Pnl1.1 luciferase vector

The mouse MITF-M promoter construct (−1143 to +48, wtCRE) was amplified via PCR using mouse genomic DNA as template, and cloned into the pGL3-basic firefly luciferase reporter vector (Promega, Madison, WI, USA). The mtCRE was constructed by deleting the CRE motif (−153/−146, 5′-TGACGTCA-3′) by using the site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). All constructs were verified via DNA sequencing. The cells were seeded in 24-well plates and transfected for 24 h with pGL3-Mitf-M (wtCRE or mtCRE) luciferase reporter, using Lipofectamine^® 2000 reagent (Invitrogen, Carlsbad, CA, USA), before treatment with α-MSH and/or PCA for a further 24 h. The cells were harvested and assayed using the Nano-Glo^® Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI, USA). The pNL1.1 luciferase vector (Promega, Madison, WI, USA) was used as a normalization control.

The human p62 promoter construct (-1,650 to +120) was amplified by PCR using human genomic DNA as template, and cloned into the pGL3-basic firefly luciferase reporter vector (Promega Corp). The construct was verified by DNA sequencing. Huh7 cells seeded in 24-well plate were transfected with pGL3-p62 luciferase reporter using Lipofectamine^® 2000 reagent (Invitrogen) for 24h before treatment with DC and/or SP600 for 24h. Cells were harvested and assayed using the Nano-Glo^® Dual-Luciferase^® Reporter Assay System (Promega Corp). A pNL1.1 luciferase vector (Promega Corp) was used as a normalization control.

The coding regions of the corresponding genes were amplified by RT-PCR using the primers listed in S1 Table. The RT-PCR products were cloned in the pCS2 expression plasmids and the mutant constructs were prepared by PCR mutagenesis using the Phusion polymerase (ThermoFisher Scientific). The TOP5 and FOP5 luciferase constructs were prepared by re-cloning the promoter regions of the previously described TOPflash and FOPflash plasmids [28 (link)] into the pNL1.1 luciferase plasmid (Promega). The AXIN2 promoter luciferase constructs were prepared by PCR using the primers listed in S1 Table. The PCR products were cloned into the luciferase pNL1.1 vector (Promega) and verified by sequencing. The constructs were verified by sequencing. The expression of the wild type and mutant KDM2A/B proteins was verified by western blot (S1 Fig). The plasmids were transfected into cells using Fugene6 (Promega) or Turbofect (ThermoFisher Scientific).