Fig 1: Structure of the EGR4 gene and splice variant expression in cells and tumour tissue. (A) Schematic diagram of the Human EGR4 gene, located on Chromosome 2, showing its 2 exon structure (top), the 3 possible mRNA transcripts (middle), and 3 hypothetical protein isoforms arising from these different transcripts (bottom). Based on: https://www.ensembl.info/known-bugs/ensembl-100/ (accessed on 15 August 2017). (B) RNAseq analysis for EGR4 exon expression in 56 different breast cancer cell lines. Reads were normalised to exon size by scaling to base-level coverage. (C) Example of Western blot results for HER2 and EGR4 expression in normal (N) and breast tumour (T) biopsies from HER2- (pt 1–4) and HER2+ (pt 5–9) patients.
Fig 2: Confirmation of the EGR4-S splice variant in cell lines and clinical samples. (A) Results from qPCR on three breast tumour patient biopsies showing the amplification cycle for a qPCR product (mean ± SEM) detected using primers binding in exon 1 vs. primers binding in exon 2 for the same tumour sample. (B) Analysis of TCGA BRCA RNA-seq dataset (n = 1085) showing distribution and median proportional change for the expression of EGR4 exon 1 and exon 2 mRNA in patients diagnosed with breast carcinoma (n = 1085). Expression of mRNA is separated according to breast cancer sub-type. (C) Representative punch biopsies from a HER2+ breast tumour at low magnification (left panels) and high magnification (right panels).
Fig 3: The immunoreactivity of pertuzumab was not compromised after NOTA conjugation and 67Cu-labeling. (A) Western blot analysis of HER2 positive HCC1954 and HER2 negative MDA-MB-231 cells (the blots are cropped for clarity. The full length blots at different exposure levels are presented in Supplementary Figure S1); (B) Lindmo assay of [67Cu]Cu-NOTA-Pertuzumab using HCC1954 cells; (C) Cell uptake assay of [67Cu]Cu-NOTA-Pertuzumab in HCC1954 and MDA-MB-231 cells. (B) was graphed with GraphPad Prism 7.0 (https://www.graphpad.com/scientific-software/prism/).
Fig 4: SPECT/CT imaging results. (A) Representative maximum intensity projection (MIP) SPECT/CT images of HCC1954 HER2+ tumor-bearing mice injected with [67Cu]Cu-NOTA-Pertuzumab (Group 2, 3, 4, and 5) at day 2 and 5 post the start of treatment (yellow arrows indicate the tumors); (B) Actual radioactivity concentration in tumors (MBq/mL) on Day 2 and 5 (without decay correction).
Fig 5: MTT assay shows that upregulation of miR-495 or silencing ERBB2 inhibits GC cell viability. * p<0.05, vs. the blank and NC groups; NC – negative control; OD – optical density; miR-495 – microRNA-495; MTT – 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ERBB2 – human epidermal growth factor receptor 2; these data was measurement data and analyzed by repeated-measures ANOVA; the experiment was repeated 3 times.
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