Gv298 vector

Full human SOST cds sequence and a human SOST sh-RNA were cloned and inserted into the PCDH-CMV-MCS-EF1-copGFP vector (GenePharma, Shanghai, China) and the GV298 vector (Genechem, Shanghai, China), respectively. Lentiviral particles were produced using three-plasmid systems, including pMD2.G, psPAX2 and the individual vectors, with NEOFECT™ DNA transfection reagent (Neofect, Beijing, China) according to the manufacturer’s instruction. For infection, HDPCs were incubated with lentiviral particles and polybrene (four μg/mL) in complete medium for 12 h. Cells with successful infection by pCDH-human-SOST were designated SOST, cells infected by sh-SOST were designated sh-SOST, and control cells infected by empty vector were designated Control and sh-Ctrl, respectively. The expression of sclerostin was quantified by real-time PCR and Western blot analysis. The mRNA and protein expression levels were significantly upregulated in the SOST group (P < 0.001, Figs. 1E and 1F), while they were knocked down by over 85% in the sh-SOST group 48 h after infection (P < 0.0001, Figs. 1G and 1H).

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).

By using PCR, CNBP cDNA (540 bp) and its truncated forms were prepared from NB specimens (Table S8), and inserted into the CV186 (Shanghai Genechem Co., Ltd., China), pET28a‐mCherry (Genprice Inc., San Jose, CA, USA), pmCherry‐N1 (Genprice Inc.) or pCMV‐3Tag‐1A (Addgene, Cambridge, MA, USA). Human MYCN expression vector was a kind gift from Dr. Arturo Sala (College of Health, Medicine and Life Sciences, Brunel University London).⁵⁴ PCR primers (Table S8) were used to amplify human SMARCC2 cDNA truncations (3645 bp) that were then subcloned into CV186 (Genechem Co., Ltd.), pCMV‐N‐MYC (Addgene) or pET28a‐EGFP (Genprice Inc.). Oligonucleotides specific for shRNAs against CNBP, MYCN, KPNB1, SMARCC2, SMARCC1 or SMARCA4 (Table S9) were ligated into GV298 vector (Genechem Co., Ltd). The dCas9‐BFP‐KRAB (Addgene) was used to produce single‐guide RNAs (sgRNAs; Table S9) against upstream or downstream regions of the transcription start sites for CNBP, BYSL, NOP58 or RRP9. Puromycin (Invitrogen) screening was undertaken for establishment of stable cells.