Additional file 1 of The novel SMYD3 inhibitor EM127 impairs DNA repair response to chemotherapy-induced DNA damage and reverses cancer chemoresistance
Creators: name: Sanese, Paola
name: De Marco, Katia
name: Lepore Signorile, Martina
name: La Rocca, Francesca
name: Forte, Giovanna
name: Latrofa, Marialaura
name: Fasano, Candida
name: Disciglio, Vittoria
name: Di Nicola, Elisabetta
name: Pantaleo, Antonino
name: Bianco, Giusy
name: Spilotro, Vito
name: Ferroni, Claudia
name: Tubertini, Matilde
name: Labarile, Nicoletta
name: De Marinis, Lucia
name: Armentano, Raffaele
name: Gigante, Gianluigi
name: Lantone, Valerio
name: Lantone, Giuliano
name: Naldi, Marina
name: Bartolini, Manuela
name: Varchi, Greta
name: Del Rio, Alberto
name: Grossi, Valentina
name: Simone, Cristiano
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Supplementary Material 1: Supplementary Figure 1. (A) Immunoblot analysis of PARP and cleaved PARP by using two different antibodies in HCT116 cells treated with BCI-121 (100 μM) or EM127 (5 μM) and/or doxorubicin (1 μM) as described in Figure 1A. ACTIN was used as a loading control (left panel). Ratio of cleaved PARP to total PARP determined by measuring the optical density of the immunoblot bands (right panel). (B) Flow cytometry analysis of Annexin V staining in HCT116 cells treated with different doses (10, 30 μM) of irinotecan for different times (24, 48, 72 h) compared with cells pre-treated with EM127 (5 μM) for 48 h and then treated with irinotecan (10 μM) for 24 h in the presence of EM127. (C) Bliss synergy surface analysis obtained with Combenefit software of HCT116 cells treated with different concentrations of EM127 (0, 1, 2.5, 5 μM) and doxorubicin (0, 5, 10, 20 μM). (D) Quantification of apoptotic cell death by Annexin V staining in HT29, SW480, and CaCO2 cells treated as described in Figure 4A. (E, F) Flow cytometry analysis of Annexin V staining in MDA-MB-231, HCC70, and MCF7 BC cell lines (E) and AGS and NCI-N87 GC cell lines (F) pre-treated or not with EM127 (5 μM) for 48 h and then treated or not with 5-fluorouracil (10 μM) or paclitaxel (100 nM) (BC cell lines) or with paclitaxel (10 nM) or oxaliplatin (10 μM) (GC cell lines) for another 24 h in the presence of EM127. *p&lt;0.05 treated vs. untreated. #p&lt;0.05 combined treatment vs. single treatments. cl.PARP = cleaved PARP; DMSO = dimethyl sulfoxide; DOXO = doxorubicin; 5-FU = 5-fluorouracil; IRINO = irinotecan; OXA = oxaliplatin; PTX = paclitaxel. Supplementary Figure 2. (A) Immunoblot analysis of the phosphorylation levels of CHK2 (at T68) and p53 (at S15) in CaCO2 and HT29 cells pre-treated or not with EM127 (5 μM) for 24 h and then exposed or not to irinotecan (10 μM) for 6 h in the presence of EM127. VINCULIN was used as a loading control. (B) Densitometric analysis of the phosphorylation levels of CHK2 (at T68) and p53 (at S15) measured by immunoblot in HCT116 cells pre-treated or not with EM127 (5, 2.5 μM) and/or KU60019 (5, 2.5 μM) for 24 h and then exposed or not to irinotecan (10 μM) for 6 h in the presence of the inhibitors. (C) Quantification of cell proliferation by CellTiter 96 Aqueous Assay in HCT116 cells pre-treated or not with EM127 (5 μM) and/or KU60019 (5 μM) for 48 h and then treated or not with irinotecan for 24 h in the presence of the inhibitors. (D) Immunostaining for 53BP1 (red) in HCT116 cells pre-treated or not with EM127 (5 μM) for 2.5 h and then exposed or not to irinotecan (10 μM) and fixed at the indicated time points. Nuclei were counterstained with DAPI (blue). (E) Flow cytometry analysis of total DNA damage in HCT116 cells pre-treated or not with EM127 (5 μM) for 12 h and then exposed or not to irinotecan (10 μM) for 6 h. Cells were stained for pATM (S1981) and γH2AX (S139) to calculate the percentage of cells with DNA damage. (F) Immunostaining for MRE11, RPA32 and RAD51 (green) in HCT116 cells pre-treated or not with EM127 (5 μM) for 2.5 h and then exposed or not to irinotecan (10 μM) for 6 h. Nuclei were counterstained with DAPI (blue).*p&lt;0.05 treated vs. untreated. #p&lt;0.05 EM127 and KU60019 combined treatment vs. single treatments. DMSO = dimethyl sulfoxide; IRINO = irinotecan. Supplementary Figure 3. Stability studies determined by DLS analysis. (A) Particle size changes of EM127@HSA nanoparticles in H2O pH 7.4 (blue line), in PBS pH 7.4 (green line), FBS 10% in PBS pH 7.4, v/v (red line); (B) Polydispersity index changes of EM127@HSA nanoparticles in H2O pH 7.4 (blue dotted line), in PBS pH 7.4 (green dotted line), FBS 10% in PBS pH 7.4, v/v (red dotted line).