Figure 7. VPS35 depletion results in prolonged activation of JAK/STAT signalling by type-I IFNs.

(a) Immunoblots for tyrosine phosphorylation levels of STAT1 (pSTAT1) in CTRL and VPS35-depleted RPE1 cells stimulated with IFN-α or IFN-β for the indicated times. Representative immunoblot out of four independent experiments. (b) Quantification of experiments performed in a: pSTAT1 and STAT1 levels were normalized to tubulin (tub) level (loading control) and the ratio (pSTAT1/tub)/(STAT1/tub) was calculated for each condition. STAT1 activation on VPS35 depletion was normalized to CTRL as 1. (c) PCA using ΔCT value in CTRL and VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation for all replicates (biological duplicates with technical duplicates each). (d) Clustering analysis based on −ΔCt values for gene expression in CTRL and VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation for all samples replicates (biological duplicates with technical duplicates each). Each square represents a value for a given gene (row) for a specific condition (column). Genes depicted in blue are expressed at low level (low −ΔCT value); genes depicted in red are expressed at high level (high −ΔCT value). (e) Genes significantly and selectively upregulated by IFN-α or IFN-β stimulation in VPS35-depleted RPE1 cells. (f) Expression of IFN-independent genes: p21 (upper panel), and CHMP2A (lower panel) in CTRL and in VPS35-depleted RPE1 cells with or without IFN-α or IFN-β stimulation. Reproducibility of experiments: panel a shows representative data for four independent experiments, b shows quantification of data for four, four and three independent experiments for each time point, respectively. Statistical analysis with two-tailed, unpaired t-test. *P<0.05 and ****P<0.0001; NS, nonsignificant. (c–e) Representative data for two independent experiments. (f) Quantification of data for three independent experiments. Graphs b and f show mean value±s.e.m.