FOXO1 knockdown is toxic for Burkitt's lymphoma (BL) cell lines. FOXO1 knockdown is toxic for Burkitt's lymphoma (BL) cell lines

We found that FOXO1 knock-down inhibits cell cycle progression in Burkitt's lymphoma (BL) cell lines BL41 and Namalwa. Overall design: Burkitt's lymphoma (BL) cell lines BL41 and Namalwa were transduced with sh_hFOXO1.H-10381 in pRSI12-U6-sh-UbiC-TagRFP-2A-Puro lentiviral vector (DECIPHER shRNA Library, Human Module I (Cellecta, https://www.cellecta.com/) obtained from BioCat, Heidelberg, Germany). On Day 4 after transduction total RNA was isolated with RNeasy mini kit (QIAGEN). Microarray analyses were performed using 200 ng of total RNA as starting material and 5.5 µg ssDNA per hybridization (GeneChip Fluidics Station 450; Affymetrix, Santa Clara, CA). The total RNAs were amplified and labeled following the Whole Transcript (WT) Sense Target Labeling Assay (http://www.affymetrix.com). Labeled ssDNA was hybridized to Human Gene 1.0 ST Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA). The chips were scanned with a Affymetrix GeneChip Scanner 3000 and subsequent images analyzed using Affymetrix® Expression Console Software (Affymetrix). Probe level data were obtained using the robust multichip average (RMA) normalization algorithm.

本研究发现，FOXO1基因敲低可抑制伯基特淋巴瘤（Burkitt's lymphoma, BL）细胞系BL41与Namalwa的细胞周期进程。

实验设计：将伯基特淋巴瘤（Burkitt's lymphoma, BL）细胞系BL41及Namalwa用携带sh_hFOXO1.H-10381的pRSI12-U6-sh-UbiC-TagRFP-2A-Puro慢病毒载体进行转导；该载体源自DECIPHER短发夹RNA（short hairpin RNA, shRNA）文库人类模块I（Cellecta，https://www.cellecta.com/），购自德国海德堡的BioCat公司。转导后第4天，使用RNeasy迷你试剂盒（QIAGEN）提取总RNA。

以200 ng总RNA为起始材料，每次杂交使用5.5 μg单链DNA（single-stranded DNA, ssDNA），采用GeneChip流体工作站450（Affymetrix，美国加利福尼亚州圣克拉拉）开展微阵列分析。总RNA按照全转录组（Whole Transcript, WT）正义靶标标记检测流程（http://www.affymetrix.com）完成扩增与标记。将标记后的ssDNA与人类基因1.0 ST Affymetrix基因芯片（Affymetrix，美国加利福尼亚州圣克拉拉）进行杂交。使用Affymetrix GeneChip扫描仪3000扫描芯片，并采用Affymetrix® Expression Console软件（Affymetrix）分析扫描得到的图像。最终采用稳健多芯片平均（robust multichip average, RMA）归一化算法获取探针水平的数据。