Additional file 1 of ANGPTL4 accelerates ovarian serous cystadenocarcinoma carcinogenesis and angiogenesis in the tumor microenvironment by activating the JAK2/STAT3 pathway and interacting with ESM1

Additional file 1:  Fig. S1. Prognostic significance of ARGs in OC patients. A Prognostic models were constructed by LASSO regression based on ARGs. B Lambda on abscissa, coefficients on ordinate. C The survival times, risk score, and signature expression in OC patients. D Overall survival analysis for OC patients with high or low risk. E ROC curves for survival analysis. F Correlation analysis between the risk score and immune cell infiltration. *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S2. Hub prognostic ARGs in OC.  Uni_cox (A) and Mult_cox (B) for 13 signatures in OC. Overall survival significance confirmed by the nomogram (C) and calibration curve (D). Drug sensitivity analysis for EGF, ANGPTL4, RUNX1, PLG, and NOTCH4 in the CTRP database (E) and GDSC database (F). PCR analysis (G) for the level of ANGPTL4 and ESM1 in SKOV3 treated with different doses of bevacizumab *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S3. The expression of ANGPTL4 in pan-carcinoma based TCGA database and GTEx database. The blue is for normal tissue samples and the red is for cancer samples.  Fig. S4. The expression of downstream genes in HeyA8 cells after ANGPTL4 overexpression.  A  ANGPTL4 expression confirmed in Hey-A8 cells by IF staining.  B  Volcano plot for DEG expression after ANGPTL4 overexpression by RNA sequencing. C  Heatmaps for DEG expression. D GSVA analysis and E  KEGG analysis for 14 DEGs.  Fig. S5. The level of free ANGPTL4 in CM. Free ANGPTL4 expression confirmed in the CM of SKOV3 and Hey-A8 cells by ELISA. *P < 0.05.  Fig. S6. ESM1 was a key factor in the downstream of JAK-STAT pathway.  A  The expression of ESM1 in SKOV3-DMSO, SKOV3-Colivelin, Hey-A8-DMSO, and Hey-A8-AG490 groups. B  The effect of ESM1 on the angiogenesis ability of OC induced by JAK inhibitor/activator. C  The effect of ANGPTL4 on ESM1 expression. D  Co-IP showed the effects of JAK activator Colivelin on the interaction between ANGPTL4 and ESM1 in SKOV3 cells. Fig. S7. The ANGPTL4/ESM1 axis promotes OC growth and angiogenesis in vivo.  A  Morphological observation, HE and IHC staining of xenografts in the vector-SKOV3, ANGPTL4 KD-SKOV3, ANGPTL4 KD-SKOV3 with ESM1 OE, vector-HeyA8, ANGPTL4 OE-HeyA8, and ANGPTL4 OE-HeyA8 with ESM1 OE groups. B  The tumor weight and volume and the IHC staining score of xenografts in the vector-SKOV3, ANGPTL4 KD-SKOV3, ANGPTL4 KD-SKOV3 with ESM1 OE, vector-HeyA8, ANGPTL4 OE-HeyA8, and ANGPTL4 OE-HeyA8 with ESM1 OE groups. *P < 0.05.

附加文件1：图S1. 卵巢癌（Ovarian Cancer, OC）患者中抗原相关基因（Antigen-Related Genes, ARGs）的预后价值。A：基于抗原相关基因构建套索回归（LASSO regression）预后模型。B：横坐标为Lambda值，纵坐标为回归系数。C：卵巢癌患者的生存时间、风险评分及特征基因表达情况。D：高、低风险组卵巢癌患者的总生存分析。E：生存分析的受试者工作特征（Receiver Operating Characteristic, ROC）曲线。F：风险评分与免疫细胞浸润的相关性分析。*P < 0.05，**P < 0.01，***P < 0.001。

图S2. 卵巢癌核心预后抗原相关基因。A为13个特征的单因素Cox（Uni_cox）回归分析，B为多因素Cox（Mult_cox）回归分析。通过列线图（nomogram）与校准曲线（calibration curve）验证总生存预后价值。在CTRP数据库（E）与GDSC数据库（F）中对EGF、ANGPTL4、RUNX1、PLG及NOTCH4进行药物敏感性分析。采用聚合酶链式反应（Polymerase Chain Reaction, PCR）分析（G）不同剂量贝伐珠单抗处理后的SKOV3细胞中ANGPTL4与ESM1的表达水平*P < 0.05，**P < 0.01，***P < 0.001。

图S3. 基于TCGA数据库与GTEx数据库的泛癌中ANGPTL4的表达情况。蓝色代表正常组织样本，红色代表肿瘤组织样本。

图S4. ANGPTL4过表达后HeyA8细胞中下游基因的表达情况。A：采用免疫荧光（Immunofluorescence, IF）染色验证Hey-A8细胞中ANGPTL4的过表达效果。B：RNA测序得到的ANGPTL4过表达后差异表达基因（Differentially Expressed Genes, DEGs）的火山图。C：差异表达基因的热图。D：对14个差异表达基因进行基因集变异分析（Gene Set Variation Analysis, GSVA），E：对其进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析。

图S5. 细胞上清液（Conditioned Medium, CM）中游离ANGPTL4的水平。采用酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）验证SKOV3与Hey-A8细胞的细胞上清液中游离ANGPTL4的表达情况*P < 0.05。

图S6. ESM1是JAK-STAT通路下游的关键调控因子。A：SKOV3-二甲基亚砜（Dimethyl Sulfoxide, DMSO）、SKOV3-Colivelin、Hey-A8-二甲基亚砜（DMSO）及Hey-A8-AG490组中ESM1的表达水平。B：ESM1对JAK抑制剂/激活剂诱导的卵巢癌血管生成能力的影响。C：ANGPTL4对ESM1表达的调控作用。D：免疫共沉淀（Co-Immunoprecipitation, Co-IP）实验验证JAK激活剂Colivelin对SKOV3细胞中ANGPTL4与ESM1相互作用的影响。

图S7. ANGPTL4/ESM1轴在体内促进卵巢癌的生长与血管生成。A：对空载对照（vector）-SKOV3、ANGPTL4敲低（KD）-SKOV3、ANGPTL4敲低-SKOV3联合ESM1过表达（OE）、空载对照-HeyA8、ANGPTL4过表达-HeyA8及ANGPTL4过表达-HeyA8联合ESM1过表达组的异种移植瘤进行形态学观察、苏木精-伊红（Hematoxylin-Eosin, HE）染色及免疫组化（Immunohistochemistry, IHC）染色。B：上述各组异种移植瘤的瘤重、体积及免疫组化染色评分*P < 0.05。