Supplementary Data from Identification and Characterization of Synthetic Viability with ERCC1 Deficiency in Response to Interstrand Crosslinks in Lung Cancer

Abstract

<p>Supplemental Figure S1. A. Clonogenic survival assays with cisplatin treatment in p53WT cell lines {plus minus} ERCC1. B. Clonogenic survival assays with cisplatin treatment in p53 null and p53mutant cell lines {plus minus} ERCC1. n = 3 independent experiments plated in triplicate for each cell line. Data plotted as average of at least three independent experiments {plus minus}SD. C. Viability assays with cisplatin treatment in p53WT cell lines {plus minus} ERCC1. D. Viability assays with cisplatin treatment in p53null and p53mutant cell lines {plus minus} ERCC1. n = 3 independent experiments plated in triplicate for each cell line. Data plotted as average of at least three independent experiments {plus minus} SD. Supplemental Figure S2. A. Cell lines utilized in the current study and status of p53, EGFR, and K-ras are listed. Mutation status was obtained from Cosmic Database (cancer.sanger.ac.uk) or cBIOportal (cbioportal.org) (except for H522 p53 status where Sanger sequencing did not identify the homozygous deletion that was previously reported. B. Western blot of additional ERCC1D clones in H460 and H1299 cells and the XPFD H1299 cells. Additional ERCC1D clones in this figure were validated by western blot and the XPFD clone was validated by sequencing. C-F. Colony survival assays of additional ERCC1D and XPFD clones. G-H. Western blot showing re-expression of ERCC1-202 in H460 and H1299 ERCC1D cells. Colony survival assay shows increased resistance to cisplatin with re-expression of ERCC1-202. n=3, plated in triplicate for each colony assay performed in this figure. Error bars represent SD of the averages of all experiments. Supplemental Figure S3. A. Clonogenic survival assays with MMC treatment in p53WT cell lines {plus minus} ERCC1. B. Clonogenic survival assays with MMC treatment in p53null and p53mutant cell lines {plus minus} ERCC1. n=2, in triplicate for all colony survival assays with MMC treatment. Error bars represent SD. C. Clonogenic survival assays of H460 and H1299 WT and ERCC1? cells with camptothecin treatment. n=3, in triplicate. Error bars represent {plus minus} SD. D. UV-C sensitivity assays in H460 and H1299 WT and ERCC1??cells. n=2, in triplicate. Error bars represent {plus minus} SD. Supplemental Figure S4. A. Sensitivity of parental and ERCC1? cells to etoposide treatment in colony survival assays. n=2, plated in triplicate for each cell line. Error bars represent {plus minus} SD. B. Sensitivity of parental and ERCC1? cells to gemcitabine treatment in colony survival assays. n=2, plated in triplicate for each cell line. Error bars represent {plus minus} SD. Supplemental Figure S5. A. p53 re-expression/disruption by CRISPR-Cas9 in H1299, H460, and H522 cells. B. Clonogenic assay of H460 ERCC1? and ERCC1?/p53* cells fixed at Day 6 and Day 12. C. p53 disruption by CRISPR-Cas9 in A549 cells. D. Clonogenic survival of A549 isogenic cells after treatment with cisplatin. n=3, plated in triplicate for each cell line. Error bars represent {plus minus} SD. E. Sequencing results of p53 editing in H460, H522, and A549 cells. F. Validation of ERCC1? in OV2008 and *C13 cells by western blot. G. Induction of p53 after cisplatin treatment. H. Clonogenic survival of OV2008 and *C13 cells after cisplatin treatment. n=2, plated in triplicate for each cell line. Error bars represent {plus minus} SD. Supplemental Figure S6. A. Western blot validation of XPA knockout by CRISPR-Cas9. Colony survival assay of H1299 WT, ERCC1D, XPFD, and XPAD after B. cisplatin and C. MMC treatment. D. Validation of second XPAD clone by western blot. E. Colony survival assay of H1299 WT and second XPAD clone with cisplatin treatment. Supplemental Figure S7. Top: Progression Free Survival of Stage 3+4 ovarian cancer patients stratified by p53 status and ERCC1 expression who received a platinum agent. Bottom: Overall Survival of Stage 3+4 ovarian cancer patients stratified by p53 status and ERCC1 expression who received a platinum agent. Data obtained the 2017 TCGA Ovarian Cancer Data Set from kmplot.com/ovca. Supplemental Table S1: shRNA, primers, crRNAs Supplemental Table S2: Antibodies</p>