insect cell line expression plasmids ppac-sp1  (Addgene inc)

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) Schematic of the establishment of the TIME.219 KSHV-infected immortalized endothelial cell line by infection of TIME cells with recombinant KSHV.219 virus. Blue and red arrows indicate the period of infection without and with puromycin selection respectively. Sample collection days are shown. Representative images of de novo infection at day 6 and stable selection (TIME.219) are shown. B) qPCR analysis of representative KSHV latent and lytic genes in the latently infected TIME.219 cell line and the BCBL-1 (PEL) cell line. N=3 biological replicates at passages 30, 35 and 40 for TIME.219 cells and passage 8, 12 and 15 for BCBL-1 cells. qPCR cycles: 40. Internal reference control GAPDH. Expression was normalized to corresponding LANA expression of TIME.219 and BCBL-1, respectively. Error bar indicate ± standard deviations of 3 experiments. Black arrows highlight the ORF75 gene. C) qPCR analysis of representative latent and lytic KSHV genes at 1, 3, and 6 days post infection (DPI) after de novo infection.

Article Snippet: The insect cell line expression plasmids were a kind gifts from Dr. Robert Tijan through Addgene (pPAC-Sp1 (12095), pPAC0 (12094)).

Techniques: Infection, Recombinant, Virus, Selection, Control, Expressing

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) qPCR analysis of representative genes of latent and lytic cycle in latently infected iSLK-BAC16 and PEL cell line, BC3. N=3 biological replicate with three qPCR technical replicate. Passage 6, 9 and 14 for ISLK-BAC16 cell line. Expression normalized to uninfected iSLK cells and uninfected BJAB cells for infected iSLK-BAC16 cells and BC3 cells ,respectively with GAPDH as internal reference. Expression of all genes were fold normalized to respective LANA expression for each cell type. Shown are the means ± standard deviations of at least 3 separate experiments.

Article Snippet: The insect cell line expression plasmids were a kind gifts from Dr. Robert Tijan through Addgene (pPAC-Sp1 (12095), pPAC0 (12094)).

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) Promoter luciferase assay of ORF75 and ORF74 (1.2 kb) promoter in HEK293T and HepG2. Data normalised to respective pGL3 vector in each cell line. Histogram represents mean with SD as error bars for three biological replicate. Assayed at 72h post transfection. B) Promoter luciferase assay of ORF75 promoter in HepG2, SLK and COS-7 cells. Assayed at 72h post transfection. C) Promoter luciferase assay of the ORF75 promoter in HepG2 cells, coupled with transient overexpression of NRF2, HIF1α, and HIF-2α degradation-resistant mutants. X and 2X indicate 1:2 and 1:4 ratios of ORF75 promoter to protein expression plasmid, respectively. D, E, F) Promoter luciferase assay of ORF75 promoter in HepG2 cells with various treatments. All treatments were done 24h post transfection. Assayed at 48h. Histogram for all except A, one experiment with two technical replicates. Error bar indicate ±SD.

Article Snippet: The insect cell line expression plasmids were a kind gifts from Dr. Robert Tijan through Addgene (pPAC-Sp1 (12095), pPAC0 (12094)).

Techniques: Luciferase, Plasmid Preparation, Transfection, Over Expression, Expressing

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) Promoter luciferase assay of ORF75 full length promoter in TIVE (endothelial cell line) and BJAB (B-cell line) cells. Results shown are the fold change over the respective pGL3 empty vector for each cell type. Data shown are ± standard deviations of 3 separate experiments. P -values (** p ≤ 0.01) are calculated using two-sided paired t -test B) Western blot analysis of Sp1, Sp3 and Sp4 protein levels from different cell lines using whole cell lysates. Black and red arrow indicates full-length and alternate SP1 forms in W.B, respectively. C) EMSA and WB analysis. Nuclear lysates of various KSHV-infected and uninfected cell lines were analysed for their Sp1 binding activity to the proximal Sp1 element of ORF75 promoter through EMSA. The lower three panels represent parallel WB of the nuclear lysates. D) Promoter luciferase assay of various mutant ORF75 promoters in Schneider Drosophila line 2 (SL2) with exogenous human Sp1 expression. Presence of various elements are shown in the promoter schematic as different shapes. Absence of a shape in the promoter schematic indicates corresponding element mutation. Data shown are presented as mean ± SD of individually nucleofected samples of one experiment. Assayed at 72h post nucleofection. P -values (**** p ≤ 0.0001, ns not significant) are calculated using ordinary one way ANOVA. E) Same as A), except four different ORF75 promoters were used for the promoter luciferase assay in BJAB and TIVE cells. Assayed at 72h post transfection. Numbers on the top of each bar indicates average fold upregulation relative to empty vector pGL3 for each cell type set as 1. Shown are the means ± standard deviations of 3 separate experiments. P -values (** p ≤ 0.01, **** p ≤ 0.0001, ns not significant) are calculated using two-sided unpaired t -test. Blots stripped and reprobed, see for more information.

Article Snippet: The insect cell line expression plasmids were a kind gifts from Dr. Robert Tijan through Addgene (pPAC-Sp1 (12095), pPAC0 (12094)).

Techniques: Luciferase, Plasmid Preparation, Western Blot, Infection, Binding Assay, Activity Assay, Mutagenesis, Expressing, Transfection

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) (Left) Promoter luciferase assay of the ORF75-T2 promoter along with co-expression of increasing amount of a plasmid expressing flag-tagged ORF75 (F-ORF75) protein. (Right) Parallel WB analysis of whole cell lysates from the promoter luciferase assay showing F-ORF75 expression. + and ++ indicate 1:2 and 1:4 ratios of ORF75 promoter to protein expression plasmid F-ORF75, respectively. B) Same as in A), except RTA was co-expressed alone or together with F-ORF75 along with the ORF75 promoter luciferase construct. C) Same as in A), except F-ORF75 was co-expressed along with 3 different length LANA promoter luciferase constructs (LANA I: 570 bp, LANA CI: 1.2 kb, LANA CP: 795 bp. D and E) F-ORF75 protein was co-expressed with two different lengths of RTA (ORF50) promoter luciferase fusion constructs. F) Same as D), except with an ORF57 promoter luciferase fusion construct. In B-F, 1:4 ratio of ORF75 promoter to protein expression plasmid (F-ORF75 or pcDNA3.1) was used. G) Schematic for the assay of KSHV gene expression in 293T-BAC16 cells transfected with F-ORF75 , RTA, or a pCDNA3.1 control. Flag-ORF75 and RTA over-expression vectors or a pcDNA3.1 control vector were transfected into 293T cells latently infected with KSHV followed by qPCR analysis of endogenous KSHV genes (ORF75, LANA, ORF57 and vIL6) 48 hr post transfection. H) RT-qPCR of select genes in either empty or ORF75 and RTA expression vector transfected cells. Fold change normalized to non-transfected 293T-BAC16 cells. Endogenous ORF75 was detected using a primer pair spanning the 5’UTR and CDS. I) Same as H) except that RTA gene expression was analyzed in F-ORF75 transfected 293T-BAC16 cells. Other information: Numbers on the top of each bar in A through F indicates average fold upregulation relative to control set as 1. Error bar indicate ± standard deviations of 3 experiments. pcDNA3.1 plasmid was used as vector control. Promoter luciferase assay in 293T cells was performed at 72h. More information on the different promoters used here is provided in .

Article Snippet: The insect cell line expression plasmids were a kind gifts from Dr. Robert Tijan through Addgene (pPAC-Sp1 (12095), pPAC0 (12094)).

Techniques: Luciferase, Expressing, Plasmid Preparation, Construct, Gene Expression, Transfection, Control, Over Expression, Infection, Quantitative RT-PCR