Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation (2020)
Figure 5

Fig 5 Loss of AGO1x triggers accumulation of endogenous dsRNA.

A, Schematic representation of the dsRNA sensing mechanism, downstream signalling events and expected phenotypes.

B, Mean (+/- s.e.m.) log2 mRNA fold-changes of dsRNAs sensors (from panel A) in the two mutant cell lines compared to control (n=3) inferred from RNA-Seq. Multiple-testing-corrected P-values for Wald tests comparing fold-changes with respect to control are depicted above each bar (*** P < 0.001).

C, Mean (+/- s.d.) fold-changes of corresponding proteins (see B), if detected in the proteomics data from the two mutant (W1A, n=2; W6A, n=1) lines relative to control (n=2). Multiple-testing-corrected P-values for unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (* P < 0.05, ** P < 0.01, *** P < 0.001).

D, Western blot detection of phosphorylated eIF2α (Ser51) and total eIF2α levels in cell lysates of control and mutant MDA-MB-231 cells. GAPDH and Tubulin serve as loading control for phosphorylated eIF2α and total eIF2α blots respectively. Numbers indicated below the blots represent the band intensities of target proteins normalized to the loading controls, with the value in the control line taken as baseline of 1.

E, Mean (+/- s.d.) fold-changes in phosphorylated peptide (phosphorylation sites are indicated in brackets) levels derived from a proteomic analysis of phosphopeptides in the two mutant lines (W1A, n=2; W6A, n=2) compared to control (n=1). Multiple-testing-corrected P-values for Wald tests (transcriptomics) or unpaired two-tailed t-tests (proteomics) comparing fold-changes with respect to control are depicted above each bar (** P < 0.01).

F, Quantification of the relative abundance (mean+/- s.e.m.) of distinct dsRNA foci in each cell type . 3D images were projected on a single plane, non-overlapping foci were counted from 100 cells of each type, and the numbers in three abundance bins (<1, 1-5, >5) are shown.

G, Representative immunofluorescence image of the control and mutant MDA-MB-231 cells stained with AGO1x (green) and J2 antibody (red) and DAPI (blue) to mark the nucleus. Loss of AGO1x signal in mutants also confirms the efficacy of CRISPR-induced mutations.

H,I, Concentration of recovered RNA (mean +/- s.e.m.) measured by Nanodrop™ (H) and relative abundance of various RNA species (mean +/- s.d.) in J2 antibody immunoprecipitates from three biological replicates of control and mutant cell lines (I). Multiple testing corrected P-values from unpaired two-tailed t-tests are depicted above each comparison (* P < 0.05, *** P < 0.001).

J, Venn diagrams showing the intersection between RNA transcripts that were consistently enriched (top) or depleted (bottom) in the dsRNA-seq relative to total RNA-seq in the two mutant cell lines compared to control (n=3).

K, Boxplots showing the proportion of G/C nucleotides in all genes and in genes depleted/enriched in dsRNA-seq compared to total RNA-seq (n=3). Shown are the P-values in the non-parametric Mann-Whitney U test (*** P < 0.001). Boxes extend from the 25th to 75th percentiles (inter-quartile range (IQR)), horizontal lines represent the median, whiskers indicate the lowest and highest datum within 1.5*IQR from the lower and upper quartiles, respectively.

L, Left: Quantification of transcript abundance (mean +/- s.e.m.) by qRT-PCR in AGO1x (black, n=6 for putative targets, n=3 for negative controls) or IgG (orange, n=2) IP from control cells. n.d., not detected. Multiple test corrected P-values for an unpaired two-tailed t-test are depicted above each comparison (* P < 0.05, ** P < 0.01, *** P < 0.001). Right: Concentration of RNA (mean +/- s.d.) obtained with each type of antibody, with P-value from an unpaired two-tailed Student's t-test.