Additional file 1 of Epigenomic perturbation of novel EGFR enhancers reduces the proliferative and invasive capacity of glioblastoma and increases sensitivity to temozolomide
Creators: name: Vincent, Craig A.
name: Nissen, Itzel
name: Dakhel, Soran
name: Hörnblad, Andreas
name: Remeseiro, Silvia
Publisher: name: figshare
Year: 2023
Subjects: subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Computational Biology

description: Additional file 1: Supplementary Figure 1. Recruitment of dCas9-KRAB repressor complex leads to enrichment of H3K9me3 at specic targeted sites in U251 cells. A-F, Bar charts depicting the enrichment of H3K9me3, as determined by ChIP-qPCR, at each genomic region (i.e., CE5B, CE6B, CE8, Promoter) and in each of the EGFR enhancer-repressed lines: iCE5B (B), iCE6B (C), iCE5B+6B (D) and iCE8 (E), alongside the iPromoter (F) and control line (A). Supplementary Figure 2. CRISPRi of EGFR enhancer CE5B+6B in Human Glioblastoma cell line U3013 downregulates EGFR gene expression, reduces cell proliferation rate, and sensitises cells to temozolomide (TMZ). A, EGFR gene expression as determined by RT-qPCR. B, Proliferation rates of the iCE5B+6B and promoter-repressed U3013 cell lines determined by live-cell imaging. Images were acquired every 4 hours and proliferation was determined by automatic cell count. Data is normalised to t=0 and presented as mean ± SEM (n=3). Statistical significance was assessed by unpaired t test (* P &lt; 0.05, ** P &lt; 0.01). C-D, Proliferation rates of control U3013 and iCE5B+6B enhancer-repressed U3013 cells determined by live-cell imaging upon treatment with 125μM TMZ in comparison with the DMSO-treated control. Images were acquired every 4 hours and proliferation was determined by automatic cell count. Data is normalised to t=0h and represented as mean ± SEM (n=4). P values were determined by unpaired t test. Supplementary Figure 3. CRISPR/Cas9-mediated deletion of EGFR enhancers in U251 cells downregulates EGFR gene expression and affects cell proliferation rates. A, Genotyping PCR of the EGFR enhancer-deleted cell lines (ΔCE5B, ΔCE6B, ΔCE5B+6B, ΔCE8) alongside the Δ Promoter and empty vector control lines (left), and schematic outline of the PCR genotyping strategy (right). Note that the wild-type CE5B+6B allele is too large to be amplied under these conditions. Full uncropped gel image presented in Supplementary Figure 6. B, EGFR gene expression levels relative to HPRT in EGFR enhancer-deleted cell lines as determined by RT-qPCR assays. Data is represented as mean ± SEM (n=3). Statistical significance as assessed by unpaired t test with Welch’s correction (* P &lt; 0.05, ** P &lt; 0.01). C, Cropped western blots showing EGFR protein expression and normalised to GAPDH protein levels (full uncropped blots in Supplementary Figure 6). D, Proliferation rates of cell lines carrying EGFR enhancer deletions or promoter deletions as determined by live-cell imaging, and in comparison to the empty vector control line. Images were acquired every 4 hours and proliferation was determined by automatic cell count. Data is normalised to t= 0h and plotted as mean ± SEM (n=3). Supplementary Figure 4. CRISPRi of the EGFR enhancer CE5B+6B and promoter compromises the invasive capacity of U251 cells. A, Representative images of the fusion between the fetal brain spheroids and the GFP+ tumour spheres generated from control, iCE5B+6B and iPromoter lines. Scale bars 750µm (0h) and 300µm (2-24h). B, Quantication of fusion events across a pool of independent tumour sphere/brain spheroid confrontation assays (n=10). C, Quantication of invasion frequency between the GFP+ tumour spheres and brain spheroids between 0h and 96h. D, Representative images of invasion/non-invasion between GFP+ tumour spheres and brain spheroids. Scale bar 100µm. Arrowheads point to areas of invasion. E-F, Line plots comparing the rate at which the respective cell lines migrate either from media containing 1% FBS to 1% FBS (no-chemoattractant negative control) or from 1% FBS to 10% FBS (chemoattractant condition). Migration was assessed by live-cell imaging taking images every hour and migration rate was determined by automatic quantification of the area of migrated cells. Data is represented as mean ± SEM (n=3). Supplementary Figure 5. Expression of key metabolic genes in the EGFR enhancer-repressed iCE5B+6B cell line. A, Results from qPCR analysis where certain metabolic genes are upregulated in the iCE5B+6B line relative to the control. Gene expression levels are relative to HPRT. Supplementary Figure 6. Uncropped western blot and gel images. A-B, Western blot on repressed cell lines (A) and deletion cell lines (B) detecting EGFR and GAPDH expression. Cropped images presented in Figure 1G and Supplementary Figure 3C, respectively. C, Uncropped genotyping gel from enhancer-deletion U251 cell lines. Cropped image presented in Supplementary Figure 3A.