RNA sensing via the RIG-I-like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency (2022)
Figure 1

Figure 1: Human LGP2 is essential for the induction of a type I IFN response upon depletion of ADAR1.

A) THP-1 monocytes were genetically engineered to knockout RIG-I, MDA5, or LGP2 using CRISPR/Cas9. Cells were differentiated towards macrophage-like cells using PMA and treated with recombinant type I IFN to upregulate RLR expression. Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=3). *, non-specific band.

B) Cells generated in (A) were differentiated using PMA and transfected with a control siRNA (siCtrl) or an ADAR1-targeting siRNA (siADAR1). The type I IFN response was monitored 56h post-transfection by RT-qPCR analysis to determine IFN-β and IFIT1 transcript expression, normalized to a housekeeping gene (ACTB). ADAR1 knockdown efficiency was monitored by ADAR1 p110 expression, normalized to ACTB and displayed relative to siCtrl. Data are means ± s.d. from a representative of three biological replicate experiments.

C) Primary human monocyte-derived macrophages were transfected with the indicated siRNAs. Cells were harvested 96h post-transfection and RT-qPCR analysis was used to monitor the type I IFN response (IFN-β, IFIT1, and ISG15 transcripts) and knockdown efficiency of ADAR1 and LGP2 (DHX58). All transcripts were normalized to ACTB. Data from two independent donors (denoted with distinct symbols) are shown with mean ± s.d..

D) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band.

E) Cells generated in (D) were transfected with siCtrl or siADAR1. The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post-transfection as in (B). Data are means ± s.d. from a representative of four biological replicate experiments.

F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).

G) HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained 72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>450 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.