Supplemental Figures 1-15 from Induction of Thioredoxin-Interacting Protein by a Histone Deacetylase Inhibitor, Entinostat, Is Associated with DNA Damage and Apoptosis in Esophageal Adenocarcinoma
Authors: Feingold Paul L. , Surman Deborah R. , Brown Kate , Xu Yuan , McDuffie Lucas A. , Shukla Vivek , Reardon Emily S. , Crooks Daniel R. , Trepel Jane B. , Lee Sunmin , Lee Min-Jung , Gao Shaojian , Xi Sichuan , McLoughlin Kaitlin C. , Diggs Laurence P. , Beer David G. , Nancarrow Derek J. , Neckers Leonard M. , Davis Jeremy L. , Hoang Chuong D. , Hernandez Jonathan M. , Schrump David S. , Ripley R. Taylor 
Published: April 2023

<p>Supplemental Figure 1: Chromatin immunoprecipitation was performed to assess the histone modification marks, H3K27Me3, H3K9Ac2, H3K4Me3, and H3K79Me2 in Flo-1 and Esc2 after glutamine withdrawal; Supplemental Figure 2: qRT-PCR normalized to b-actin measured TXNIP mRNA expression after Flo-1, Esc2, OE33, and CP-C were treated for 72 hours with L-Glutamic acid γ-(p-nitroanilide) hydrochloride (GPNA) and grown in 96-well plates; Supplemental Figure 3: Realtime Seahorse extracellular flux analysis of extracellular acidification rate (ECAR) measures glycolytic activity based on sequential glucose, oligomycin (oligo), and 2-DG injections at the time points indicated; Supplemental Figure 4: YSI analyzer measured glucose uptake over a 5-day period in Flo-1 and Esc2 with overexpression of TXNIP; Supplemental Figure 5: qRT-PCR normalized to b-actin measured mRNA levels of GLUT1, GLUT4, HK2, and LDH-A in Flo-1 and Esc2 overexpressing TXNIP versus controls; Supplemental Figure 6: Oxidative consumption ratio (OCR) measured mitochondrial function with sequential injections of oligomycin (oligo), FCCP, and rotenone/antimycin a (R/A) at the time points indicated in Flo-1, Esc2, and OE33 overexpressing TXNIP versus controls; Supplemental Figure 7: Oxidative consumption ratio (OCR) measured mitochondrial function with sequential injections of oligomycin (oligo), FCCP, and rotenone/antimycin a (R/A) at the time points indicated in Esc2 cells with lentiviral shRNA knockdown of TXNIP; Supplemental Figure 8: Proliferation was measured using Cyquant DNA quantification kits over 5 days in Esc2 cells with lentiviral shRNA knockdown of TXNIP versus controls; Supplemental Figure 9: Esc2 cells overexpressing TXNIP versus controls were grown in 96-well plates with or without N-acetyl-cysteine (NAC); Supplemental Figure 10: Esc2 cells overexpressing TXNIP were grown in 96-well plates at normoxic conditions (20%) or hypoxic conditions (5%); Supplemental Figure 11: Colonies of Flo-1 overexpressing TXNIP versus controls were grown at normoxic conditions (20%) or hypoxic conditions (5%) in soft agar and were stained with crystal violet, photographed, and counted; Supplemental Figure 12: YSI analyzer measured lactate secretion at 24 and 48 hours in Flo-1 overexpressing TXNIP; Supplemental Figure 13: In Flo-1 and Esc2 cells treated with cisplatin, entinostat, or the combination, quantification of Annexin V apoptosis assay based on stage of apoptosis; Supplemental Figure 14: Representative pictures from Flow Cytometry results of Esc2 cells with lentiviral shRNA knockdown of TXNIP versus controls after treatment with cisplatin, entinostat, or the combination; Supplemental Figure 15: Chromatin immunoprecipitation was performed to assess the histone modification mark, H3K9Ac2, in Flo-1 after treatment with entinostat, cisplatin, or the combination of both.</p>