Journal: PLoS Pathogens

Article Title: ZBTB2 represses HIV-1 transcription and is regulated by HIV-1 Vpr and cellular DNA damage responses

doi: 10.1371/journal.ppat.1009364

Figure Lengend Snippet: (A) Jurkat cells were transfected with a HIV-1 promoter-driven reporter plasmid upstream of gaussian luciferase and the herpes simplex virus tk promoter upstream of cypridinia luciferase and the protein encoding plasmids indicated at the bottom. The effects on the basal and TAT activated expression of the WT HIV-1 LTR promoter and the control tk promoter in the presence or absence of ZBTB2 or a ZBTB2 variant with a deletion in the POZ domain (a.a. 1–79, ΔPOZ) are shown). The data shown are the average mean chemiluminescent reporter values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. (B) SUPT1 cells transfected with siRNAs targeting ZBTB2 or non-targeting siRNA. Two days post transfection, cells were challenged with VSV-G pseudotyped HIV-1 vectors with non-sense mutations in envelope, and Vpr with firefly luciferase in the NEF locus. Infections were done with the Vpr deletion virus or virions transcomplemented with a Vpr expression plasmid in the producer cells. A sample of cells were harvested for total protein (see C) at this time. 48 hours post infection HIV-1 transcription was assayed for by measuring firefly luciferase and cell viability was monitored by the ATP assay CellTiter-glo (Promega). The data shown are the average mean values obtained in an experiment performed with quadruplicate samples and are representative of three independent experiments. (C and D) Western blots demonstrating loss of ZASC1 or ZBTB2 protein accumulation in (C) SUPT1 cells transfected with siRNAs targeting ZBTB2 or non-targeting (NT) siRNA or (D) ZASC1 and ZBTB2 knockout cells generated by CRISPR/CAS9 in SUPT1 and Jurkat T cell lines. (E) Co-immunoprecipitation assays of endogenous proteins. WT Jurkat, ZASC1 or ZBTB2 knockout cells (1X10 7 ) were lysed and incubated with ZASC1, ZBTB2 or non-specific (NS) rabbit IgG. Co-immunoprecipitating complexes were detected by western blotting. (F) Cells (5X10 4 /well) were seeded in quadruplicate a 96 well plate, incubated for 48 hours and assayed for cell viability using the ATP assay CellTiter-glo (Promega). (G) Cells were passaged for the indicated time, counted, plotted and fitted to a growth curve using and the doubling time and 95% confidence interval was calculated using Prism graphing software. Error bars indicate the standard deviation of the data in all panels. ANOVA analysis was performed and for P-values < 0.05 a Tukey’s HSD was performed and relevant P-values reported.

Article Snippet: A cellular viability assay that measures ATP levels (Cell titer glo, Promega, Madison WI) showed no differences between siZBTB2 and non-targeting siRNA control ( light grey bars).

Techniques: Transfection, Plasmid Preparation, Luciferase, Virus, Expressing, Control, Variant Assay, Infection, ATP Assay, Western Blot, Knock-Out, Generated, CRISPR, Immunoprecipitation, Incubation, Software, Standard Deviation