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. 2020 Feb 4;11:42. doi: 10.3389/fmicb.2020.00042

FIGURE 4.

FIGURE 4

Necrostatin-1 or necrostatin-2 inhibits viral production. (A) The chemical structure of necrostatin-1. (B) VP1 expression in cellular lysates was determined after growth in control medium or different doses of necrostatin-1 medium at 36 h post-infection. The results are representative of three independent experiments. Histone is shown as loading control. (C) At 36 h post-infection, intracellular CA6 RNA levels were detected in control, necrostatin-1 (150 μM)-treated RD cells by real-time quantitative PCR. The results were standardized to GAPDH mRNA as a control and normalized to 1.0 in Con-treated cells. ***P < 0.001. (D) Total progeny viruses collected at 36 h post-infection of CA6 (MOI = 5) were titrated using RD cells. The results indicate the means ± SD of three independent experiments. *P < 0.05. (E) The chemical structure of necrostatin-2. (F) VP1 expression in cellular lysates was determined after growth in control medium or different doses of necrostatin-2 medium at 36 h post-infection. The results are representative of three independent experiments. Histone is shown as loading control. (G) At 36 h post-infection, intracellular CA6 RNA levels were detected in control, necrostatin-2 (180 μM)-treated RD cells by real-time quantitative PCR. The results were standardized to GAPDH mRNA as a control and normalized to 1.0 in Con-treated cells. ***P < 0.001. (H) Total progeny viruses collected at 36 h post-infection of CA6 (MOI = 5) were titrated using RD cells. The results indicate the means ± SD of three independent experiments. **P < 0.01. (I) At 36 h post-infection, extracellular, intracellular, and total CA6 RNA levels were detected in control, necrostatin-1 (150 μM)-treated RD cells by real-time quantitative PCR. The results of extracellular and intracellular were standardized to corresponding cell number and normalized to 1.0 in Nec-1-treated cells. ***P < 0.001. (J) At 36 h post-infection, extracellular, intracellular, and total virions were detected in control, necrostatin-1 (150 μM)-treated RD cells by TCID50. The results of extracellular and intracellular were standardized to corresponding cell number and normalized to 1.0 in Nec-1-treated cells. ***P < 0.001, **P < 0.01. NC, no significant difference; Con, control treatment; Nec-1, necrostatin-1; Nec-2, necrostatin-2.