Figure 3

Identification of SMARCA2-regulated MxA cofactors. (a) A549-MxA were transfected with an siRNA targeting SMARCA2 (Dharmacon D-017253-01) or a non-targeting control siRNA (siNT). 72 h post siRNA transfection cells were infected with H5N1 strain A/Thailand/1(KAN-1)/2004 at an MOI of 1 and harvested using DNA/RNA shield (Zymo Research). RNA extraction, rRNA depletion and analysis of the respective transcriptome was performed by Zymo Research as part of the EpiQuest TM service. (b) Gene ontology term enrichment analysis of host mRNAs differentially regulated by depletion of SMARCA2 in infected A549-MxA cells. (c) Host cell factors whose mRNA abundance was strongly decreased after SMARCA2 knockdown were silenced in A549-MxA cells using siRNA pools. 66 h after siRNA transfection A549-MxA cells were either treated, or not, with IFN-α (25 U/mL) and infected 6 h later with H7N7-RL reporter virus (MOI = 0.3). After 24 h virus reporter activity was measured using Renilla-Glo (Promega) substrate (3 technical replicates). The heatmap indicates increased (green) or decreased (red) viral replication as compared to the non-targeting siRNA control (white). (d) Knockdown and infection was performed as in (c) but experiments were extended to A549-shMxA cells and only siRNAs resulting in strong increase of viral reporter activity were used for knockdown (see (c)) (6 technical replicates). (e) To assess MxA-dependent effects, the ratios of reporter virus activities (from (d)) between A549-MxA cells and A549-shMxA cells were calculated and are presented as a heat map (High ratios in green and low ratios in red). Log2FC = Log2 Fold Change.