Short hairpin rna shrna

Short hairpin RNA (shRNA) targeting the coding sequence of MITF and control shRNA were purchased from Santa Cruz. Lentiviruses encoding control shRNA and MITF shRNA were produced in HEK-293T cells by transient transfection of lentiviral-based vectors and their packaging vectors psPAX2 and pMD2.G. The virus was collected, filtered through a 0.45 µm syringe filter after 48 h, and the M397 cells were spin-infected with viral supernatant supplemented with 10 µg/mL polybrene at 900 g and 30 °C for 90 min. The transduced cells were selected using puromycin, starting at 3 days post transduction.

The establishment method of targeted gene‐deletion cell lines that participated in this study were constructed as described previously.^[17, ^29] Briefly, THLE2 cell lines with ZDHHC1‐ZDHHC24 ablation were established by CRISPR‐Cas9 system. The designed small guide RNA (sgRNA) for human ZDHHCs gene were established and cloned into lentiCRISPR v2 vectors(Addgene, Watertown, MA, USA) to create the Cas9/sgRNA‐loaded lentivirus. Small guide RNA (sgRNA) for establishment of knockout THLE2 cells were obtained from Santa Cruz Biotechnology, Inc., as described in Table S2, Supporting Information. Oligonucleotide for the sgRNAs were packaged into lentiCRISPR v2 vectors digested with BsmBI restriction enzyme. The obtained cell clones‐containing gene deficiency were distinguished by western blotting. Mouse primary hepatocyte with Zdhhc3 deletion cells were directly isolated from global Zdhhc3‐deficient mice. For the establishment of mouse primary hepatocytes with Zdhhc1‐Zdhhc25 knockdown, short hairpin RNA (shRNA) for knockdown expression of Zdhhcs were obtained from Santa Cruz Biotechnology, Inc., as described in Table S3, Supporting Information, were packed into adenovirus (Addgene, Watertown, MA, USA), respectively.