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Additional file 7: Fig. S5. Effect of PD-L1 and MT Oas2 on primary tumor initiation and expansion in parous mice. Examples of tumor growth curves of individual mice. The 2nd/3rd and 4th mammary glands were palpated twice weekly and tumor growth was estimated by measurement of the major and minor axis of individual tumors using calipers. Y axis is placed at the day of tumor detection on the x axis as day 0. Black diamonds show a treatment with IP IgG or PD-L1 antibodies and red diamonds indicate parity. WT = wild type, MT = MT Oas2 MT/MT = homozygous MT Oas2.

Additional file 8: Fig. S6. Effect of PD-L1 and MT Oas2 on primary tumor initiation and expansion in parous mice. Two-way Kaplan–Meier survival analysis for the indicated periods and endpoints, and for the indicated genotypes and treatments (PD-L1 or IgG IP), of mouse cohorts. P values and hazard ratios (HR) calculated by the log-rank test using GraphPad Prism. n = 8 WT PD-L1, 8 WT IgG, 14 MT PD-L1, 15 MT IgG.

Additional file 6: Fig. S4. Flow cytometry gating strategy for monocytes and neutrophils known as myeloid-derived suppressor cells. Series of gates used to quantify monocytes and neutrophils using flow cytometry.

Additional file 5: Fig. S3. Effects of MT Oas2 on T cells in parous mice. A screening of various T-cell parameters was undertaken in mammary tumors, lymph nodes, spleen and thymus. Error bars are standard error of the mean and p values calculated by Student’s t test (all non-significant). Axes represent % of total cells passing the previous gate. Genotypes, WT/WT homozygous wildtype Oas2, MT/MT homozygous mutant Oas2.

Additional file 1: Table S1. Antibodies, concentration and antigen retrieval conditions for immunohistochemistry. All reagents were from Leica BOND or DAKO for automated or manual IHC (as specified). Visualization of antigen–antibody complexes was performed using the DAB + liquid substrate chromogen system (K3467).

Additional file 3: Fig. S1. Effect of MT Oas2 on primary tumor expansion in parous mice. Examples of tumor growth curves of individual mice. The left- and right-hand side 2nd/3rd and 4th mammary glands were palpated twice weekly and tumor growth was estimated by measurement of the major and minor axis of each gland using calipers. Y axis is placed at parity indicated on the x axis as day 0. Numbers show the total number of glands with tumors detected, the number that showed a period of no growth, and the number that showed regression at or following parity. W, wild type; M, MT; W/W, homozygous wild type; M/M, homozygous MT.

Additional file 4: Fig. S2. Effect of MT Oas2 on primary tumor initiation and expansion in nulliparous and parous mice. Two-way Kaplan–Meier survival analysis for the indicated periods and endpoints, and for the indicated genotypes, WT wildtype Oas2, MT mutant Oas2, and parity status of mouse cohorts (20 per group). P values and hazard ratios (HR) calculated by the log-rank test using GraphPad Prism. n = 19 MT parous, 21 WT parous, 20 MT nulliparous and 19 WT nulliparous.)

Additional file 2: Table S2. Antibodies used for flow cytometry. Panel name, antibody name by antigen recognized and conjugated fluorochrome, the antibody supplier and catalogue number and final diluted concentration for each is shown.

Additional file 1: Figure S1. Analysis of ID4 protein expression across a panel of breast and ovarian cell lines. (A) Western blotting analysis of ID4 protein expression across a panel of breast (top) and ovarian (bottom) normal and cancer cell lines. A rabbit monoclonal antibody specific to ID4 (Biocheck, BCH-9/82–12) was used for detection [11]. Two isoforms of ID4 are detectable across the panel. β-Actin is shown as a loading control. Modifications to images indicated with vertical black line. Cell lines selected for further analysis are indicated with a black square. Breast cancer subtypes and ovarian cancer histotypes indicated [82, 83]. (B) Western blot validation of RIME protocol. MDA-MB-468 cells were processed using the RIME protocol. Antibodies targeting ID4 (pooled polyclonal antibodies) and IgG (rabbit species matched control) control were used for immunoprecipitation. Western blot analysis for ID4 protein expression, using an independent ID4 monoclonal antibody, following immunoprecipitation. Figure S2. ID4 ChIP-sequencing analysis of HCC70 cell line identifies reproducible ID4-chromatin binding sites. (A) Table summarising three biological replicates of ID4 ChIP-seq analysis in HCC70 cell line. (B) 10,000 bp resolution image of ID4 ChIP-seq technical replicate #1 binding to MALAT1 gene. Red sequencing reads are aligned to the positive strand (5′ - 3′), and blue to the negative strand of DNA (3′ - 5′). ID4 binding to (C) left to right: GBA, FAIM, MIRLET7BHG, NEAT1 and ZFP36L1. The chromosomal location, size of the gene and Refseq, human reference genome, are displayed at the top of the image. Reads have been aligned to the human reference genome Hg19 and peaks called using MACs peak calling algorithm (v2.0.9) [38]. Images contain ChIP-seq coverage data and the peaks called for each ID4 technical replicate and the consensus peaks called for all three ID4 ChIP-seq biological replicate for selected gene regions. ID4 binding is shown in comparison to IgG and Input data for the same region. Data visualised using IGV [56, 57]. Transcription Start Site (TSS) indicated with black arrow. Figure S3. ID4 ChIP-exonuclease sequencing analysis of HCC70 and HCC1954 cell lines reproduces ChIP-seq analysis. (A) Table summarising ChIP-exonuclease sequencing analysis of ID4 and IgG binding events in HCC70 and HCC1954 breast cancer cell lines. ID4 binding normalised as for ChIP-seq analysis. ChIP-exo analysis of the HCC70 and HCC1954 breast cancer cell lines showing ID4 binding to NEAT1, MALAT1 and GBA (B), ZFP36L1 and ELF3 (C), and KDM4C and ERRFI1 (D). Reads have been aligned to the human reference genome Hg19 and peaks called using MACs peak calling algorithm (v2.0.9) [38]. Images contain ChIP-exo coverage data and the peaks called for each ID4 technical replicate and the consensus peaks called for both ID4 ChIP-exo technical replicate for selected gene regions. ID4 binding is shown in comparison to IgG and input data for the same region. Data visualised using IGV [56, 57]. The chromosomal location and size of the gene are displayed at the top of the image. Below this, Refseq, human reference genome, displays the gene corresponding to particular genomic loci. Transcription Start Site (TSS) indicated with black arrow. Figure S4. Validation of ID4 binding to specific loci in HCC70, MDA-MB-468 and HCC1954 cell lines. (A) Schematic of primer binding across ELF3 gene region. Primers 1–5 are scattered along the length of the ELF3 gene ID4. ChIP-qPCR analysis in (B) HCC70 (n = 2–8), (C) MDA-MB-468 (n = 2) and (D) HCC1954 (n = 2) cells. Multiple primers were designed to tile across the large ID4 binding sites. ID4 binding normalised to input DNA and to a region not bound by ID4 (negative region) and represented as fold-change over IgG control. 1-Way-ANOVA of ID4 binding across all primers compared to negative region: HCC70 p = 0.0001, MDA-MB-468 p = 0.549, HCC1954 p = 0.519. Figure S5. ID4 knockdown using SMARTChoice inducible lentiviral knockdown system. Representative western blot showing ID4 and β-Actin protein expression in wild-type HCC70 cell line and HCC70 cells stably transfected with SMARTChoice vectors containing doxycycline-inducible shRNAs targeting ID4 (shID4 #1, #2 and #3) or non-targeting control region (shNTC). Cells treated with and without DOX for 72 h. (B) Western blotting densitometry quantification of ID4 protein expression across six biological replicates of ID4 knockdown following treatment with and without DOX for 72 h. ID4 expression normalised to β-ACTIN and no DOX control. Error bars measure standard error, n = 4–6. (C) qRT-PCR analysis of ID4 RNA expression matched to cells in (B). Data normalised to B2M housekeeping gene and no DOX control. Error bars measure standard error, n = 4–6. Student’s t-test compares shNTC with the other modified cell lines. ** p