Fig 2: Expression of the IRF1 and DTX3L proteins/mRNAs in breast cancer cell-lines and tissues. (A) IRF1 (Interferon-Responsive-Factor-1) and DTX3L (Deltex-E3-Ubiquitin-Ligase-3L) mRNAs were measured by PCR (Polymerase Chain Reaction) in the indicated 41 cell-lines (column graphs). Each value is the mean ± standard deviation (SD) of 3 replicate cultures and the results are normalized for the 18S mRNA. The Western blots illustrate the levels of IRF1 and DTX3L proteins. The 16 cell-lines belonging to our experimental panel are indicated in blue (basal cell-lines) and red (luminal cell-lines). The basal or luminal phenotype of all the other cell-lines is indicated by blue and red dots, respectively. Each well contains an equivalent amount of protein. CAL-851 and BT474 cells (internal loading controls) are marked in green. The asterisk underneath the indicated Western blots marks the cell-lines for which bands are not detectable. The positivity of each cell-line to HER2 (HER2+) or ER (ER+) as well as the negativity of each cell line to HER2, ER and PR (progesterone receptor) is indicated by the Triple-neg (Triple-negative) designation. For the whole Western blot figures, please see Supplementary Information. (B) and (C) The IRF1 as well as DTX3L mRNA and protein levels are compared following clustering of the cell-lines according to the luminal (Lum) and basal (Bas) phenotype or the presence/absence of the ER (Estrogen Receptor) and HER2 proteins as defined in (A). Each point represents a single cell-line. The quantitative protein data were obtained from the densitometric analysis of the Western blots shown in (A), following normalization with the internal loading controls. * Significantly different following the Student’s t-test, corrected for multiple testing (Benjamini–Hochberg) (p < 0.05). ** (p < 0.01). (D) and (E) The Box Plots show the IRF1 and DTX3L mRNA levels of breast cancer tissue samples determined from the RNA-seq data available in the TCGA database (1108 cases). The breast cancer cases are classified according to the PAM50 algorithm into 5 distinct groups: Luminal A = LumA, HER2-positive = HER2+, Luminal B = LumB, Normal-like = Nor-like, Basal = Bas. Each point represents an individual case. * Significantly different following pairwise t-test comparison adjusted for multiple testing according to the Benjamini–Hochberg correction method (p < 0.05). ** (p < 0.01).

Fig 3: Effects of ATRA on the immunophenograms, endogenous retroviral elements and viral mimicry upstream regulators of breast cancer cell-lines. (A) The indicated cell-lines were treated with vehicle (DMSO) or ATRA (1 μM) for 24 h. The circle plots show the effects exerted by ATRA on the immunophenograms derived from the RNA-seq data. The ATRA-score values are shown in parenthesis. (B,C) The indicated breast cancer cell-lines were exposed to vehicle (DMSO) or ATRA (1 μM) for 24 h. (B) The box-plots show the effects exerted by ATRA on the expression of individual RNAs deriving from endogenous retroviruses (ENV). For each cell-line, the points represent the mean (n = 3) ATRA/DMSO fold-change values of the single RNAs determined. Within the luminal and basal groups, cell-lines are positioned according to a decreasing ATRA-score from left to right, as indicated. * Significant ATRA-dependent up- or downregulation (p-value < 0.1); *** Significant ATRA-dependent upregulation (p-value < 0.001). (C) The diagrams illustrate the correlations between the effects exerted by ATRA on ENV-derived mRNAs and the ATRA-scores determined in luminal and basal cell-lines. ATRA/DMSO fold-change ENV RNAs = fold-change of the median expression values of the 42 classes of ENV RNAs determined in each cell-line following treatment with ATRA (1 μM) or vehicle (DMSO) for 24 h. The median fold-changes are expressed in linear values and were calculated from the logarithmic values shown in (B) and Supplementary Table S5. The R2 values of the correlations are indicated. (D) Left: SK-BR-3 cells were treated with DMSO or ATRA (1 μM) for 24 h. Cell extracts were subjected to Western blot analysis for TBK1, Serine/172 phosphorylated TBK1 (TBK1S172), p65 and Serine/536 phosphorylated p65 (p65S536). Right: SK-BR-3 cells were treated with DMSO or ATRA (1 μM) for the indicated amount of time. Total cell extracts were subjected to Western blot analysis for TBK1, TBK1S172 and tubulin (TUB) as a loading control. TBK1S172/TBK1 = ratio of the densitometric results obtained for the phosphorylated TBK1 and total TBK1 bands (p65S536/p65 = ratio of the densitometric results obtained for the phosphorylated p65 and total p65 bands). (E) SK-BR-3 cells were treated with the TBK1 inhibitor, MRT67307, in the presence/absence of ATRA at the indicated concentrations for 24 h. Cells were pre-treated with the TBK1 inhibitor for 1 h. Total cell extracts were subjected to Western blot analysis for IRF1, DTX3L and tubulin (TUB). IRF1/TUB = ratio of the densitometric results obtained for the IRF1 and Tubulin bands, DTX3L/TUB = ratio of the densitometric results obtained for the DTX3L and Tubulin bands. For the whole Western blot figures, please see Figure S19.

Fig 4: DTX3L interaction influences PARP14 ADP-ribosylation.. A) ADP-ribosylation assay using NAD+-biotin and 1 μg PARP14 constructs containing catalytic domain (CAT) or WWE or 14D/KHL domain (14DWC). 1 μg DTX3L D3-D4 was added before reaction was initiated with -NAD+ -biotin. Arrows indicate ADP-ribosylation products. B) 1 M Neutral hydroxylamine was incubated with the reaction products of the ADP-ribosylation assay to identify of modification of acidic residues for each PARP14 construct and in the presence of DTX3L KHL2-5 (expected position indicated by arrow). C) 190 nM PARP14 DWC truncation was included in an ADP-ribosylation assay in the presence of PARP9/DTX3L. Biotinylated ADP-ribosylation products (red) and the DTX3L species are detected with a specific antibody (green) such that ADP-ribosylated DTX3L appears yellow. D) Similarly, the upper band detected in the ADP-ribosylation assay was identified as PARP9 using a specific antibody (green).

Fig 5: DTX3L domains and interactions with PARP9 and PARP14. A) Predicted aligned error (PAE) plot of full length DTX3L indicates regions of known domains (RING/CT) and four ‘domains’ in the N-terminus (D1-D4). B) Domain boundaries used in this study and truncations used in interaction mapping (summarised in Table 1). C) Co-immunoprecipitation (Co-IP) assay using over-expressed constructs in HEK293T cells using GFP-TRAP beads with GFPf fusions of 9C and 14C co-expressed with untagged full length (FL) DTX3L or a truncated (D1-4) containing no RING-CT domains. Inputs and bead eluates were blotted for DTX3L (top) and flag (bottom). D) Anti-FLAG Co-IP of GFPf, 9F and 14C co-expressed with HA tagged DTX3L D4 (KLH4-KLH5).