ExtData_Figure_3_replicates

<em>(A-C) HEK293T ishXrn1 cells were treated with doxycycline for 3-4 days to induce knock down of Xrn1, then transfected with luciferase reporters containing 99 bp insertions from the indicated genes, and where indicated, with WT PA-X from the PR8 strain (A-C), the catalytic mutant PR8 PA-X D108A (A), PR8 PA with a mutation to reduce frameshifting and prevent PA-X production (PA(fs)) (A), herpes simplex virus 1 (HSV-1) vhs or Kaposi’s sarcoma-associated herpes virus (KSHV) SOX (B), or WT PA-X from the Perth influenza strain (C). RNA was extracted and used to run 5’ RACE. (D) Xrn1 knock out A549 cells were infected with WT PR8, Perth or pH1N1pdm09 or the corresponding PA(∆X) mutants, or mock infected. 5’ RACE was then performed using primers specific for </em>STOML2<em> or </em>YKT6<em> ~250-300 nt downstream of the predicted cut sites. The PCR products were run on an agarose gel. For all gels, the DNA bands were purified and sequenced to confirm their identities. </em> <em>R# --&gt; replicate number; LG### --&gt; experiment number.</em> <em>.sgd files are the original source files acquired with the Syngene Imager. </em>

(A-C) 将HEK293T ishXrn1细胞用多西环素处理3-4天以诱导Xrn1基因敲低，随后转染携带指示基因来源的99 bp插入片段的荧光素酶报告质粒；按实验需求分别共转染PR8毒株野生型PA-X（A-C组）、PR8毒株PA-X的催化突变体D108A（A组）、带有可降低移码效率并阻断PA-X产生的突变的PR8 PA（PA(fs)，A组）、单纯疱疹病毒1型（HSV-1）vhs或卡波西肉瘤相关疱疹病毒（KSHV）SOX（B组），以及珀斯流感毒株的野生型PA-X（C组）。提取细胞总RNA后开展5'末端快速扩增（5' RACE）实验。
(D) 将Xrn1基因敲除的A549细胞分别感染野生型PR8、珀斯流感毒株、pH1N1pdm09毒株或其对应的PA(∆X)突变株，或设置模拟感染对照组。随后在预测剪切位点下游约250-300 nt的位置，使用针对STOML2或YKT6的特异性引物开展5'末端快速扩增（5' RACE）实验。将聚合酶链式反应（PCR）产物进行琼脂糖凝胶电泳。所有凝胶中的DNA条带均被纯化并测序，以确认其分子身份。
R# 代表重复次数；LG### 代表实验编号。.sgd文件为使用Syngene成像仪获取的原始源文件。