Additional file 1 of HIGD2A silencing impairs hepatocellular carcinoma growth via inhibiting mitochondrial function and the MAPK/ERK pathway
Creators: name: Huang, Kuiyuan
name: Liu, Ziying
name: Xie, Zhanglian
name: Li, Xiaoran
name: Zhang, Haixing
name: Chen, Yu
name: Wang, Yiran
name: Lin, Zimo
name: Li, Chuanjiang
name: Liu, Hongyan
name: Zhang, Xiaoyong
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Table S1. The shRNA sequences of HIGD2A and the primer sequences used in real-time PCR analysis. Figure S1. Diagnostic and prognostic value of HIGD2A expression and its correlation with clinical features. A Representative immunohistochemistry images of HIGD2A expression in normal liver tissues and liver cancer tissues from the HPA. B The protein level of HIGD2A in normal hepatocyte L02 cell line and different liver cancer cell lines. C–F HIGD2A expression in different status of T stage (C), pathologic stage (D), vascular invasion (E) and OS event (F). G–J Kaplan–Meier plots of OS for HIGD2A expression levels in subgroups including T stage: T1 (G), N stage: N0 (H), M stage: M0 (I) and pathologic stage: stage I (J). K Nomogram for OS prediction, with T stage, N stage, M stage, histologic grade and expression of HIGD2A applied as parameters. L Calibration curves of the nomogram for 1-, 3-, 5-year survival prediction. *P &lt; 0.05, **P &lt; 0.01. Figure S2. Knockdown of HIGD2A impedes MHCC97H cells proliferation and migration. A Western blot assay for total HIGD2A protein expression in HIGD2A-knockdown MHCC97H cells. B Left, colony formation of MHCC97H cells transfected shCtrl or shHIGD2A.1. Right, quantification of colony formation based on three independent assays. C The proliferation ability of MHCC97H cells infected with shCtrl lentivirus or shHIGD2A.1 lentivirus was measured by CCK8 assay at the indicated time points. D, E Flow cytometry analysis of Annexin V/7-AAD double stained HepG2, Huh7 and MHCC97H cells transfected shCtrl or shHIGD2A.1. Representative flow cytometric plot (D) and the proportion of apoptosis cells (E). F Annexin V/7-AAD apoptosis assay of L02 cells after HIGD2A gene silencing. Left, representative flow cytometric plot. Right, the proportion of apoptosis cells. G Left, cell migration ability of MHCC97H cells transfected shCtrl or shHIGD2A.1 was analyzed by Transwell assay. Right, quantitative data of migrated cells. Results shown are mean ± SEM. an unpaired t-test was used. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ns, not significant. Scale bar, 100 µm. Figure S3. HIGD2A knockdown inhibited the proliferation and migration of HCC cells in vitro. A, B Colony formation experiments for the effect of HIGD2A knockdown with shRNA on the proliferation of HepG2 and Huh7 cells. C, D The effect of HIGD2A knockdown on the growth of HepG2 and Huh7 cells was detected by CCK8 assays. E The effect of HIGD2A knockdown on cell apoptosis was detected by western blot. F, G Transwell chamber was used to evaluated the effect of HIGD2A knockdown on the migration of HepG2 and Huh7 cells. Results shown are mean ± SEM. an unpaired t-test was used. *P &lt; 0.05, **P &lt; 0.01 and ***P &lt; 0.001. Scale bar, 100 µm. Figure S4. Depletion of HIGD2A induces mitochondrial stress in MHCC97H. A, B Intercellular ATP level in MHCC97H (A) and L02 (B) cells transfected with shCtrl or shHIGD2A.1. C Immunofluorescent (IF) images showing mitochondria morphology in MHCC97H transfected with shCtrl or shHIGD2A.1. Left, representative IF images (magenta, MitoTracker; yellow, DAPI) and 3D mitochondria reconstruction of shCtrl- and shHIGD2A-infected MHCC97H cell. Scale bar, 5 μm; Sphericity heat map, 0.326–0.915. Right, sphericity analysis of 3D reconstructed mitochondria (results are presented as mean ± SD). D Left, oxygen consumption rate (OCR) in MHCC97H transfected with shCtrl or shHIGD2A.1 was measured by seahorse analyzer. Right, basal respiration rate and maximal respiration capacity are shown. E Comparison of Basal Respiration among L02 and HCC cells. Results shown are mean ± SEM. A Mann–Whitney test was used. *P &lt; 0.05, **P &lt; 0.01 and ***P &lt; 0.001; ns, not significant.