Additional file 1 of Activation of ACLY by SEC63 deploys metabolic reprogramming to facilitate hepatocellular carcinoma metastasis upon endoplasmic reticulum stress
Creators: name: Hu, Chenyu
name: Xin, Zechang
name: Sun, Xiaoyan
name: Hu, Yang
name: Zhang, Chunfeng
name: Yan, Rui
name: Wang, Yuying
name: Lu, Min
name: Huang, Jing
name: Du, Xiaojuan
name: Xing, Baocai
name: Liu, Xiaofeng
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Computational Biology

description: Additional file 1: Figure S1. ACLY is upregulated by SEC63 in response to ER stress in HCC cells. A The heatmaps displaying the differentially expressed ARGs from TCGA-LIHC, ICGC-LIRI-JP, and GSE101728 cohorts. B The expression level of the indicated genes was analyzed in the indicated cohorts. C The overall survival curve of the HCC patients with different ACLY mRNA levels from ICGC was analyzed by Kaplan-Meier analyses. D Triple immunofluorescene (IF) staining for ACLY (red), SEC63 (green), and nuclei (DAPI, blue) was performed in Huh7 and HepG2 cells. Scale bar, 10 μm. E GST pulldown assay with purified fragments as indicated. Figure S2. SEC63 is phosphorylated at T537 after ER stress. A Western blot analysis of SEC63 in cell lysates of Huh7 cells treated as indicated. B Relative SEC63 expression levels in HepG2 and Huh7 cells were determined by immunofluorescence assay using Leica LAS X confocal software (n = 100). C The predication analysis of SEC63 localization by COMPARTMENTS. D Cell fraction was performed and western blot was employed for analyzing the indicated protein. E Dot blot analysis was performed for validating T537-p antibody. F Cell lysates from Huh7 cells were subjected to Co-IP using the indicated antibody. G Cell fraction was performed and the indicated proteins were determined by western blot. Figure S3. SEC63 increases the stability of ACLY. A Total RNA extracted from the Huh7 cells treated with TM (5 μg/mL) or TG (1 μM) were subjected to RT-qPCR as indicated. B HepG2 and Huh7 cells were treated with TM (5 μg/mL) or TG (1 μM) and immunofluorescence was performed. Scale bar, 10 μm. Figure S4. SEC63 is associated with prognosis of HCC patients. A The mRNA level of SEC63 was analyzed as indicated. B The overall survival curve of the HCC patients with different SEC63 mRNA levels from ICGC database was analyzed by Kaplan-Meier analyses. C The overall survival and disease-free survival curves of HCC patients were plotted against the SEC63 and/or ACLY mRNA levels based on the prognosis data of TCGA-LIHC database using the Kaplan-Meier method. The difference between the survival curves was analyzed using the log-rank test. D Overall survival curve of HCC patients was plotted against the SEC63 and/or ACLY mRNA levels based on the prognosis data of ICGC database using the Kaplan-Meier method. The difference between the survival curves was analyzed using the log-rank test. Figure S5. SEC63 promotes HCC cell metastasis relying on ACLY. A HepG2 or Huh7 stable cell lines with overexpression of SEC63 were generated. Western blot was performed for analyzing the expression level of SEC63. B Huh7 or PLC/PRF/5 stable cell lines with knockdown of SEC63 were generated. The knockdown effect was determined by western blot. C-F The cells were treated as indicated and cell proliferation was assessed by MTT assay. G Migration assay or invasion assay was performed as indicated. Migration or invasion rate was further quantified (n = 3). H Wound healing assay was performed as indicated. Wound closure rate was quantified (n = 3). I Western blot analysis of the indicated proteins in 12 individual paired HCC tissues used in Figure 4I. Figure S6. SEC63 modulates ACLY-related metabolic and epigenetic reprogramming. A The cellular acetyl-CoA level was evaluated in the indicated cells (n = 4). B The cellular free fatty acids, cholesterol level, and triglyceride levels were evaluated in the indicated cells (n = 4). C The potential interacting proteins of SEC63 related to ER stress were identified by liquid chromatography-tandem mass spectrometry. D Huh7 cells were transfected as indicated. Then, total RNA was extracted and RT-qPCR was performed using the SNAI1 primers (n = 3). E The ctrl cells or ACLY-depleted cells were treated with WM1119. Western blot was performed for the indicated proteins. F The correlation analyses were analyzed as indicated using TCGA HCC dataset. G The overall survival or disease-free survival curves were analyzed as indicated using TCGA HCC dataset. H The overall survival curve was plotted as indicated using ICGC dataset. Table S1. Correlation of SEC63/ACLY expression with clinicopathologic status in 139 cases of patients with HCC. Table S2. Reagents and antibodies. Table S3. shRNA sequence. Table S4. Primer used for RT-qPCR.