Additional file 1 of HGF-mediated elevation of ETV1 facilitates hepatocellular carcinoma metastasis through upregulating PTK2 and c-MET

Additional file 1: Supplementary materials. FigureS1. (A) ETV1expression in LIHC and correlation of ETV1 expression with overallsurvival were analyzed in LIHC according to the data of The Cancer Genome Atlas(TCGA). (B) CellCounting Kit-8 (CCK8) assay assessing the cell proliferation of theETV1-overexpressing PLC/PRF/5 cells and ETV1-knockdown MHCC97H cells. (C) Colony formation assay showing the proliferationof the indicated HCC cells. Therepresentative photos were shown and the cell numbers were quantified. (D-F) Tumorgrowth of the indicated HCC cells was assessed by subcutaneous xenograft tumormodels. The tumor volume and weight were shown in (D) and (E), the representative images of Ki67 were shownin (F). n = 5 in each group. (G) The correlation between ETV1 expression and PTK2 or MET expression inTCGA-LIHC and GEO database. *p < 0.05, ****p < 0.0001. Data were shown as Mean ± SD. Figure S2. ETV1 binding sites withinthe promoter regions of PTK2. Thesequences highlighted in yellow represent the three binding sites of ETV1 onthe PTK2 promoter, and the arrow represents the transcription initiation sites.The   mutagenesis of the promoter sequencewere annotated. Figure S3. ETV1binding sites within the promoter regions of MET. The sequenceshighlighted in yellow represent the four binding sites of ETV1 onthe MET promoter, and the arrow represents the transcription initiation sites.The mutagenesis of the promoter sequence were annotated. Figure S4. (A-C) Western blotverifying PTK2 and MET knockdown effect in PLC/PRF/5-ETV1 cells and ELK1knockdown effect in PLC/PRF/5 cells. FigureS5. (A) The expression levels of MTDH, RHOA, TCF4 and MCL1 were determined in the indicated cells by real-time PCR. (B) Westernblotting assays of MTDH, RHOA, TCF4 and MCL1 in the indicated cells transfected with lentivirus. (C) The migratingand invasive capability of the indicated cells was determined via transwellassay. Figure S6. (A) The level of ETV1 in the PLC/PRF/5 cells upon HGFtreatment with/without ERK1/2 knockdown. Figure S7. Transcription factors binding siteswithin the promoter regions of ETV1. The sequences highlighted in blue represent the four binding sites ofELK1 on the ETV1 promoter. The yellow highlighted sequences representthe one binding site of ETS1 onthe ETV1 promoter. The sequenceshighlighted in grey represent the binding site of SP1 on the ETV1promoter. The red highlighted sequences represent the binding site of NF-ΚB1 onthe ETV1 promoter. The pink highlighted sequences represent the bindingsequence of STAT3 on the ETV1 promoter. The arrows representtranscription start sites. The mutagenesis of the promoter sequence wereannotated. Figure S8. (A)Representative IHC staining of ETV1 is shown. (B) Pearsoncorrelation analyses between ETV1 IHC score and the levels of serum HGF in HCCpatients. n=30. (C) The correlation between ETV1 expression and HGF expression inTCGA-LIHC and GEO database.(D) The correlation between ETV1expression and ELK1 expression in TCGA-LIHC and GEO database. Figure S9. Effectof ERK1/2 inhibitor on HGF-mediated HCC cell migration and invasion. (A)Transwell assays displayed the migratory and invasive capacity of theindicated cells upon HGF treatment. (B) Transwell assays displayed themigratory and invasive capacity of the indicated cells. Supplementary Table S1. List of genesdifferentially expressed in PLC/PRF/5-ETV1 versus PLC/PRF/5-Control cells usinga human liver cancer PCR array. Supplementary Table S2. List of genes differentially expressed in MHCC97H-shETV1 versusMHCC97H-shControl cells using a human liver cancer PCR array. Supplementary Table S3. Primer sequences usedin the study. Supplementary Table S4. Knockdown shRNA sequencesused in this study. Supplementary Table S5. Correlation between PTK2 expression and clinicopathologicalcharacteristics of HCCs in two independent cohorts of human HCC tissues. Supplementary Table S6. Correlationbetween c-MET expression and clinicopathological characteristics of HCCs in twoindependent cohorts of human HCC tissues

DataSheet_1_Identification of necroptosis-related subtypes, development of a novel signature, and characterization of immune infiltration in colorectal cancer.docx

IntroductionNecroptosis, a type of programmed cell death, has recently been extensively studied as an important pathway regulating tumor development, metastasis, and immunity. However, the expression patterns of necroptosis-related genes (NRGs) in colorectal cancer (CRC) and their potential roles in the tumor microenvironment (TME) have not been elucidated.MethodsWe explored the expression patterns of NRGs in 1247 colorectal cancer samples from genetics and transcriptional perspective. Based on a consensus clustering algorithm, we identified NRG molecular subtypes and gene subtypes, respectively. Furthermore, we constructed a necroptosis-related signature for predicting overall survival time and verified the predictive ability of the model. Using the ESTIMATE, CIBERSORT, and ssGSEA algorithms, we assessed the association between the above subtypes, scores and immune infiltration. ResultsMost NRGs were differentially expressed between CRC tissues and normal tissues. We found that distinct subtypes exhibited different NRGs expression, patients’ prognosis, immune checkpoint gene expression, and immune infiltration characteristics. The scores calculated from the necroptosis-related signature can be used to classify patients into high-risk and low-risk groups, with the high-risk group corresponding to reduced immune cell infiltration and immune function, and a greater risk of immune dysfunction and immune escape. DiscussionOur comprehensive analysis of NRGs in CRC demonstrated their potential role in clinicopathological features, prognosis, and immune infiltration in the TME. These findings help us deepen our understanding of NRGs and the tumor microenvironment landscape, and lay a foundation for effectively assessing patient outcomes and promoting more effective immunotherapy.</p