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. 2014 Nov 26;89(3):1925–1931. doi: 10.1128/JVI.02262-14

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FIG 4 — Knocking down SAMD9 rescues MYXV-M062R-null (vMyxM062RKO) and C7LK1L double-knockout VACV (VACV-C7LK1L-DKO) by disengaging the antiviral granule formation. (A) SAMD9 is responsible for the antiviral granule formation stimulated by either vMyxM062RKO (a) or VACV-C7LK1L-DKO (b) but not for the antiviral granule formation stimulated by E3L-knockout VACV (VACV-E3LKO-_LtdTr) (c). Antiviral granule formation was investigated at 19 h p.i. for vMyxM062RKO and at 5 h p.i. for VACV infection of normal HeLa cells or SAMD9 knockdown (SAMD9KD) at a MOI of 10 and a MOI of 5, respectively. After IF staining of SAMD9, G3BP1, and nuclei, images were captured with a Nikon c2 confocal microscope. Scale bar, 50 μm. (B) Knocking down SAMD9 expression rescues M062R-null MYXV replication. HeLa cells stably expressing shRNAs targeting SAMD9 (SAMD9KD) showed significantly reduced SAMD9 protein levels compared with control HeLa cells (con). A total protein of 40 μg was separated on SDS-PAGE for Western blotting against SAMD9 and β-actin. Multiple-step growth curve analysis of MYXV-M062R-null was conducted on control HeLa (con) or SAMD9 knockdown (SAMD9KD) HeLa cells. Duplicate of infection at each time point per virus was conducted. Titration was performed on BSC40 cells in triplicate at each dilution. The growth curve shown is one view representative of the results of two independent experiments. ffu, focus-forming units. (C) Knocking down SAMD9 expression rescues viral replication of VACV-C7LK1L-DKO. A549 cells stably expressing control shRNA (A549-con) or shRNAs targeting SAMD9 (A549-SAMD9KD) were engineered by infecting cells with lentivirus containing corresponding shRNAs. Western blot analysis was conducted with a total protein of 40 μg from either A549-con or A549-SAMD9KD by probing against SAMD9 and β-actin. The experiment designed to construct the multiple-step growth curve of VACV-C7LK1L-DKO was performed by infecting control or knockdown cells at a low MOI of 0.1 followed by harvesting at different time points. At each time point, samples from triplicate infections were collected for titration on BSC40 cells. The growth curve shown is a view representative of the results of two independent experiments.

FIG 4 — Knocking down SAMD9 rescues MYXV-M062R-null (vMyxM062RKO) and C7LK1L double-knockout VACV (VACV-C7LK1L-DKO) by disengaging the antiviral granule formation. (A) SAMD9 is responsible for the antiviral granule formation stimulated by either vMyxM062RKO (a) or VACV-C7LK1L-DKO (b) but not for the antiviral granule formation stimulated by E3L-knockout VACV (VACV-E3LKO-_LtdTr) (c). Antiviral granule formation was investigated at 19 h p.i. for vMyxM062RKO and at 5 h p.i. for VACV infection of normal HeLa cells or SAMD9 knockdown (SAMD9KD) at a MOI of 10 and a MOI of 5, respectively. After IF staining of SAMD9, G3BP1, and nuclei, images were captured with a Nikon c2 confocal microscope. Scale bar, 50 μm. (B) Knocking down SAMD9 expression rescues M062R-null MYXV replication. HeLa cells stably expressing shRNAs targeting SAMD9 (SAMD9KD) showed significantly reduced SAMD9 protein levels compared with control HeLa cells (con). A total protein of 40 μg was separated on SDS-PAGE for Western blotting against SAMD9 and β-actin. Multiple-step growth curve analysis of MYXV-M062R-null was conducted on control HeLa (con) or SAMD9 knockdown (SAMD9KD) HeLa cells. Duplicate of infection at each time point per virus was conducted. Titration was performed on BSC40 cells in triplicate at each dilution. The growth curve shown is one view representative of the results of two independent experiments. ffu, focus-forming units. (C) Knocking down SAMD9 expression rescues viral replication of VACV-C7LK1L-DKO. A549 cells stably expressing control shRNA (A549-con) or shRNAs targeting SAMD9 (A549-SAMD9KD) were engineered by infecting cells with lentivirus containing corresponding shRNAs. Western blot analysis was conducted with a total protein of 40 μg from either A549-con or A549-SAMD9KD by probing against SAMD9 and β-actin. The experiment designed to construct the multiple-step growth curve of VACV-C7LK1L-DKO was performed by infecting control or knockdown cells at a low MOI of 0.1 followed by harvesting at different time points. At each time point, samples from triplicate infections were collected for titration on BSC40 cells. The growth curve shown is a view representative of the results of two independent experiments.