Additional file 1 of Anti-tumor effects of dual PI3K-HDAC inhibitor CUDC-907 on activation of ROS-IRE1α-JNK-mediated cytotoxic autophagy in esophageal cancer
Creators: name: Jian, Zheng
name: Han, Yichao
name: Zhang, Wentian
name: Li, Chengqiang
name: Guo, Wei
name: Feng, Xijia
name: Li, Bin
name: Li, Hecheng
Publisher: name: figshare
Year: 2022
Subjects: Biochemistry
Medicine
Cell Biology
Molecular Biology
Pharmacology
Chemical Sciences not elsewhere classified
Cancer

description: Additional file 1: Figure S1. a. Clinical characteristics were summarized in Table S1 b. Representative computerized tomography (CT) images from one of patient with esophageal cancer before and after surgery were presented. Figure S2. a. ESCC cell lines were treated with variable concentrations of CUDC‐907 in 96‐well plates in a time-dependent manner. The CCK-8 assay was performed to determine viable cells. b. Apoptosis rates were determined by Annexin V/PI assay after ESCC cells were exposed to CUDC-907 (5 ~ 50 nM) for 24 h. c. Cell cycle profile analysis in ESCC cells exposed to CUDC-907(10 and 30 nM) for 24 h. d. Migration assays were tested by a wound healing assay in ESCC cells (KYSE30 and KYSE150) exposed to CUDC-907(10 and 30 nM) for 24 h. Scale bar: 100 μm. e. Invasion assays were performed with ESCC cells after being exposed to CUDC-907(10 and 30 nM) for 24 h by Transwell chambers. Scale bar: 100 μm. f. statistical analysis of migration assay and invasion assay. g. Immunofluorescence analysis of γ-H2AX foci were performed in in ESCC cells (KYSE30 and KYSE150) exposed to CUDC-907(10 and 30 nM) for 24 h. Nuclei were visualized by using DAPI. Scale bar: 20 μm. h. Levels of proteins related to process of apoptosis, cell cycle, DNA damage, and epithelial–mesenchymal-transition (EMT) were measured via western blot analysis after ESCC cells (KYSE30 and KYSE150) exposed to CUDC-907(10 and 30 nM) for 24 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Error bars are ± SD. *, P &lt; 0.05; **, P &lt; 0.01; ***, P &lt; 0.001. Figure S3. a, b, c. Protein levels associated with PI3K-Akt pathway and HDACs were evaluated via western blot after ESCC cells were exposed to CUDC-907 (10 nM and 30 nM) for 24 h. Tubulin and GAPDH were employed as loading control. Figure S4. a.Formation of autophagosomes showed by cellular fluorescence image. CUDC-907-etreated ESCC cells (KYSE-30 and KYSE-150) obtained punctate profile of GFP-LC3B. b. Autophagy-related protein expressions of Beclin-1, Atg5, Atg7, Atg12, LC3B-I/II, and P62 were determined by western blot analysis. c. Western blot analysis of p-mTOR/mTOR, p-ULK1/ULK1, Beclin1, Atg5, Atg7, and LC3B-I/lC3-II in CUDC-907 (30 nM, 48 h)-treated KYSE-450 and KYSE-510 cells with rapamycin (5 nM) or MHY1485 (10 μM) treatment for 48 h. d. Western blot analysis of p-ULK1/ULK1, Beclin1, and LC3B-I/lC3-II in CUDC-907 (30 nM, 48 h)-treated KYSE-450 and KYSE-510 cells with siULK1 or negative control treatment for 48 h. e. Analysis of cell viability and LDH release were done by CCK-8 in CUDC-907 (30 nM, 48 h)-treated KYSE-450 and KYSE-510 cells with rapamycin or MHY1485 treatment for 48 h. Error bars are ± SD. *, P &lt; 0.05; **, P &lt; 0.01; ***, P &lt; 0.001. Figure S5. a. Heatmap of selective seven upregulated and seven downregulated genes in ESCC cells. b. Intracellular iron levels in KYSE-450 and KYSE-510 after CUDC-907 treatment. c. ChIP-qPCR assay to reveal whether down-expressed LCN2 was regulated by H3 acetylation after CUDC-907 treatment. d. An online prediction website to predict LCN2 potential transcriptional factors ( https://www.grnpedia.org/trrust/ ). e. Protein levels associated with NF-κB pathway were evaluated via western blot after ESCC cells were exposed to CUDC-907 (10 and 30 nM). Error bars are ± SD. *, P &lt; 0.05; **, P &lt; 0.01; ***, P &lt; 0.001. Figure S6.a, c. The expression levels of IRE1α and LCN2 in KYSE-450 and KYSE-510 cells with siLCN2 or siIREα and negative control treatment for 48 h were confirmed by western blot. b. Western blot analysis of LCN2 in KYSE-450 and KYSE-510 cells with OE-LCN2 and negative control treatment for 48 h. d. Western blot analysis of expression of p-PERK/PERK and p-eIF2α/ eIF2α. e. Treatment schema. The vehicle group (N = 6) worked as control group and these mice were fed with 30% Captisol, whereas the treatment group (N = 6), which was 200 mg/kg CUDC-907 group, were fed with 200 mg/kg CUDC-907 dissolving in 30% Captisol. Dose regimen was followed by 5-days-on and 2-days-off via oral gavagea and started on day 10. f. Tumor growth inhibition upon CUDC-907 treatment in KYSE-450 ESCC xenografts. Curve of mice body weight were presented. Additional materials S1 and S2.