foxm1 coding sequence  (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

Journal: Reproduction (Cambridge, England)

Article Title: mTORC1-dependent suppression of autophagic activity in somatic cell nuclear transfer mouse embryos

doi: 10.1530/REP-25-0338

Figure Lengend Snippet: Preimplantation autophagic activity compared in IVF and SCNT embryos. (A) Representative fluorescent images of IVF and SCNT embryos at various times after fertilization or activation. From left to right, 24, 48, 72, and 96 h later were shown, IVF embryos on the upper row, and SCNT embryos on the lower row. GFP-LC3 (green), RFP-LC3ΔG (red). (B) Preimplantation autophagic activity in IVF and SCNT embryos. The x -axis shows the elapsed time, and the y -axis shows the relative RFP/GFP values for each embryo based on the 24-h time point. IVF (blue), SCNT (orange), lighter-colored areas indicate SEM. Two-tailed t -tests were performed at 36, 48, 60, and 72 h. IVF n = 35, SCNT n = 6. (C) Autophagic activity in IVF and SCNT embryos that were treated to enhance gene expression from 24 to 72 h after insemination or activation. The x -axis shows the elapsed time, and the y -axis shows the relative RFP/GFP values for each embryo based on the 24-h time point. IVF (blue), Kdm4d-overexpressed (red), TSA-treated (green), lighter-colored areas indicate SEM. IVF: n = 12, Kdm4d: n = 38, TSA: n = 18. (D) Autophagic activity up to 48 h after activation in SCNT embryos treated to enhance gene expression. Kdm4d-overexpressed (red), TSA-treated (green), lighter-colored areas indicate SEM. The broken lines indicate embryos that were subjected to the respective treatments, but embryonic development was arrested after the 4-cell stage. Two-tailed t -tests were performed at 48 h for each treatment group. Kdm4d arrest n = 22, TSA arrest n = 35.

Article Snippet: Briefly, the pcDNA3.1 plasmid carrying the full-length mouse Kdm4d sequence (61553, Addgene, USA) was linearized by XbaI, and it was used as a template for in vitro transcription using mMESSAGE mMACHINE T7 ULTRA Transcription kits (AM1345, Thermo Fisher Scientific).

Techniques: Activity Assay, Activation Assay, Two Tailed Test, Gene Expression