lookout mycoplasma qpcr detection kit  (Merck & Co)

Figure Legend Snippet: (A) Reanalysis of ChIP-Seq data (E-MTAB-7788) showing enrichment of BZLF1 on E2F1 promoter. Bottom panel indicates the MACS2 identified peaks (Sites 1–3) for BZLF1/AP-1 binding on E2F1 promoter. (B) BZLF1 homologue AP-1 binding motif identified on the MACS2 peaks of E2F1 promoter region. (C) ChIP-qPCR data showing recruitment of BZLF1 on E2F1 promoter upon EBV lytic cycle reactivation by TPA-NaBu treatment for 72 h in P3HR1 cells. (D) Immunoblot analysis of whole cell lysates (WCLs) from EBV + P3HR1 cells reactivated to lytic cycle replication as similar to (C). (E) HEK293 cells transiently transfected with empty vector or increasing concentrations of flag-tagged BZLF1 expression plasmid for 36 h were harvested and subjected to immunoblot analysis. (F) Luciferase reporter activity and the corresponding immunoblot analysis of the wild-type E2F1 promoter in the presence of increasing concentrations of BZLF1 expression plasmid in transiently transfected HEK293 cells. (G) Schema showing three wild-type BZLF1/AP-1 binding sites (Sites 1–3) and their corresponding mutations (Muts 1–3) on E2F1 promoter for cloning into pGL3 luciferase reporter vector. (H) Luciferase reporter activity of the wild-type and the mutant E2F1 promoters in the presence of either vector control or BZLF1 expression plasmid in HEK293 cells. A fraction of the total protein was evaluated by immunoblot analysis. (I) Schema showing different structural domains of BZLF1 for cloning in flag-tagged expression vector. (J) Luciferase reporter activity and the corresponding immunoblot analysis of the E2F1 promoter in the presence of empty vector, wild-type (WT) or transactivation domain deleted (ΔTAD) BZLF1 expression plasmids in HEK293 cells. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Techniques Used: ChIP-sequencing, Binding Assay, ChIP-qPCR, Western Blot, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Cloning, Mutagenesis, Control

Figure Legend Snippet: (A) ChIP-Seq analysis of E2F1 binding in two EBV transformed lymphoblastoid cell lines – LCL#1 and LCL#89. Tracks are aligned with the annotated EBV genome shown at the bottom. (B) ChIP-qPCR analysis of E2F1 occupancy at different EBV promoters and genomic regions. Anti-E2F1 ChIP was performed on chromatin extracted from LCL#1 and LCL#89, followed by qPCR using primers specific for Wp, Cp, Qp, LMP1p, LMP2p, OriLytL, OriLytR and Zp regions. Data are presented as % input. (C) E2F1 ChIP-Seq tracks and the corresponding MACS2 identified peaks on BZLF1 promoter region (Zp) in LCL#1 and LCL#89. Bottom panel indicates different Zp elements and three putative E2F1 binding motifs obtained from JASPAR database in Zp. (D) Schema showing three wild-type E2F1 binding sites (Sites 1–3) and their corresponding mutations (Muts 1–3) on Zp for cloning into pGL3 luciferase reporter vector. (E) Luciferase reporter activity and the corresponding immunoblot analysis of the wild-type (WT) and mutant (Mut) Zp in the absence and presence of flag-tagged E2F1 expression vector in HEK293 cells. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Techniques Used: ChIP-sequencing, Binding Assay, Transformation Assay, ChIP-qPCR, Cloning, Luciferase, Plasmid Preparation, Activity Assay, Western Blot, Mutagenesis, Expressing

Figure Legend Snippet: (A) Whole transcriptome analysis of P3HR1 stably expressing E2F1 sh-RNA (P3HR1-sh-E2F1) in the absence and presence of doxycycline (-/ + DOX) were either left untreated or treated with TPA/sodium butyrate (NaBu) for 72 h. Left panel indicates heatmap analysis of top 100 downregulated genes. Right panel bar diagram indicates most significantly affected pathways ( p < 0.05, FDR < 0.05) based on top 100 downregulated genes in the RNA-Seq data of E2F1 knockdown and EBV lytic cycle reactivation. (B) Heat map visualization of c-Myc transcripts in the P3HR1 RNA-Seq data. (C) Two-tailed unpaired Student’s t-test and two-sided Pearson’s correlation were employed to analyse the association between E2F1 and c-Myc transcripts in 19 EBV + BL lines from DepMap portal. Right panel indicates heatmap representation of E2F1 and c-Myc expressions in P3HR1 and Jiyoye cells. (D) qRT-PCR analysis of cDNA isolated from P3HR1-sh-E2F1 cells without or with doxycycline (-/ + DOX) treatment. (E) Immunoblot analysis of P3HR1-sh-E2F1 cells without or with doxycycline (-/ + DOX) treatment. (F) qRT-PCR analysis of cDNA isolated from Jiyoye cells transiently transfected with either control vector or flag-tagged E2F1 expression plasmid. (G) Immunoblot analysis of Jiyoye cells transiently transfected with either control vector or flag-tagged E2F1 expression plasmid. (H) EBV + LCL#89 and Raji ( GSE76191 ) ChIP-Seq tracks of E2F1 occupancy at c-Myc gene locus. (I) ChIP-qPCR analysis of E2F1 occupancy at c-Myc promoter region in LCL#89 and P3HR1 cells. (J) Schema showing two wild-type E2F1 binding sites (Red, Sites 1–2) and their corresponding mutations (Blue, Muts 1–2) on c-Myc promoter region for cloning into pGL3 luciferase reporter vector. (K) Luciferase reporter activity and the corresponding immunoblot analysis of the wild-type (WT) and mutant (Mut) c-Myc promoter in the absence and presence of flag-tagged E2F1 expression vector in HEK293 cells. (L) Immunoblot analysis of Jiyoye cells transiently transfected with either control vector or flag-tagged c-Myc expression plasmid. (M) Immunoblot analysis of P3HR1 cells expressing either control (sgCon) or two c-Myc specific sgRNAs. (N) Reanalysis of RNA-Seq data ( GSE140653 ) of E2F1 transcripts in Akata cells expressing either control (sgCon) or c-Myc specific sgRNA. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Techniques Used: Stable Transfection, Expressing, RNA Sequencing, Knockdown, Two Tailed Test, Quantitative RT-PCR, Isolation, Western Blot, Transfection, Control, Plasmid Preparation, ChIP-sequencing, ChIP-qPCR, Binding Assay, Cloning, Luciferase, Activity Assay, Mutagenesis