FIGURE 3 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

DHX9 is overexpressed in breast cancer and suppresses PKR activation. A, Schematic showing the domain structure of PKR, ADAR1, DHX9 and other helicases identified by proximity labeling in Fig. 1, dsRBD refers to the dsRNA binding domain. B, Pearson and Spearman correlation coefficients for the correlation between ADAR1 expression at the RNA level and the expression of each indicated helicase at the RNA level, data from breast tumors within TCGA. Scatterplots showing the correlation between ADAR1-p110 (C), or ADAR1-p150 (D), and DHX9 expression in normal breast or breast tumors. E, Expression of DHX9 at the RNA level in normal breast, non-TNBC or TNBC tumors. F, Waterfall plot showing DHX9 dependency of breast cancer cell lines using data from DepMap, ER = estrogen receptor–positive cell lines, ERRB2 = HER2-positive cell lines. G, Foci formation assay following knockdown of DHX9 with two different shRNAs in four TNBC cell lines. Cells were plated for foci formation 2 days after transduction and foci were stained after 10 days. G, Representative immunoblot following knockdown of DHX9 with two different shRNAs in four TNBC cell lines, same cells as used in G. Protein lysates were collected from cells 4 days after transduction with lentivirus encoding the shRNAs listed. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. I, Quantification of PKR phosphorylation as determined by the immunoblot in H. Quantification of protein expression for other proteins of interest can be found in Supplementary Fig. S4A–S4E. Bars represent the average of at least three replicates, error bars are ± SD. *, P P P P values determined by Dunnett test.</p

Supplementary Figure 9 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Figure 9</p

Supplementary Figure 5 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Figure 5</p

Supplementary Tables from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Tables</p

Supplementary Figure 13 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Figure 13</p

Supplementary Figure 2 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Supplementary Figure 2</p

FIGURE 5 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Induction of a viral mimicry phenotype upon knockdown of DHX9 and ADAR1 in MCF-7. A, Volcano plot showing changes in RNA expression upon knockdown of DHX9 and ADAR1 in MCF-7, a volcano plot for SK-BR-3 is in Supplementary Fig. S6F. RNA was isolated from cells 5 days after transduction with the shRNAs listed. Fold change of RNA expression shown in A was determined using an interaction term between ADAR1 and DHX9 knockdown, volcano plots for fold change of RNA expression for single knockdown of ADAR1 or DHX9 is in Supplementary Fig. S6A–S6B and S6D–S6E. B, GO terms identified by gene set enrichment analysis of the RNA-seq data in A. C–E, Heat maps and summary box plots showing RNA expression changes in MCF-7 and SK-BR-3 upon knockdown of ADAR1 and/or DHX9. C shows RNA expression for core ISGs (22, 35), D shows ATF4 targets, and E shows NFκB targets with ISGs removed. Genes are clustered by RNA expression across all four samples. Clustering was performed for each gene set (ISGs, ATF4 targets, or NFκB targets) independently. For more information, see GitHub repository link in Data and Code Availability.</p

FIGURE 8 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Rescue of PKR activation by ADAR1-p110 and ADAR-p150. A, Representative immunoblot showing the phenotype of ADAR1 and DHX9 knockdown with ADAR1-p110 or ADAR1-p150 overexpression in SK-BR-3. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. B, Fold change of PKR phosphorylation at Thr-446 upon ADAR1 and DHX9 knockdown with ADAR1 isoform overexpression, quantified from immunoblots in A. C, Representative foci formation phenotype of ADAR1 and DHX9 knockdown ADAR1 isoform overexpression. Quantification of relative foci area is shown in D. Quantification of protein expression for other proteins of interest can be found in Supplementary Fig. S11A–S11F. Timepoints for collecting protein lysates and foci formation are the same as described in Fig. 4 for SK-BR-3. Bars represent the average of at least three replicates, error bars are ± SD. ***, P P values determined by Dunnett test.</p

FIGURE 2 from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Validation of putative protein–protein interactions identified by proximity labeling. Immunoprecipitation of DHX9 (A–C), DDX54 (D–E), or DDX17 (F–G) followed by immunoblot for ADAR in breast cancer cell lines. Immunoblot of immunoprecipitation eluates and inputs from SK-BR-3 (A, D, and F), MCF-7 (B, E, and G), and MDA-MB-231 (C). Input represents 5% of the lysate used for immunoprecipitation. The IgG lanes represent immunoprecipitation eluates from pulldown with anti-rabbit IgG antibody. The lanes labeled DHX9, DDX54, and DDX17 indicate the eluates from immunoprecipitation with antibodies against those proteins, respectively. The IgGHC label indicates the band corresponding to the IgG heavy chain from the antibody used for immunoprecipitation. Uncropped immunoblots for A–G can be found in Source Data Figures. Immunofluorescence for ADAR1 and DHX9 (H), DDX54 (I), or DDX17 (J) in SKBR3 or MCF-7.</p

Source Data Figures from Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells

Source Data Figures</p