shRNA-mediated knockdown of AURKA or AURKB decreases the transformed phenotype of KRAS-positive lung cells. Unless otherwise indicated, A549 and H358 stable cells with inducible expression of 2 different shRNAs targeting AURKA (shAKA#1 and shAKA#2), AURKB (shAKB#1 and shAKB#2) or a non-targeting shRNA (shCtrl) were either treated with 2 μg/mL doxycycline (DOX) for 5 days to induce shRNA expression or left untreated (MOCK). a Protein lysates of doxycycline-treated ( +) and untreated ( −) cells were submitted to western blotting with the indicated antibodies. TUBA) anti-α-tubulin. b Growth curve analysis of the indicated cells. All cells were treated with 2 μg/mL doxycycline (DOX) for the indicated times. c The indicated cells were plated for clonogenic assays as described in methods and treated for 21 days with 2 μg/mL doxycycline (DOX). Colonies formed were stained with crystal violet and counted. Images shown are representative of three independent experiments. d The indicated treated (DOX) or untreated (MOCK) cells were stained with BrdU and propidium iodide (PI) as described in methods, and cell cycle analysis was performed by flow cytometry. e Protein lysates of A549 and H358 stable cells with inducible expression of 2 different shRNAs targeting AURKA (shAKA#1 and #2), AURKB (shAKB#1 and #2) or a non-targeting shRNA (shCtrl), treated ( +) or not (-) with 2 μg/mL doxycycline (DOX) for 5 days, were submitted to western blotting with the indicated antibodies. C3) anti-caspase 3. f Anchorage-independent growth was evaluated by plating the indicated cells in soft agar as described in methods. Cells were then treated for 21 days with 2 μg/mL doxycycline (DOX) or left untreated (MOCK). Colonies formed were stained with MTT and counted. Images shown are representative of three independent experiments. In all cases, statistical significance was determined when appropriate by Student’s t-test (*p < 0.05, **p < 0.01) and the groups being compared are indicated by horizontal bars.

shRNA 介导的 AURKA 或 AURKB 敲低会降低 KRAS 阳性肺细胞的转化表型。除非另有说明，否则对具有 2 种不同靶向 AURKA（shAKA#1 和 shAKA#2）、AURKB（shAKB#1 和 shAKB#2）或非靶向 shRNA (shCtrl) 的 shRNA 诱导表达的 A549 和 H358 稳定细胞进行处理用 2 μg/mL 多西环素 (DOX) 5 天诱导 shRNA 表达或不处理 (MOCK)。a将多西环素处理的 (+) 和未处理的 (-) 细胞的蛋白质裂解物用所示抗体进行蛋白质印迹分析。TUBA) 抗α-微管蛋白。b所示细胞的生长曲线分析。所有细胞均用 2 μg/mL 强力霉素 (DOX) 处理指定时间。c按照方法中的描述将指定细胞铺板用于克隆形成测定，并用 2 μg/mL 多西环素 (DOX) 处理 21 天。将形成的菌落用结晶紫染色并计数。所示图像代表三个独立实验。d按照方法中所述，用 BrdU 和碘化丙啶 (PI) 对指定的处理 (DOX) 或未处理 (MOCK) 细胞进行染色，并通过流式细胞术进行细胞周期分析。e A549 和 H358 稳定细胞的蛋白裂解物，可诱导表达 2 种不同的靶向 AURKA（shAKA#1 和 #2）、AURKB（shAKB#1 和 #2）或非靶向 shRNA (shCtrl) 的 shRNA，经处理 (+)或不使用 2 μg/mL 多西环素 (DOX) 5 天，然后使用所示抗体进行蛋白质印迹分析。C3) 抗半胱天冬酶 3。f如方法中所述，通过将所示细胞铺板在软琼脂中来评估锚定非依赖性生长。然后用 2 μg/mL 多西环素 (DOX) 处理细胞 21 天或不处理 (MOCK)。形成的集落用MTT染色并计数。所示图像代表三个独立实验。在所有情况下，在适当的情况下通过学生t检验（* p  < 0.05，** p  < 0.01）确定统计显着性，并且所比较的组由水平条表示。