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. 2018 Oct 29;92(22):e00825-18. doi: 10.1128/JVI.00825-18

FIG 4.

FIG 4

Knockout of PJA1 facilitates transcription of ectopic DNA. (A and B) To measure the effect of endogenous PJA1 on the regulation of episomal gene activation and DNA virus replication, the Cas9-mediated PJA1 knockout cell line 293T(PJA1-KO) and the control cell line with the pSpCas9-2A-Puro-MCS vector integrated were generated. Shown are bright-field images (A) and PJA1 protein levels (B) for 293T control cells and 293T(PJA1-KO) cells. (C) 293T control cells and 293T(PJA1-KO) cells were plated in a 96-well plate at a density of 1,000 cells/well in 100 μl of culture medium in triplicates. Ten microliters of a CCK-8 solution was added to each well of the plate, and the plate was incubated for 2 h in an incubator. The absorbance was measured at 450 nm. (D and E) 293T control cells and 293T(PJA1-KO) cells were plated in 24-well plates in triplicate and then transfected with 0.2 μg HBV Enh1-Luc (D) and 0.2 μg Tp53-Luc (E) for 24 h, and luciferase activity and relative luciferase mRNA levels were measured. (F) Tp53 protein levels in 293T control cells or 293T(PJA1-KO) cells were detected by Western blot analysis. (G) 293T control cells and 293T(PJA1-KO) cells were plated in 12-well plates and transfected with 0.2 μg pHBV-Enh1-Luc with or without 2 ng pCAGGS-HA-PJA1B for 24 h. Luciferase activities of HBV Enh1-Luc and protein levels of PJA1 and GAPDH were measured. (H) 293T control cells and 293T(PJA1-KO) cells were plated in 6-cm dishes and transfected with 5 μg pHBV-Enh1-Luc for 24 h. NSE4 binding to the episomal reporter or the GAPDH gene was monitored by ChIP assays using anti-NSE4 antibody. The amount of immunoprecipitated DNA was determined by RT-qPCR. (I and J) 293T control cells and 293T(PJA1-KO) cells were plated in 12-well plates overnight and then infected with HSV-1 at an MOI of 0.1 for the indicated times. HSV-1 US11 gene mRNA (I) and ICP27 gene mRNA (J) levels were determined by RT-qPCR. h.p.i, hours postinfection. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. ***, P < 0.001.