Supplementary figures and tables from Zinc Metallochaperones Reactivate Mutant p53 Using an ON/OFF Switch Mechanism: A New Paradigm in Cancer Therapeutics
Authors: Yu Xin , Kogan Samuel , Chen Ying , Tsang Ashley T. , Withers Tracy , Lin Hongxia , Gilleran John , Buckley Brian , Moore Dirk , Bertino Joseph , Chan Chang , Kimball S. David , Loh Stewart N. , Carpizo Darren R. 
Published: April 2023

<p>Supplementary figures and tables Supplementary Fig. S1. Quantitative RT-PCR (qPCR) validation of the expression of MT1A, MT2A, and ZnT1 in TOV112D cells after ZMC1 treatment. Supplementary Fig. S2. Quantitative RT-PCR (qPCR) validation of the knockout of MT1A and MT2A in the CRISPR-cas9 treated TOV112D cells. The ZnT1 expression was measured by qPCR and showed a six-fold increase in the KO cells. Supplementary Fig. S3. FluoZin-3 fluorescent imaging in the TOV112D cells with knockdown of either MT1A or MT2A by siRNA transfection followed by treatment of 1 ï�­M ZMC1 for 0 - 8 hours. The scale bar = 50 ï�­m. The transfection efficiency was measured by qPCR and shown on the right. Supplementary Fig. S4. Sensitivity of TOV112D cells with siRNA knockdown of MT2A and MT1X was measured by cell viability assay (Calcein-AM assay). The cells were transfected by siRNA followed by treatment of serial dilutions of ZMC1 for 72 hours. The EC50s were shown on the right. Supplementary Fig. S5. The TOV112D cells with various time of exposure of the serial dilutions of ZMC1 and then the drug-free medium was replaced for the incubation up to 72 hours. Cell viability measurement was performed using Guava ViaCount. Supplementary Fig. S6. In vivo Maximum tolerated dose (MTD) studies and efficacy in subcutaneous tumors from KPC mouse tumor cell line. (A) MTD studies were perfomed by treating the mice with various doses of ZMC1 by IP for 7 days. The survival is shown in Kaplan-Meier curve. (B) In MTD studies, the health, behavior and the body weight was monitored every day. The body weight change is shown. Supplementary Fig. S7. Pharmacodynamic analysis of the ZMC1 in tumors from KPC-172 and KPC-270. The tumors were harvested one hour after mice received the last dose of ZMC1 (5 mg/kg once daily for three days). Immunohistochemistry staining of the tumor tissues was performed using Cleaved caspase-3 (CC3). (A) Representative IHC staining is shown. (B) The quantitation is shown. Supplementary Fig. S8. Sensitivity of TOV112D cells to Zn-1 was evaluated by cell viability assay (MTS assay). The cells were treated with serial dilutions of ZMC1 or Zn-1 for 72 hours. Supplementary Fig. S9. LC-MS/MS analysis of ZMC1 and Zn-1. (A) ZMC1 monomer. (B) Zn-1 (the complex). A peak in MS (right panel) at 532, indicating the intact complex. Supplementary Fig. S10. In vivo Maximum tolerated dose (MTD) studies for Zn-1. (A) MTD studies were perfomed by treating the mice with various doses of Zn-1 by IP for 9 days. The survival is shown in Kaplan-Meier curve. (B) In MTD studies, the health, behavior and the body weight was monitored every day. The body weight change is shown. Supplementary Fig. S11. The gene expression of p53 target genes Puma, Noxa and p21 was increased in p53R172H but not in p53R270H tumor tissues. The mice bearing subcutaneous tumors from cell lines KPC-p53R172H vs. KPC-p53R270H were treated with one dose of ZMC1 by IV for 24 hours. The gene expression was measured by qRT-PCR to the RNA extracted from the tumor tissues. The statistical analysis was performed as unpaired, two-tailed t test with confidence intervals of 95%. **, p value < 0.0001. Supplemental Table S1. The 16 most regulated genes of 37 zinc regulatory genes in TOV112D cells after ZMC1 treatment by RNAseq analysis Supplemental Table S2. Efflux ratio of ZMC1 was measured using the Caco-2 permeability assay.</p>