Figure 4: Microtubule destabilization leads to a decrease in type I IFN production.

(a) 786-0 cells were pretreated with 100 nM colchicine or vehicle and infected with VSVΔ51 at MOI 0.01 or mock. Sixteen hours later, qPCR was performed with cDNA from nuclear and cytoplasmic fractions. Data represent the fold change in IFN-β, normalized to GAPDH, relative to uninfected controls. (b) 786-0 were treated as above and infected at MOI 3 or mock. Eight hours later, fractionated lysates were probed with the indicated antibodies. (c) 786-0 cells were pretreated with colchicine and challenged with 0 or 500 U ml⁻¹ IFNβ. One hour later, fractionated cell lysates were probed with the indicated antibodies. 786-0 cells were treated as in a and 24 h later (d) supernatants were collected and IFN-β quantified by ELISA, (e,f) 786-0 pretreated as in a and cell lysates probed for the indicated antibodies. (g,h) 786-0 cells were treated as in a and at 24 h post infection, whole-cell lysates were collected and run on a 10–50% sedimentation gradient and absorbance measured over each fraction. (h) cDNA was generated from fractions and the abundance of mRNAs encoding IFNα, IFNβ and VSV M in each fraction assessed with PCR. For a,d, error bars represent s.e. from at least three replicate experiments and data analysed by analysis of variance (ANOVA) for statistical significance (P<0.05). For b,c,e,f, representative blots from three experiments are shown and apparent molecular sizes (kDa) of the prestained protein ladder (26616, Thermo Scientific) according to electrophoresis conditions are marked on the left.