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. 2021 Jul 20;12:4427. doi: 10.1038/s41467-021-24724-2

Fig. 2. MARCH8 inhibits IAV replication in vitro.

Fig. 2

a A549—Vector and A549—MARCH8 cells were constructed and MARCH8 expression determined by western blotting. b, c A549—Vector and A549—MARCH8 cells were infected with WSN virus for indicated times. Viral titers of supernatants were quantified by plaque assay. d A549 cells were transfected with siRNA targeting MARCH8. MARCH8 expression was examined by western blotting. e, f A549 cells transfected with siNC or siMARCH8 were infected with WSN virus. Viral titers of supernatants were quantified by plaque assay. g The endogenous MARCH8 in A549 cells was knocked out through the use of lentiviral CRISPR-Cas9. MARCH8 expression was determined by western blotting. h, i Control (Ctrl) or MARCH8-knockout (KO MARCH8) A549 cells were infected with WSN virus. Supernatants were harvested and viral titers were detected at indicated times. b, e, h MOI = 1. c, f, i MOI = 0.002. j, k Ctrl and KO MARCH8 A549 cells were infected with WSN virus. At 24 h post infection, viral NP protein expression was measured by flow cytometry (j) and confocal microscopy (k). Scale bar, 200 μm. Data represent averages of independent biological replicates and are presented as means ± SD (b, e, and h, n = 4, c, f, i, and j, n = 3). **P < 0.001, n.s., not significant, unpaired two-tailed Student t-test, without any adjustments for multiple comparisons.