Journal: bioRxiv

Article Title: KSHV genome harbors both constitutive and lytically induced enhancers

doi: 10.1101/2024.01.30.577957

Figure Lengend Snippet: Functional assays reveal both constitutive and lytically induced enhancers within the KSHV genome . (A) The KSHV-FAIRE-STARR library was transfected in BCBL1 cells and luciferase assay was performed. Cells were left untreated following the transfection (uninduced), or were treated with TPA and NaB for 24 h. (B) iSLK.219 cells were transfected same as in A and induced with 1μg/ml Doxycycline and 1.25mM Sodium Butyrate for 24 hours, followed by only Doxycycline (1μg/ml) induction for another 24 hours before collecting the lysates for dual luciferase reporter assays. Relative Luciferase units represent the luciferase units that were normalized with a renilla control. Black bars indicate uninduced cells and red bars are the induced counterpart. Data represent Mean ± S.D. of n=3 biological replicates in 3 experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: Purified amplicons were cloned into STARR-seq luciferase reporter vector within the XbaI and FseI sites using Gibson assembly master mix (NEB Cat. No. E5510).

Techniques: Functional Assay, Transfection, Luciferase

Journal: bioRxiv

Article Title: KSHV genome harbors both constitutive and lytically induced enhancers

doi: 10.1101/2024.01.30.577957

Figure Lengend Snippet: KSHV genomic enhancers are k-RTA responsive . (A) iSLK cells were transfected with the KSHV-FAIRE-STARR library and treated with 1μg/ml Doxycycline for 24 h before collecting the lysates for dual luciferase reporter assays. Black bars indicate uninduced cells and red bars are the induced counterpart. (B) HCT116 cells were transfected with putative enhancers with or without expression vector for RTA, and luciferase assays were performed. (C) Schematic illustrations of RTAp and PANp in promoter and enhancers position. Reporter plasmids were transfected into iSLK cells as indicated on the left and treated with 1μg/ml Doxycycline for 24 h before collecting the lysates for dual luciferase reporter assays. Data represent Mean ± S.D. of n=3 biological replicates in 3 experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: Purified amplicons were cloned into STARR-seq luciferase reporter vector within the XbaI and FseI sites using Gibson assembly master mix (NEB Cat. No. E5510).

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: KSHV genome harbors both constitutive and lytically induced enhancers

doi: 10.1101/2024.01.30.577957

Figure Lengend Snippet: KSHV Terminal Repeats are RTA responsive . (A) iSLK.219 cells were transfected with clone 21 and induced with 1μg/ml Doxycycline (Treatment A), 1μg/ml Doxycycline with 1.25mM Sodium Butyrate for 24 hours (Treatment B), or 1μg/ml Doxycycline with 1.25mM Sodium Butyrate for 24 hours followed by only 1μg/ml Doxycycline induction for another 24 hours (Treatment C). (B) iSLK cells were transfected with clone 21 and induced with 1μg/ml Doxycycline (Treatment A), same as in A. (C) Logo binding motifs as revealed by Motif search analysis in MEME suite using RTA responsive viral genomic enhancers, and LANA binding consensus sequence on human chromosome. (D) Sequence of LBS within TR, where LBS1-3 are marked as boxes below the sequence. The MEME-predicted common RTA-LANA binding motif is marked by the red rectangle. Mutated nucleotides used in reporter assay are marked in red above the TR sequence. (E) iSLK cells were transfected with the TR sequence or the mutated as presented in D cloned downstream to the luciferase. Clone 21 (TRx7) served as a positive control. Cells were treated with vehicle (DMSO) or 1μg/ml Doxycycline for 24 h before luciferase assay. (F) iSLK cells were transfected with clone 21 together with a constant amount of LANA expression vector or empty vector and then treated with increasing doses of Dox (50ng, 250ng, 500ng, and 1000ng/ml). Data represent Mean ± S.D. of n=3 biological replicates. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: Purified amplicons were cloned into STARR-seq luciferase reporter vector within the XbaI and FseI sites using Gibson assembly master mix (NEB Cat. No. E5510).

Techniques: Transfection, Binding Assay, Sequencing, Reporter Assay, Clone Assay, Luciferase, Positive Control, Expressing, Plasmid Preparation