apn promoter sequences  (Addgene inc)

Journal: Nature Communications

Article Title: UHRF1 restricts HCoV-229E infection through epigenetic silencing of the viral receptor APN

doi: 10.1038/s41467-025-64977-9

Figure Lengend Snippet: A UHRF1 does not regulate the expression of exogenous HA-tagged APN and ACE2 from transiently transfected plasmids. Representative Western blotting images from three independent experiments were shown. B Bisulfite sequencing of CpG sites in APN proximal promoter from control and UHRF1 -knockout A549 cells. The methylation (Meth) rate was calculated as the ratio of methylated sites to the total number of sites tested. C A549 cells were treated with 5-AZA for 3 days, and relative APN mRNA levels were determined by qRT-PCR. D , E Huh7 stably expressing DNMT3A were generated by lentivirus transduction. Relative APN mRNA levels were determined by qRT-PCR ( D ), and infectivity was detected by flow cytometry after infection with HCoV-229E (MOI 0.01, 24 h) ( E ). F , G In vitro methylation and dual-luciferase reporter assays. The methylation status of luciferase reporter plasmid was verified by HpaII/MspI digestion ( F ). Representative image from three independent experiments was shown ( F ). The luciferase activity of unmethylated (Unmeth) or methylated (Meth) luciferase reporter plasmid co-transfected with internal control pRL-TK was measured at 24 h post-transfection. Results were normalized to the unmethylated plasmid ( G ). H Electrophoretic mobility shift assay (EMSA). Nuclear extracts were incubated with biotin-labeled unmethylated or methylated APN promoter probe to detect DNA-protein complexes. Representative images from three independent experiments were shown. I , J Chromatin immunoprecipitation (ChIP) assay with c-Maf expression. qPCR was performed to detect c-Maf binding to the transcription factor (TF) binding site ( I ) or CpG island ( J ) of the APN proximal promoter. K Schematic diagram of UHRF1 truncations. L , M UHRF1 -knockout A549 stably expressing wild-type or truncated UHRF1 were established by lentivirus transduction and verified by western blotting ( L ). Representative images from three independent experiments were shown ( L ). Cells were infected with HCoV-229E (MOI 0.5, 24 h) at day 10 post-transduction, and the infectivity was determined by flow cytometry ( M ). Error bars represent standard deviations from three independent experiments ( n = 3), and each performed in duplicate. One-way ANOVA with Sidak’s test ( C , M ); unpaired, two-sided t-test ( D , E , G , I – J ); mean ± s.d.; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The APN promoter sequences (−153 to −1) were cloned into the pGL3 Basic vector (Addgene ##212936), resulting in the plasmid pGL3-APN-Luc.

Techniques: Expressing, Transfection, Western Blot, Methylation Sequencing, Control, Knock-Out, Methylation, Quantitative RT-PCR, Stable Transfection, Generated, Transduction, Infection, Flow Cytometry, In Vitro, Luciferase, Plasmid Preparation, Activity Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Chromatin Immunoprecipitation, Binding Assay