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. 2020 May 20;117(23):13044–13055. doi: 10.1073/pnas.2000625117

Fig. 4.

Fig. 4.

Gene-specific effect of SM on EBV promoters is mediated by XPB. (A) Effect of SM on SM-dependent and -independent promoters. AGSiZ cells were transfected with either control (Csi) or SM siRNA (SMsi) and EBV lytic replication was induced 3 h post-siRNA transfection. Twenty-four hours after lytic induction, SM-dependent or SM-independent promoter reporter constructs were transfected in parallel with reporter plasmids. Cells were lysed and SM-dependent late (BcLF1 and BDLF1), SM-independent late (BGLF1) and early (BALF2) promoter activities were measured by luciferase assay. Data are normalized to luciferase activity in uninduced control siRNA transfected cells and shown as the RQ. All transfections were done in biological triplicates and luciferase activity was measured in technical triplicates. Error bars represent the SEM. (B) Efficacy of SM KD. Western blots were performed using the above protein lysates and anti-SM serum. The blot was stripped and reprobed with antitubulin as a loading control. (C) Effect of SPR on SM-dependent and -independent promoters. AGSiZ cells were treated with SPR at time of lytic induction with doxycycline. Twenty-four hours after induction, cells were transfected with SM-dependent or -independent promoter constructs as stated previously. Cells were lysed and SM-dependent late (BcLF1 and BDLF1), SM-independent late (BGLF1), and early (BALF2) promoter activities were measured as in A. (D) Effect of SPR on XPB expression. Western blots were done using the above protein lysates and anti-XPB antibody. The blot was stripped and reprobed with antitubulin. (E) Effect of XPB depletion on SM-dependent and SM-independent promoters. AGSiZ cells were transfected with either control (Csi) or XPB siRNA (XPBsi) and EBV lytic replication was induced 3 h post-siRNA transfection. Twenty-four hours after lytic induction, SM-dependent or SM-independent promoter reporter constructs were transfected in parallel. Cells were lysed and SM-dependent late (BcLF1 and BDLF1), SM-independent late (BGLF1), and early (BALF2) promoter activities were measured by luciferase assay as in A. (F) Efficacy of XPB KD. Western blots were done using above protein lysates and anti-XPB antibody. The blot was stripped and reprobed with antitubulin as a loading control. The error bars indicate the SEM from three replicates. *P = 0.0001 to 0.042; NS, P = 0.12 to 0.56.