Constructing PER1 Reporter Plasmids and ALKBH5 Lentivirus

The full-length of PER1 sequence was cloned into a PmirGLO dual luciferase expression vector (Promega, Madison, WI, USA) containing Renilla luciferase (R-luc) and firefly luciferase (F-luc) to construct a wild-type PER1 reporter plasmid. To build the PER1 mutant reporter plasmid, adenosine bases within the m6A consensus sites were replaced by cytosine, and the amplified ALKBH5 promoter region with wild-type or mutated P53-binding sites was subcloned into a pGL3 basic vector (Promega). Second, the pGMLV-PA6 vector (Genomeditech, Shanghai, China) was employed to construct the ALKBH5-expressing lentivirus (Lv-ALKBH5). shALKBH5 containing ALKBH5-targeting shRNA was constructed by pGMLV-SC5RNAi lentiviral vector (Genomeditech). The target sequence of ALKBH5 was 5′-GAAGCTTCAATGGTCTCCTTA-3′. A scrambled shRNA targeting 5′-TTCTCCGAACGTGTCACGT-3′ was used as a negative control. Stably transfected cells were selected with puromycin (Sigma-Aldrich, St Louis, MO, USA). In addition, lentiviral vectors expressing human PER1 (Lv-PER1), P53 (Lv-P53), YTHDF2-specific shRNA (shYTHDF2), empty vectors (vector), and plasmids containing scrambled shRNA (scramble) were constructed as previously described [25 (link)–27 ].