Journal: Clinical and Translational Medicine
Article Title: Transfer of metastatic traits via miR‐200c in extracellular vesicles derived from colorectal cancer stem cells is inhibited by atractylenolide I. Transfer of metastatic traits via miR‐200c in extracellular vesicles derived from colorectal cancer stem cells is inhibited by atractylenolide I
Figure Lengend Snippet: Characterization of colorectal CSCs isolated from LoVo and HT29 cells. A, Flow cytometric sorting of CSCs using CD44 and EpCAM as markers. The percentage of parental LoVo and HT29 cells expressing both CD44 and EpCAM was approximately 15%, representing the proportion of CSCs. CSCs isolated from parental LoVo and HT29 cells (denoted as LoVo‐CSCs and HT29‐CSCs, respectively) exhibited high expression of both markers ( > 90%). Relative proliferation of (B) LoVo‐CSCs and (C) HT29‐CSCs was inhibited by ATL‐1 at 200 μM for up to 72 h, compared to that of control (+PBS) CSCs. The protein expression of stemness markers OCT‐4, SOX‐9, and Nanog, relative to GAPDH, in (D) LoVo‐CSCs and (E) HT29‐CSCs was downregulated by ATL‐1 at 200 μM after 48 h of culture, compared to that of control (+PBS) CSCs. Transwell assay of the (F) migration and (G) invasion of LoVo‐CSCs demonstrated decreased cell counts in both cases when ATL‐1 was administered at 200 μM, compared to those of control (+PBS) CSCs. H, The percentage of apoptotic LoVo‐CSCs and HT29‐CSCs was elevated in the presence of ATL‐1 at 200 μM, compared to that of control (+PBS) CSCs. The data represent the mean ± SD of three independent technical replicates ( t ‐test); * P
Article Snippet: Thereafter, the membranes were incubated overnight at 4°C with rabbit primary antibodies against the following proteins: octamer‐binding transcription factor 4 (OCT‐4, 1:1000, AB18976, Abcam, Cambridge, UK), sex‐determining region Y‐box (SOX‐9, 1:1000, AB185966, Abcam), Nanog (1:1000, AB106465, Abcam), CD63 (1:1000, PAB33929, Bioswamp), CD81 (1:1000, PAB33928, Bioswamp), TSG101 (1:1000, PAB32949, Bioswamp), PI3K (1:1000, AB191606, Abcam), phospho‐PI3K (p‐PI3K, 1:1000, AB182651, Abcam), Akt (1:1000, AB8805, Abcam), p‐Akt (1:1000, AB38449, Abcam), mTOR (1:2000, AB32028, Abcam), p‐mTOR (1:1000, AB84400, Abcam), and GAPDH (1:5000, PAB36264, Bioswamp).
Techniques: Isolation, Expressing, Transwell Assay, Migration