Additional file 1 of Aerobic glycolysis enhances HBx-initiated hepatocellular carcinogenesis via NF-κBp65/HK2 signalling
Creators: name: Chen, Lingjun
name: Lin, Xianyi
name: Lei, Yiming
name: Xu, Xuan
name: Zhou, Qi
name: Chen, Yan
name: Liu, Huiling
name: Jiang, Jie
name: Yang, Yidong
name: Zheng, Fengping
name: Wu, Bin
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Mental Health
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Figure S1. NF-κB family members in HCC. a-e The overall survival rates of NF-κBp65, RelB, cRel, NF-κB1 and NF-κB2 in HCC patients were analysed using the Gene Expression Profiling Interactive Analysis (GEPIA) online database. f The expression of NF-κBp65 mRNA in HBV-related HCC and normal liver tissues was analysed by real-time PCR (n=10 per group). Values are the mean ± SD. *P &lt; 0.05 using Student’s t test. g, h The NF-κBp65 and p-p65 indices of 10 HBV-related HCC samples and 10 normal liver tissue samples were quantified by ImageJ software. Values are the mean ± SD. *P&lt; 0.05, **P &lt; 0.01 using Student’s t test. Figure S2. NF-κBp65 was associated with the poor prognosis in HCC patients. a Representative images of NF-κBp65 staining in HCC and paracancerous tissue microarrays. The expression level was evaluated according to the immunoreactivity score. b, c The maximal tumoursize (cm) in different NF-κBp65 expression level of HCC and paracanceroustissues in non-HBV-related HCC and HBV-related HCC. Values are the mean ± SD. P&lt; 0.05 by Student’s t test. d, e Survival analysis in non-HBV-related HCC and HBV-related HCC with different NF-κBp65 expression. P &lt; 0.05 by Kaplan-Meiersurvival analysis. Figure S3. HBx induced NF-κBp65 expression and phosphorylation in vitro and in vivo. a Quantification of HBx, NF-κBp65 and p-p65 IHC staining of liver tissues from WT and HBx-TG mice at 6 months and 18 months. Values are the mean ± SD (n=6 for each group). *P &lt; 0.05 using Student’s t test. b, c Quantification of NF-κBp65 and p-p65 protein in LO2 and HepG2 cells transfected with vector, pHBV 1.3 or pHBV 1.3 x-null. Values are the mean ± SD. *P &lt; 0.05 compared with the vector group, #P&lt; 0.05 compared with the HBV-transfected group using one-way ANOVA. d Quantification of HBx and NF-κBp65 in LO2, HepG2, HepG2.2.15 and Hep3B cell lines. Values are the mean ± SD. *P &lt; 0.05 compared with LO2 cells, #P &lt;0.05 compared with HepG2 cells using one-way ANOVA. e Quantification of HBx, NF-κBp65 and p-p65 protein in LO2 and HepG2 cells transfected with the HA-HBx or vector plasmid. f Quantification of NF-κBp65 and HBx protein in HepG2.2.15 and Hep3B cells transfected with the flag-p65 or vector plasmid. g By incubation with HBV-infected patient serum, HepG2-NTCP cells were infected with HBV virions, and the level of HBV DNA in the cell supernatant was tested. The control group was incubated with healthy volunteers’ serum. **P &lt; 0.01 using Student’s t test. h, i Western blotting analysis and quantification of NF-κBp65 and p-p65 protein in HepG2-NTCP cells infected with HBV virions. Values are the mean ± SD (n=3 for each group). *P &lt; 0.05 using Student’s t test. j Quantification of the NF-κBp65 nuclear translocation index in LO2 and HepG2 cells stably transfected with the HA-HBx lentivirus and vector lentivirus. k Quantification of HBx, NF-κBp65 and p-p65 protein in the cytoplasm and nucleus in LO2 and HepG2 cells stably transfected with the HA-HBx lentivirus and vector lentivirus. Values are the mean ± SD. *P &lt; 0.05 using Student’s t test. l Flag-p65 and HA-HBx plasmids were transfected into HepG2 cells. Co-IP was used to detect the interaction between HBx and NF-p65 in HepG2 cells. Figure S4. HBx enhanced aerobic glycolysis in hepatocellular carcinogenesis. a The lactate content was measured in human normal liver tissues and HBV-related HCC tissues. n=6 per group. Values are the mean ± SD. *P &lt; 0.05 using Student’s t test. b, c Quantification of HBx, GLUT1, HK2 and LDHA protein in LO2 and HepG2 cells transfected with HA-HBx or vector plasmid. Values are the mean ± SD. *P &lt; 0.05 using Student’s t test. Figure S5. Inhibitionof glycolysis restrained HBx-induced proliferation. LO2 and HepG2 cells with or without stable expression of HA-HBx were treated with 2-DG (10 mM) for 24 h. a, c PCNA mRNA in different groups was detected by real-time PCR. b, d Western blot analysis of HBx and PCNA protein expression in cells and quantification of the relative PCNA protein expression. The experiment was repeated three times. All values are the mean ± SD. One-way ANOVA was used. *P &lt; 0.05 compared with the vector group. #P &lt; 0.05 compared with the HBx group. Figure S6. HBx reprogramed glycolytic metabolism via NF-κBp65/HK2 signalling in hepatocellular carcinogenesis. a, b Quantification of NF-κBp65-Flag, GLUT1, HK2 and LDHA protein expression in HepG2.2.15 and Hep3B cells transiently transfected with NF-κBp65 plasmids. c, d Quantification of NF-κBp65-Flag, GLUT1, HK2, LDHA and PCNA protein expression in HepG2.2.15 and Hep3B cellstransfected with HK2 siRNA or combined with the NF-κBp65 plasmid. *P &lt; 0.05 by one-way ANOVA. e, f  HK2 mRNA levels in HepG2.2.15 and Hep3B cells transiently transfected with NF-κBp65 plasmids or NF-κBp65 siRNA. All values are the mean ± SD. *P &lt; 0.05 by Student's t test. Table S1. Clinicopathological features in 10 HBV-related HCC cases and 10 liver haemangioma cases. Table S2. Clinicopathological features in 31 non-HBV-related HCC cases and 82 HBV-related HCC cases in tissue microarrays. Table S3. Primers of genes for quantitative PCR. Table S4. Primers of genes for ChIP‒qPCR.