Additional file 1 of Downregulation of CD151 restricts VCAM-1 mediated leukocyte infiltration to reduce neurobiological injuries after experimental stroke

Additional file 1: Supplementary method. RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR). Table S1. Detailed information of lentivirus CD151 shRNA sequences. Figure S1. Different lentivirus CD151 shRNA transfection effectiveness in vitro. Figure S2. Lentivirus CD151 shRNA transfection effectiveness assessment in vivo. Figure S3. Lentivirus transfection effectiveness in vitro. Figure S4. CD151 expression assessment at observation time points in vivo and in vitro. Figure S5. p38 and NF-κB activation were restrained in vivo after CD151 knockdown. The MAPK kinase (i.e., p38, JNK, and ERK) activation was evaluated (a, b, c) using infarcted hemisphere (n = 6 per group) or enriched endothelial cells (g, h) from infarcted hemisphere (n = 3 per group), * and **vs. LV Vehicle + MCAO indicate p < 0.05 and 0.01, respectively. The NF-κB pathway activation evaluated using the (d) IκB α degeneration and the p65 translocation from the (e) cytoplasm to the (f) nucleus in infarcted hemisphere (n = 6 per group), *, ** and *** vs. LV Vehicle + MCAO indicate p < 0.05, 0.01 and 0.001, respectively. Figure S6. Anisomycin increased the phosphorylation of both p38 and JNK in BMVECs. Cultured primary BMVECs were treated with 1μM anisomycin for 3 h (n = 3 per group). An increase in the phosphorylation of p38 (a) and JNK (b) were observed, ** vs. control group indicate p < 0.01.

附加文件1：补充方法。RNA提取与逆转录聚合酶链反应（reverse transcriptase-polymerase chain reaction，RT-PCR）。表S1：慢病毒CD151短发卡RNA（short hairpin RNA，shRNA）序列详细信息。图S1：不同慢病毒CD151 shRNA的体外转染效率。图S2：慢病毒CD151 shRNA体内转染效率评估。图S3：慢病毒体外转染效率评估。图S4：体内外各观测时间点CD151表达水平评估。图S5：CD151敲低后体内p38与核因子κB（nuclear factor κB，NF-κB）活化受到抑制。采用梗死侧半球（每组n=6）或梗死侧半球来源的富集内皮细胞（g、h，每组n=3）评估丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）激酶（即p38、JNK、ERK）的活化情况（a、b、c）；与空载体慢病毒（LV Vehicle）+大脑中动脉闭塞（MCAO）组相比，*和**分别表示P<0.05和P<0.01。采用梗死侧半球（每组n=6）的（d）IκBα降解与（e）细胞质内p65向（f）细胞核转位情况评估NF-κB通路活化水平；与LV Vehicle + MCAO组相比，*、**和***分别表示P<0.05、P<0.01和P<0.001。图S6：茴香霉素可上调脑微血管内皮细胞（brain microvascular endothelial cells，BMVECs）中p38与JNK的磷酸化水平。将原代培养的BMVECs以1μM茴香霉素处理3小时（每组n=3），可观察到（a）p38与（b）JNK的磷酸化水平升高；与对照组相比，**表示P<0.01。