Supplementary Data and Methods from AraC-FdUMP[10] Is a Next-Generation Fluoropyrimidine with Potent Antitumor Activity in PDAC and Synergy with PARG Inhibition
S1. (A) Representative graph of 5-day picogreen survival assay with PDX139, a patient derived xenograft line. (B) Representative images of 10-day colony growth inhibition assays with crystal violet staining in indicated PDAC cell lines. (C) Graph of changes in MIA-PaCa 2 flank-tumor volume after initiation of CF10 treatment (200mg/kg 3x/week) as compared to vehicle (saline) control. (D) Graph of individual flank-tumor volumes (n= 4 mice per arm) after 21 days of F10 treatment (200mg/kg 3x/week) as compared to vehicle (saline), Graph of changes in tumor volume after initiation of F10 treatment as compared to vehicle control, and graph of changes in relative weight after initiation of F10 treatment vs vehicle. (E) Representative images of Ki67 and cleaved caspase-3 IHC staining of tumor tissues from F10 experiment with quantifications of IHC staining. For each in-vitro experiment, three independent experiments were conducted, and errors bars represent standard error of the means. S2. Representative immunofluorescence images with an antibody specific for Top1 cleavage complexes (Top1cc) in MIA-PaCa 2 cells with either (A) no treatment or treated with (B) 10nM F10 for 24 hours (C) 10nM CF10 for 24 hours (D) 10nM 5FU for 24 hours (E) 1uM topotecan for 1 hour. S3. Representative immunofluorescence images with an antibody specific for Top1 cleavage complexes (Top1cc) in PANC1 cells with either (A) no treatment or treated with (B) 10nM F10 for 24 hours (C) 10nM CF10 for 24 hours (D) 10nM 5FU for 24 hours (E) 1uM topotecan for 1 hour. S4. (A) Quantification of Top1cc images of MIA-PaCa 2 cells from supplementary figure 2 (B) Quantification of Top1cc images of PANC1 cells from supplementary figure 3 For each experiment, three independent experiments were conducted, and errors bars represent standard error of the means. Significance was tested via two-way ANOVA with Bonferroni correction denoted by *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. S5. (A) Representative Bliss synergy plots from MIA-PaCa 2 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5-FU. (B) Representative Bliss synergy plots from ASPC1 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5-FU. (C) Representative bliss synergy plots of indicated MIA-PaCA 2 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5-FU. (D) Representative Bliss synergy plots from ASPC1 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5-FU. S6. (A) Representative cell viability plots used for bliss synergy analysis from MIA-PaCa 2 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5FU. (B) Representative cell viability plots used for bliss synergy analysis from ASPC1 cells concurrently treated with serial dilutions of thymidine and F10, CF10, and 5FU. (C) Representative cell viability plots used for Bliss synergy analysis from MIA-PaCa 2 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5FU. (D) Representative cell viability plots for Bliss synergy analysis from ASPC1 cells concurrently treated with serial dilutions of uridine and F10, CF10, and 5FU. S7. (A) Representative cell viability plots used for Bliss synergy analysis from MIA-PaCa 2 cells concurrently treated with serial dilutions of CF10 plus Olaparib, CF10 plus PDDX-04 and F10 plus PDDX-04. (B) Representative cell viability plots used for Bliss synergy analysis from PANC1 cells concurrently treated with serial dilutions of CF10 plus Olaparib, CF10 plus PDDX-04 and F10 plus PDDX-04. (C) Representative Bliss synergy plots from MIA-PaCa 2 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. (D) Representative Bliss synergy plots from PANC1 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. (E) Representative cell viability plots from MIA-PaCa 2 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. (F) Representative cell viability plots from PANC1 cells concurrently treated with serial dilutions of AraC plus PDDX and F10 plus AraC. S8. (A) 5-day survival assay of MIA-PaCa 2 cells treated with CF10 or F10 after siRNA knockdown of PARG compared to control scrambled siRNA with western blot validation of knockdown. (B) Representative western blot for PARG, PAR, yH2AX, and cleaved capase-3 with appropriate loading controls in MIA-PaCa 2 cells treated with CF10 after 48 hours of either siControl or siPARG knockdown with quantification of three independent experiments. Supplementary Table S1: Commercially available chemical compounds Supplementary Table S2: Western blot antibodies