Fig. 5: Knockdown of OTULIN enhances KSHV lytic reactivation in BCBL-1 cells. | Nature Communications

Fig. 5: Knockdown of OTULIN enhances KSHV lytic reactivation in BCBL-1 cells.

From: Linear ubiquitination regulates the KSHV replication and transcription activator protein to control infection

Fig. 5: Knockdown of OTULIN enhances KSHV lytic reactivation in BCBL-1 cells.

a BCBL-1 cells were transfected with control siRNA (siCntrl) or three different siRNAs against OTULIN (siOTULIN 1, 2 and 3). Knockdown efficiencies of OTULIN in BCBL-1 cells were measured by RT-qPCR. n = 3 biological replicates and 2 technical replicates, mean ± s.d., two-sided t test. b BCBL-1 cells from (a) were treated with TPA plus NaB for 24 h. The mRNA levels of RTA, ORF59, ORF9, and ORF8.1 were determined using RT-qPCR. n = 2 biological replicates and 2 technical replicates, mean ± s.d., two-sided t test, * P = 0.02880, 0.00475, 0.00568, 0.015 (siCntrl vs. siCntrl+TPA+NaB) for RTA, ORF59, ORF9, and ORF8.1. #, P = (0.01953, 0.00380, 0.03093), (0.01167, 0.00975, 0.01240), (0.00778, 0.00557, 0.00764), (0.00265, 0.00877) for RTA, ORF59, ORF9, and ORF8.1. (c) As in (b), but the DNA in the culture media was extracted and qPCR was used to measure KSHV virions, n = 2 biological replicates and 2 technical replicates, mean ± s.d., two-sided t test. d, e OTULIN knockdown enhances RTA binding to the ORF57 and ORF59promoters. BCBL-1 cells were transfected with siCntrl or siOTULIN followed by treatment with TPA plus NaB. The cells were subjected to ChIP assay. The quantities of ORF57 (d) or ORF59 (e) promoter DNA in the precipitates were determined by qPCR. d, n = 2 biological replicates and 2 technical replicates, mean ± s.d., two-sided t test. e, n = 2 biological replicates and 2 technical replicates, mean ± s.d., two-sided t test. f–h OTULIN knockdown alters the intracellular localization of RTA. BCBL-1 cells were transfected with siCntrl or siOTULIN followed by treatment with TPA plus NaB. Cells were detected by immunoblotting (f) and immunofluorescence (g, h). n = 3 independent experiments. Subcellular localizations of RTA (in green) and of OTULIN (in red) are shown in (g) and (h). All cell nuclei in the fields were stained with Hoechst (blue). Scale bar: 10 μm. Image is representative in total n = 3 independent experiments. Source data are provided as a Source Data file.

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