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  • br Screen enrichment analysis br The top resistant

    2020-03-24


    2.4. Screen enrichment analysis
    The top resistant genes were identified by having a q value of <0.01 and log2 fold change >2 in the cisplatin-treated group compared to the vehicle control group. The Database for Annotation, Visualization and Integrated Discovery (DAVID), which uses a modified Fisher's exact test followed by Benjamini-Hochberg multiple hypothesis testing correction [24], was used to determine the enriched gene ontology (GO) term biological processes within the Herboxidiene top resistant genes [25]. In addition to GO term enrichment, Gene Set Enrichment Analysis (GSEA), which uses the Kolmogorov-Smirnov statistic, was used to determine the enrichment of the KEGG MMR pathway across the entire ranked gene list [26,27].
    For each sample, 25 mg of protein was loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA) followed by a Western blot (see Supplementary Table 2 for antibodies). Membranes were imaged using the Odyssey Fc Imaging System (LI-COR Biosciences, Lincoln, NE, USA) following application of ECL (MilliporeSigma).
    2.6. Caspase activation
    Cells were cotreated with the indicated concentration of cisplatin and 4 mM CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) according to manufacturer instructions. Phase contrast and green fluorescence were imaged every 3 h following initial treatment using the Incucyte Zoom system (Essen Bioscience, Ann Arbor, USA) with a 4 objective. The displayed results are cumulative
    green cell counts normalized to total cell confluency. We compared the induction in cisplatin-treated MSH2 knockdown Herboxidiene with that in treated nontargeting cells using a repeated-measure one-way analysis of variance (ANOVA; independent variable is knockdown).
    2.7. Quantitative reverse transcriptase PCR
    Total RNA was extracted using the NucleoSpin RNA kit (Machery-Nagel). Synthesis of cDNA was performed with the iScript cDNA Synthesis Kit (BioRad). The SsoFast Evagreen supermix (BioRad) was used for quantitative reverse transcriptase PCR assays (see Supplementary ]GIF$DT)1.gi([
    Table 3 for primer sequences). Target genes were normalized to mean GAPDH expression and a two-way ANOVA was performed, where the two factors were knockdown and treatment.
    2.8. Analysis of bladder tumors
    All human MIBC data were from The Cancer Genome Atlas (TCGA) [16]. Clinical, treatment, and survival data were downloaded using the TCGAbiolinks R package [28]. One hundred patients were treated with platinum-based (cisplatin or carboplatin) chemotherapy, 12 of whom were in the neoadjuvant setting, but molecular characterization was performed
    Fig. 1 – A whole-genome CRISPR screen to identify mediators of cisplatin resistance in a bladder cancer cell line. (A) Experimental outline of the screen and analysis. (B) A heatmap displaying median-centered counts for 9757 differentially abundant sgRNAs. (C) Counts from the screen for the top
    10 resistant sgRNAs. (D) A volcano plot displaying the log2 fold change and adjusted p value for all sgRNAs identified in the screen. A threshold of adjusted p < 0.05 is indicated in the plot.
    prior to any chemotherapy treatment [16]. Level 4, batch-normalized reverse-phase protein array (RPPA) was from the study by Li et al. [29] and MSH2 mRNA (RNA Seq V2 RSEM) expression was from the studies by Gao et al. [30] and Cerami et al. [31]. TCGA bladder cancer patients receiving cystectomies were identified using data from the Broad GDAC Firehose [32]. We used neoantigens published by Robertson et al. [16]. Overall, 340 TCGA patients had RPPA and clinical data (Supplementary Table 4). Overall survival was analyzed using the survival, survminer, and ggplot2 R packages [33–35]. Patient characteristics were compared using a Student t test or Fisher's exact test. The primary endpoint is overall survival, which was defined as the time from diagnosis to death from any cause. Patients were censored at the point of their last follow-up (last known time the patient was alive). For all survival analyses, each arm was censored after the at-risk population had <10 patients. Statistical differences between two patient groups were evaluated with the log-rank test.