Pgmlv sc5rnai lentiviral vector

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 ].

PAX6 was silenced in A549 cells with siRNA (RiboBio Co., Ltd., Guangzhou, China), according to the manufacturer’s instructions; the target sequences were as follows: si-h-PAX6_001, GCGACTCCAGAAGTTGTAA; si-h-PAX6_002, GCAGACGGCATGTATGATA; si-h-PAX6_003, GCTTCACCATGGCAAATAA. The corresponding negative control was purchased from RiboBio Co., Ltd. To stably knock down PAX6 in cells, siRNA targeting the si-h-PAX6_002 coding sequence 5′-GCAGACGGCATGTATGATA-3′ was designed and inserted into a pGMLV-SC5RNAi lentiviral vector (Genomeditech Co., Ltd, Shanghai, China); a scramble siRNA was used as a negative control.
To overexpress PAX6 in cells, an expression construct was generated by subcloning PCR-amplified, full-length human PAX6 cDNA into a pGMLV-CMV-PAX6 lentiviral vector (Genomeditech); an empty vector was used as the negative control. These procedures were performed, as described previously.^{24 (link)} The knockdown and overexpression efficiencies were evaluated by quantitative reverse transcription PCR (RT-qPCR) and western blotting.