNAs (sgRNAs) were designed using Guide Design Resources (http://crispr.mit.edu) to target upstream or downstream region relative to transcription start site of MZF1 (Table S2), and inserted into dCas9-VPR or dCas9-BFP-KRAB (Addgene), respectively. Stable cell lines were screened by administration of neomycin or puromycin (Invitrogen).