Additional file 2 of Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis

Additional file 2: Fig. S1 a, Expression levels of different class I HDAC genes in different pediatric small-round-blue-cell tumors, carcinomas and normal tissues by box plot presentation using a comparative study of the amc onco-genomics software tool ( https://hgserver1.amc.nl/cgi-bin/r2/main.cgi ). The number of samples in each cohort is given in brackets. b, Differential expression levels of class I HDAC genes in primary EwS at different tumor sites by box plot presentation using the GSE63157 study set and the amc onco-genomics software tool. The number of samples in each cohort is given in brackets, ND: not determined. p-value 10 mm in diameter were considered positive and sacrificed. Kaplan-Meier plots of individual experiments with six mice per group are shown. Log-rank test was used to test for differences in survival. Fig. S6 a, Proliferation of SK-N-MC and CHLA-10 after treatment with Doxorubicin and/or HDACi (MS-275 or FK228) was analyzed with the xCELLigence system. Cell impedance was measured every 4 hours. Data are shown as mean ± SEM (hexaplicates/group; p-value 0.0001). b, Heatmaps of pathways and ontology terms that are enriched among up- and downregulated genes. The plots are based on gene lists for two EwS cell lines (EW7, SK-N-MC), containing the 300 strongest differentially expressed genes after FK228, Vincristine or combined treatment, compared to solvent controls, respectively. The strongest enriched ontology terms are depicted in the heatmaps. The cells are colored by p-value. Grey cells indicate that a term is not significantly enriched in a gene list. Hence, the heatmap shows common and unique enrichments for the two cell lines. c, Spheroid growth was monitored in Greiner bio-one CELLSTAR® Cell-Repellent Surface 96-well round bottom plates. Left, CHLA-10 or EW7 cells were plated in Matrigel-containing medium and cells were treated for 48 hours with the inhibitors as indicated. Results were compared to solvent controls. Right, primary EwS tumor cells derived from PDX mice. Cell viability was measured with CellTiter Glo® Luminescent assay (quadruplets/group). Fig. S8 a, Western blot analysis of apoptosis susceptibility after FK228 or MS-275 and/or A-395 treatment, respectively. Protein levels measured by antibodies against, PARP, CASP3, and GAPDH as loading control. CHLA-10, EW7 or SK-N-MC cells were treated for 48 hours with inhibitors. b, Left, heat map of 824 genes, 3-fold differentially expressed in different EwS tumor samples (CHLA-10 and SK-N-MC) at the end of treatment, are shown. Right, zoomed in heat map with 132 genes contains only those genes with a p-value 1; in blue, genes with p-value 1; and in black, genes with p-value ≥ 0.05 and absolute log FC ≤ 1. Positive log FCs indicate higher expression of the gene in the treated cell lines. D, GSEA enrichment plots of up- and downregulated gene sets after combined A-395 and FK228 treatment. NES: normalized enrichment score. GSEA: http://www.broadinstitute.org/gsea/index.jsp .

补充文件2：图S1a：采用AMC肿瘤基因组学软件工具（amc onco-genomics software tool，https://hgserver1.amc.nl/cgi-bin/r2/main.cgi）开展比较分析，以箱线图展示不同儿童小圆蓝细胞肿瘤、癌组织及正常组织中各类I类组蛋白去乙酰化酶（class I HDAC）基因的表达水平。各队列的样本量标注于括号内。图S1b：利用GSE63157数据集及AMC肿瘤基因组学软件工具，以箱线图展示不同肿瘤部位原发性尤因肉瘤（EwS）中I类HDAC基因的差异表达水平。各队列的样本量标注于括号内，ND：未检测（not determined）。直径10mm的p值被判定为阳性并处死。每组6只小鼠的单次实验的Kaplan-Meier生存曲线如图所示，采用log-rank检验分析生存差异。图S6a：采用xCELLigence实时细胞分析系统分析SK-N-MC与CHLA-10细胞经多柔比星及/或组蛋白去乙酰化酶抑制剂（HDACi，MS-275或FK228）处理后的增殖情况。每4小时检测一次细胞阻抗，数据以平均值±标准误（SEM）表示（每组六重复；p值=0.0001）。图S6b：上调及下调差异基因富集的通路与基因本体术语的热图。该热图基于两种尤因肉瘤细胞系（EW7、SK-N-MC）的基因列表构建，分别对应FK228、长春新碱或联合处理后与溶剂对照组相比，排名前300的差异表达基因。热图中展示了富集程度最高的基因本体术语，单元格颜色由p值决定，灰色单元格代表某术语在对应基因列表中无显著富集。因此该热图可体现两种细胞系共有的与特有的富集特征。图S6c：在Greiner bio-one CELLSTAR® 细胞排斥表面96孔圆形底板中监测球体生长情况。左侧：将CHLA-10或EW7细胞接种于含基质胶（Matrigel）的培养基中，按指示用抑制剂处理48小时，结果与溶剂对照组进行比较。右侧：从患者来源异种移植小鼠（PDX mice）中分离的原发性尤因肉瘤肿瘤细胞，采用CellTiter Glo® 荧光素酶细胞活力检测试剂盒检测细胞活力（每组四重复）。图S8a：分别检测FK228或MS-275及/或A-395处理后的细胞凋亡易感性的蛋白质免疫印迹分析结果。以抗多聚ADP核糖聚合酶（PARP）、抗半胱氨酸天冬氨酸蛋白酶3（CASP3）抗体及作为内参的抗甘油醛-3-磷酸脱氢酶（GAPDH）抗体检测蛋白水平。CHLA-10、EW7或SK-N-MC细胞经抑制剂处理48小时。图S8b：左侧：展示824个基因的热图，这些基因在治疗结束后的不同尤因肉瘤肿瘤样本（CHLA-10与SK-N-MC）中呈现3倍差异表达。右侧：132个基因的放大热图，仅包含p值为1的基因（蓝色标注），以及p值≥0.05且绝对对数倍数变化（log FC）≤1的基因（黑色标注）。正对数倍数变化代表处理组细胞系中该基因表达水平更高。图S8d：联合使用A-395与FK228处理后，上调与下调基因集的基因集富集分析（GSEA）富集曲线。标准化富集得分（normalized enrichment score，NES）。GSEA：http://www.broadinstitute.org/gsea/index.jsp。