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. Author manuscript; available in PMC: 2022 Apr 5.
Published in final edited form as: Cell Rep. 2022 Mar 8;38(10):110411. doi: 10.1016/j.celrep.2022.110411

Figure 6. SMC6 associates with EBV genomic R-loop regions in the absence of BNRF1.

Figure 6.

(A) Confocal immunofluorescence analysis of P3HR-1 ZHT/RHT cells stained with S9.6, anti-SMC6-HA, or DAPI. S9.6 recognizes R-loops and double-stranded RNA (dsRNA). Cells were not induced for exogenous SMC6-HA (−dox) or for lytic reactivation (−4-HT/NaB). Representative of 3 independent experiments.

(B) Confocal analysis of P3HR-1 ZHT/RHT cells treated with dox (5 μg/mL) to induce exogenous SMC6 expression and with 4-HT/NaB for 9 h to induce EBV lytic reactivation, as indicated. Cells were then stained with S9.6, anti-HA-SMC6, or DAPI. Representative of 3 independent experiments.

(C) Confocal analysis of cells as in (B) that were pre-incubated with RNaseH (5.5 U/μg DNA) prior to staining with S9.6, anti-HA, or DAPI. Representative of 3 independent experiments.

(D) Confocal analysis of cells as in (B) that were stained with the anti-dsRNA antibody rJ2 or DAPI. Representative of 3 independent experiments.

(E) Confocal analysis of cells as in (B) that were pre-incubated with RNaseH (5.5 U/μg DNA) prior to staining with the anti-dsRNA antibody rJ2 or DAPI. Representative of 3 independent experiments.

(F) Confocal analysis of cells as in (B) that were stained with S9.6, anti-F-actin, or DAPI. Representative of 3 independent experiments.

(G) Confocal analysis of cells as in (B) that were stained with the anti-dsRNA antibody rJ2, anti-HA-SMC6, anti-F-actin, or DAPI, as indicated. Representative of 3 independent experiments.

(H) Confocal analysis of cells as in (B) that were pre-incubated with RNaseH (5.5 U/μg DNA) prior to staining with the anti-dsRNA antibody rJ2, anti-HA-SMC6, anti-F-actin, or DAPI. Representative of 3 independent experiments.

(I) Immunoblot analysis of 1% input and S9.6-immunopurified complexes from P3HR-1 ZHT/RHT cells induced for exogenous HA-SMC6 and treated with 4-HT/NaB and phosphonoacetic acid (PAA; 200 μg/mL) for 9 h, as indicated. Samples were treated with RNaseH (5.5 U/μg DNA) or benzonase (5 U/μg DNA) prior to immunoprecipitation (IP), as indicated.

(J) Mean ± SEM values from 3 replicates of ChIP-qPCR analysis of SMC6 occupancy at the EBV genomic oriLytR region. Anti-HA ChIP was performed on chromatin from EBV+ Akata cells with dox induced (5 μg/mL) SMC6-HA or GFP-HA expression following 48 h of αIgG crosslinking, followed by qPCR using oriLytR region-specific primers. *p < 0.05.