Additional file 1 of Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer
Creators: name: Baker, Laura A.
name: Holliday, Holly
name: Roden, Daniel
name: Krisp, Christoph
name: Wu, Sunny Z.
name: Junankar, Simon
name: Serandour, Aurelien A.
name: Mohammed, Hisham
name: Nair, Radhika
name: Sankaranarayanan, Geetha
name: Law, Andrew M. K.
name: McFarland, Andrea
name: Simpson, Peter T.
name: Lakhani, Sunil
name: Dodson, Eoin
name: Selinger, Christina
name: Lyndal Anderson
name: Goli Samimi
name: Hacker, Neville F.
name: Elgene Lim
name: Ormandy, Christopher J.
name: Naylor, Matthew J.
name: Simpson, Kaylene
name: Nikolic, Iva
name: O’Toole, Sandra
name: Kaplan, Warren
name: Cowley, Mark J.
name: Carroll, Jason S.
name: Molloy, Mark
name: Swarbrick, Alexander
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: 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