Journal: Scientific Reports
Article Title: (−)-Epigallocatechin gallate inhibits stemness and tumourigenicity stimulated by AXL receptor tyrosine kinase in human lung cancer cells
doi: 10.1038/s41598-020-59281-z
Figure Lengend Snippet: Involvement of AXL in tumour sphere formation and ALDH activity in H1299-sdCSCs. ( A ) Treatment of H1299-sdCSCs with GAS6 (250 ng/ml) significantly increased phosphorylated AXL (p-AXL Y702) (p-AXL/AXL) compared with that in control cells, as analysed by western blotting. α-Tubulin was used as a control. This blot is a part of Supplementary Fig. . Tumour sphere formation was stimulated by treatment with GAS6 (100 ng/ml) for 3 weeks. The number of tumour spheres is shown as a percentage of the control. Three independent experiments were conducted. ( B ) Knockdown of AXL with siAXL-1 and siAXL-2 inhibited tumour sphere formation. Cells were treated with siAXLs or siControl for 2 days and then cultured in serum-free medium for 3 weeks. Images show representative tumour spheres. Percentages of tumour spheres of non-treated cells are shown in the graph. The results are the mean ± SD of three independent experiments. ( C ) Knockdown of AXL with siAXL-1 and siAXL-2 reduced the percentage of ALDH-positive cells. ALDH-positive cells in H1299-sdCSCs treated with siControl, siAXL-1, or siAXL-2 were detected by Aldefluor flow cytometry assay. The upper figure shows the representative results. The graph shows the average percentage of ALDH-positive cells in three independent experiments. Full-length blots/gels are presented in Supplementary Information WB-1 and -2. ( D ) Representative images of ALDH1A1 immunocytochemical staining in non-treated, siControl-, siAXL-1-, and siAXL-2-treated H1299-sdCSCs. Numbers indicate MFI of 15 tumour spheres from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.
Article Snippet: Anti-AXL (R&D Systems, Minneapolis, MN, USA), anti-p-AXL (Y702) (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-SLUG (Cell Signaling Technology, Inc.), anti-ALDH1A1 (Santa Cruz, Biotechnology, Inc., Santa Cruz, CA, USA), and anti-α-tubulin (Cell Signaling Technology, Inc.) antibodies were used for the experiments.
Techniques: Activity Assay, Control, Western Blot, Knockdown, Cell Culture, Flow Cytometry, Staining
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