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. 2021 Oct 1;12:5772. doi: 10.1038/s41467-021-26061-w

Fig. 4. RNF213 counteracts HSV-1 infection.

Fig. 4

a HeLa cells were infected up to 72 h with eGFP-expressing recombinant herpes simplex virus 1 (HSV-1) at MOI 0.1. Forty eight hours prior to infection, cells were transfected with a pool of siRNAs targeting RNF213 (siRNF213) or a pool of scrambled siRNAs (siScramble) as control. The viral load was determined by monitoring the GFP signal in each condition every 24 h to generate a viral growth curve (right panel, representative viral growth curve from a single experiment, n = 4 technical replicates, curve connecting AVG, two-tailed unpaired t-test comparing siRNF213 to siScramble, a.u. arbitrary units). The area under the curve (AUC) was calculated for each growth curve and the average AUC of three independent experiments is shown relative to the siScramble control (left panel, AVG ± SEM, n = 3 independent experiments, two-tailed unpaired t-test). b HSV-1 infection experiment performed as in a, except that 16 h prior to infection cells were treated with interferon (IFN)-α (right panel, representative viral growth curve from a single experiment, n = 4 technical replicates, curve connecting AVG, two-tailed unpaired t-test comparing siRNF213 to siScramble; left panel, AVG ± SEM, n = 3 independent experiments, two-tailed unpaired t-test). Knockdown of RNF213 leads to significantly higher HSV-1 infection levels. c Immunoblots against RNF213, HSV-VP5, and MXA with tubulin as loading control confirmed knockdown of RNF213, HSV-1 infection and interferon-α treatment, respectively, in the experiments shown in a, b. d HSV-1 infection experiment performed as in a, except that 24 h prior to infection at MOI 0.05 cells were transfected with plasmids encoding 3xFLAG-RNF213 or MXB or with an empty vector (mock) as control (right panel, representative viral growth curve from a single experiment, AVG ± SEM, n = 4 technical replicates, curve connecting AVG, two-tailed unpaired t-test comparing RNF213 or MXB overexpression to mock). The average AUC of two independent experiments is shown relative to the mock control (left panel, AVG ± SEM, n = 2 independent experiments, two-tailed unpaired t-test). Overexpression of RNF213 leads to significantly lower HSV-1 infection levels. e Immunoblots against FLAG, HSV-VP5, and MXB with tubulin as loading control confirmed HSV-1 infection and expression of FLAG-RNF213 and MXB in the experiments shown in d. In a, b, e asterisks indicate p values with *p < 0.05, **p < 0.01, and ****p < 0.0001. Source data are provided as a Source Data file.