Chronic Active B Cell Receptor Signaling in Diffuse Large B Cell Lymphoma. Homo sapiens

To determine gene expression of DLBCL upon NF-kB inhibition or knockdown of BCR signaling components. Overall design: Total RNA (Trizol reagent; Invitrogen, Carlsbad, CA) was prepared from 8 HBL-1 samples following incubation with 25 µmol/L MLN120B (Millennium Pharmaceuticals, Cambridge, MA) for 2, 3, 4, 6, 8, 12, 16, and 24 hours. In addition, 16 HBL-1 samples were infected with retroviral vectors expressing various shRNAs (CARD11, SYK, BTK, CD79A) in a doxycycline-inducible fashion, selected with puromycin, treated with doxycycline for 24 or 48 hr and then harvested for total RNA. Uninduced cultures were prepared in parallel as control samples. Biological replicates were performed for the CARD11, BTK, and CD79A samples.

本研究旨在探究弥漫大B细胞淋巴瘤（DLBCL）在NF-κB抑制或B细胞受体（BCR）信号通路组分敲降后的基因表达变化。实验设计概况：采用Trizol试剂（Invitrogen，美国加利福尼亚州卡尔斯巴德市）提取总RNA，样本来自8株HBL-1细胞，经25 μmol/L MLN120B（Millennium Pharmaceuticals，美国马萨诸塞州剑桥市）分别孵育2、3、4、6、8、12、16及24小时后收集。此外，另有16株HBL-1细胞样本，通过多西环素诱导型逆转录病毒载体转染表达靶向CARD11、SYK、BTK、CD79A的短发夹RNA（shRNA），经嘌呤霉素筛选后，分别用多西环素处理24或48小时，随后收集细胞提取总RNA；同步设置未诱导的细胞培养物作为对照样本。针对CARD11、BTK及CD79A靶向样本设置了生物学重复。