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Supplemental Figures and Tables Supplemental Figure 1. Elevated NQO1 expression in patients with HNC correlates with poor overall survival to standard therapy. Overall survival associated with NQO1 gene expression was assessed in 286 head and neck cancer patients using the PROGgeneV2 online webtool with data sourced from The Cancer Genome Atlas (TCGA) through the following URL: (http://watson.compbio.iupui.edu/chirayu/proggene/database/?url=proggene) [1]. Patient cohorts were divided into “High” (145 patients) and “Low” (141 patients) NQO1 gene expression groups based on median gene expression. The raw data from this analysis was plotted as a Kaplan-Meier overall survival plot in GraphPad Prism and statistical significance was calculated using the log-rank test. Overall survival data indicated that patients with high NQO1 expressing tumors have significantly reduced survival compared to low expressing HNC cancers (p = 1 X 10-4). Supplemental Figure 2. Functional shRNA-NQO1 knockdown rescues β-lapinduced cell death. (A) Lentiviral shRNA against NQO1 was used to stably reduce the protein abundance of NQO1 in FaDu cells and a representative western blot for several different independent NQO1 knockdowns is presented. A non-targeting scrambled shRNA (shSCR) was used to control for the lentiviral vector and GAPDH served as the loading control. (B) β-Lap-induced lethality is rescued in FaDu shNQO1 knockdown clones. Relative survival of shSCR and shRNA-NQO1 knockdown cells #6 and #7 were evaluated after various doses of β-lap exposure (µM, 2 h) as depicted on the x-axis demonstrating a β-lap induced NQO1 dependent cell death (***p < 0.001). Supplemental Figure 3. β-Lap-induced NQO1 dependent DNA damage and NAD+ depletion. Time-dependent increase in DNA strand breaks in FaDU cells was demonstrated by the alkaline comet assay in the presence of β-lap alone (5 µM, 2 h) or co-treatment with β-lap and DIC (50 μM). (A) Shown are representative micrographs of studies performed at least three times and (B) the graphed comet tail lengths were measured using Komet 5.5 software showing means ± SE from three experiments. Li et al Student’s t tests were performed to assess significance (***p < 0.001). (C) NAD+ levels are dramatically reduced by exposure to β-lap at the indicated doses and this response is blocked by co-treatment with DIC (50 μM) in FaDu and SqCC/Y1 cells. Student’s t tests were performed to assess for significance (***p < 0.001). Supplemental Figure 4. Combination sublethal doses of β-lap and low dose IR induce NQO1 dependent anti-cancer lethality. Relative cell survival assays were performed by treating with sublethal doses of β-lap at the specified concentration (M 2 h) immediately after 2 Gy exposure, with or without co-treatment of DIC (50 M) in SqCC/Y1 cells. The triple asterisks represent statistically significant differences between the β-lap (2.5 M) + 2 Gy vs. β-lap (2.5 M) + 2 Gy + DIC (50 M) treatments (***p < 0.001). A lethal dose of β-lap (6 M), with or without DIC (50 M) served as controls. Supplemental Figure 5. Cooperative antitumor efficacy using a combination of IR and β-lap to treat HNC xenograft models. Mice bearing 30 mm3 SqCC/Y1 HNC xenografts with high levels of NQO1 expression (A and B are SCID-NOD data with 10 mice for each treatment group; C and D are Athymic nu/nu phenotype with 5 mice for each treatment group) were treated with 2 or 4 Gy every other day for five treatments (10 and 20 Gy total, respectively). β-Lap-HPβ-CD 10 mg/kg was intravenously injected by tail-vein immediately following IR treatment. Vehicle alone (HPβ-CD) served as a control cohort. Results (means ±SE) are representative of repeated similar experiments (n=10 for each group). Student’s t-tests (*** p < 0.001) were performed comparing treated vs control groups. (A and C) Tumor volume measurements and (B and D) Kaplan-Meier overall survival over the indicated number of days is graphed for control (HPβ-CD), β-lap-HPβ-CD 10 or 20 mg/kg alone, 2 or 4 Gy alone and a combination of 2 or 4 Gy plus β-lap-HPβ-CD 10 mg/kg for the NOD-SCID cohorts. The nu/nu cohorts did not receive β-lap-HPβ-CD 20 mg/kg alone, 4 Gy IR alone, or any combination that utilized these doses. Log-rank analyses were performed comparing survival curves using various β-lap-HPβ-CD + IR regimens (*** p < 0.001 for the combined treatment compared to each single treatment). 2 Li et al Figure 6. Expression of Ki-67 after combination of β-lap and IR in HNC xenograft models. Ki-67 (Santa Cruz) IHC staining was performed on 5 μm thick SqCC/Y1 HNC formalin fixed paraffin embedded slides via two-step assays as previously described (22). Cytoplasmic and nuclear staining was independently evaluated by two Pathologists (Drs. Lin and Liu) in a blinded manner. Shown are nuclear staining of Ki-67 in the control (HPβ-CD), β-lap-HPβ-CD 10 mg/kg alone, 2 Gy alone and a combination of 2 Gy plus β-lap-HPβ-CD 10 mg/kg. Supplemental Table 1. NQO1 enzyme activity and abrogation of activity in 41 HNC cell lines and primary human IMR90 fibroblasts. β-Lap-50% lethal dose (LD50, µM), and LD50 for co-treatment with β-lap plus DIC, β-lap plus BAPTA, and β-lap plus Catalase for HNC cancer cell lines and IMR90 primary fibroblasts with varying NQO1 expression and activity levels. Supplemental Table 2. Equitoxic doses comparing single to combined treatment in HNC cells. Equitoxic doses comparing single to combined treatment in HNC cells are listed in values of equivalent doses (ED) calculated using parameters obtained from fitting the data from three independent experiments with Carter’s model. Supplemental Table 3. Individual patient gender, age, subsite, stage, histology and IHC scoring for NQO1 and Catalase. Reference: 1. Goswami CP, Nakshatri H: PROGgene: gene expression based survival analysis web application for multiple cancers. Journal of clinical bioinformatics 2013, 3(1):22. 3