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. 2015 Aug 24;10(8):e0136708. doi: 10.1371/journal.pone.0136708

Fig 5. Inflammatory component produced by Vero cells during DENV propagation is responsible for IL-1β induction.

Fig 5

(A) Schematic depicting the different depletion and control fractions for depleting antibody-bound virions from DIV crude supernatant by using magnetic protein G beads. (B) Immunoblot for human IgG Fc, and two DENV structural proteins, E protein and prM, to assess efficiency of antibody-mediated DENV depletion from DIV crude supernatant. “*” denotes all 3 antibodies were used in combination at 1 μg/ml each. 3 μg/ml of each antibody was used for individual antibody conditions. (C) Control supernatants (Lane C) and residual supernatants (Lane R) from panel B were inoculated onto mobilized monocytes. Monocyte supernatants were collected at 24 hpi, and secreted IL-1β was measured by ELISA. (D) Mock and DIV crude supernatants were incubated with PBS or anti-DENV NS1 antibody prior to addition of protein G beads and subsequent depletion. Top: Residual supernatants (R) and bead-bound fractions (B) were assessed for efficiency of NS1 depletion by immunoblot. Bottom: Residual supernatants were inoculated onto mobilized monocytes. At 4 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (E) Mock and DIV crude supernatants were incubated with PBS, DNase, or Riboshredder prior to inoculation onto mobilized monocytes. At 4 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (F) Mobilized monocytes were inoculated with mock supernatant, live DIV crude supernatant, or DIV crude supernatant that had been incubated for 30 minutes at 56°C to heat inactivate (HI) it. At 24 hpi, monocyte supernatants were collected and assessed for secreted IL-1β. (G) DIV crude supernatant was centrifuged in an Amicon centrifugal filtration unit with a 100-kDa molecular-weight cutoff to generate fractions smaller and larger than 100 kDa. Fractions were brought to equal volumes and inoculated onto mobilized monocytes. At 24 hpi, monocyte supernatants were collected, and IL-1β secretion was measured. Tests used: Two-Way ANOVA with Bonferroni’s post-test (D), One-Way ANOVA with Dunnett’s post-test (F).