Additional file 1 of SEPT2 crotonylation promotes metastasis and recurrence in hepatocellular carcinoma and is associated with poor survival
Creators: name: Zhang, Xin-yue
name: Liu, Ze-xian
name: Zhang, Yi-fan
name: Xu, Li-xia
name: Chen, Meng-ke
name: Zhou, Yu-feng
name: Yu, Jun
name: Li, Xiao-xing
name: Zhang, Ning
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Virology

description: Additional file 1: Figure S1. Crotonylome in HCC cells (A)IHC analysis showed the crotonylation level are similar in HBV positive and negative HCC. (B) Migration and invasion assays showing that MHCC-97H cells had higher invasive potential than MHCC-97L cells. The data are presented as the means ± SD. *p &lt; 0.05, **p &lt; 0.01 (Student’s t test). (C-D) Motif analysis of all identified crotonylated sites. (E)Scatter diagram showing differentially expressed proteins in the MHCC-97H and MHCC-97L cell lines (p&lt;0.05). (F) The total intensity of crotonylation was higher in MHCC-97H cell line (p&lt;0.05). Figure S2. Crotonylome in differential invasive HCC cell lines (A-B) KEGG analysis: (A) Reactome analysis (B) differential crotonylated proteins. (C) Heatmap of known metastatic-related proteins in MHCC-97H and MHCC-97L cell lines. Figure S3. WB analysis of total crotonylation of HCC cell lines after NaCr treatment. Figure S4. Crotonylation was positively correlated with HCC cell migration and invasion (A)Cell Morphological changes in SMMC7721 cell line after 25mM of NaCr treatment. (B) Migration and Invasion assays showed SMMC7721 cells had higher invasive potential after 25mM of NaCr treatment. Data presented as mean ± SD. *p &lt; 0.01, **p&lt;0.001. (Student’s t test). (C) Wound heal assays showed SMMC7721 cells had greater migration capacity after 25mM of NaCr treatment. Data presented as mean ± SD. *p &lt; 0.01. (Student’s t test). (D) WB analysis of the changes in the expression of EMT-related proteins after 25mM of NaCr treatment. Figure S5. Lysine 74 crotonylation of SEPT2 was identified (A)Scatter diagram based on the false discovery rate (FDR) of protein expression and crotonylation. (B) SEPT2 K318 is less evolutionarily conserved in seven species compared with K74. K318 of SEPT2 was highlighted in red. (C) Coomassie blue of purified Flag-tagged SEPT2. (D) Dot blotting assay of site-specific antibody of SEPT2-K74 crotonylation (K74Cr). (E)WB analysis of overexpression of Flag-tagged SEPT2-WT and K74R, pan-crotonylation in cells and crotonylation on SEPT2 with or without NaCr treatment. (F) WB analysis of SEPT2 acetylation (Kac) and succinylation (Ksucc) in cells with or without NaCr treatment. Figure S6. SEPT2-K74R Inhibited Cell Migration and Invasion in vitro (A-B) Overexpression of SEPT2-K74R inhibited the ability of cell migration and invasion in SMMC7721 cells with and without 25mM of NaCr treatment. Data presented as mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p&lt;0.0001 (One-way ANOVA). Figure S7. SEPT2-K74R and NaCr Inhibited Cell Oroliferation and Cell Cycle. CCK8 analysis (A), colony formation assay (B) and cell cycle assay (C) of SEPT2-WT and SEPT2-K74R overexpression cells with and without NaCr treatment. Figure S8. SEPT2-K74R Inhibited Cell Migration and Invasion in tail vein injection mode (A)Flowchart of the tail vein injection model. Huh7 stable cells were injected into the tail vein of NCG mice (8 mice each group). Mice were sacrificed 8 weeks after injection. (B) Mice in SEPT2-K74R group had better nutrition. (C) Representative images of liver metastases in tail vein injection model. Figure S9. SEPT2-K74R Inhibited Cell Migration and Invasion in orthotopic liver tumor implantation mouse model (A)Quantitative comparison of tumor burden by calculating liver weight to body weight ratio of each mouse in orthotopic liver tumor implantation mouse model. (B) Quantitative comparison of the number of liver metastases in left lobe. Mice in the SEPT2-K74R group had fewer liver metastasis in orthotopic liver tumor implantation mouse model. (C) Representative images of lung metastases in orthotopic liver tumor implantation mouse model. Data presented as mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p&lt;0.0001 (One-way ANOVA). Figure S10. SEPT2-K74R Inhibited Cell Migration and Invasion in splenic vein injection mouse model (A)Flowchart of the splenic vein injection mouse model. Huh7 stable cells were injected into the tail vein of NCG mice (7 mice each group). Mice were sacrificed 8 weeks after injection. (B) Representative images of liver metastases in the splenic vein injection mouse model. Blue arrows showed the liver metastases. (C) Representative images of liver metastases in the splenic vein injection mouse model. Tissues were stained by HE. (D) Quantitative comparison of tumor burden by calculating liver weight to bod weight ratio of each mouse in the splenic vein injection mouse model. (E) Quantitative comparison of the number of liver metastases in each group. Mice in the SEPT2-K74R group had fewer liver metastasis. Data presented as mean ± SD. *p &lt; 0.05 (One-way ANOVA). Figure S11. SIRT2 decrotonylated SEPT2. (A)SIRT2 decreased crotonylation of SEPT2. Whole-cell lysates were immunoprecipitated with anti-Flag antibody, and precipitated proteins were detected by anti-Flag and anti-pan-kcr antibodies. (B) SIRT2 interacts with SEPT2. Whole-cell lysates were immunoprecipitated with control IgG and anti-Flag antibody, and precipitated proteins were detected by anti-SEPT2 and anti-Flag-SIRT2 antibodies. (C) SIRT2 interacts with SEPT2 in vitro. Purified GST-tagged SIRT2 protein were immunoprecipitated with Purified Flag-tagged SEPT2 and GST antibody-conjugated beads. (D-E) WB analysis of crotonylation of SEPT2. SEPT2 crotonylation was detected with different amount of SIRT2 (D) and SIRT2 interference (E). (F) WB analysis of crotonylation of SEPT2 after AGK2 treatment. (G)CBP and P300 interact with SEPT2 and could crotonylate SEPT2. Figure S12. SEPT2-K74R downregulated P85α (A) qRT-PCR analysis of relative fold change of P85α mRNA expression in NC, SEPT2-WT (WT) and SEPT2-K74R (K74R) groups. Data presented as mean ± SD. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p&lt;0.0001 (One-way ANOVA). (B) SIRT2 overexpression decreased P85α stability. SNU449 cells were treated with CHX and P85α protein was determined by WB (up panel). The down panel showed relative protein level of different groups. Error bars represent ±SD of triplicate experiments. T. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p&lt;0.0001 (One-way ANOVA). NC, negative control. (C) BafA1 increased P85α stability under SEPT2-K74R overexpression. (D) WB analysis of the expression levels of NCAD, ECAD, P85α, Phospho-AKT （S473-AKT）, AKT in mouse tumor tissues. Figure S13. Crotonylation facilitated cell invasive capability in SEPT2-K74Cr -P85α-Akt pathway (A-B) P85α rescued the function of SEPT2 under NaCr treatment. Knock down of P85α inhibited cell migration under NaCr treatment; Overexpression of P85α in SEPT2-K74R rescued the ability of cell migration in Huh7 (A) and SNU449 (B) cells. Figure S14. (A) IHC assays in adherent SNU449 cells. We used IHC staining to examine the K74-cr level in stably overexpressed SEPT2 WT and SEPT2-K74R adherent HCC cells, as positive and negative control respectively. (B) Quantitative comparison of the Pan-Kcr IHC scores in metastasis tissue and primary HCCs in Tissue microarray (HLivH060CD03). Table S1. Distribution of Patients characteristics by survival status. Table S2. Distribution of Patients characteristics by recurrence status. Table S3. Univariate Cox Regression Analysis of Potential Recurrence Factors for HCC Patients.