Fig 1: CBFA2T2 is required for migration and invasion activity of RCC cells. a Immunoblot assay of CBFA2T2 and ß-actin proteins in 786-O and A-498 cells treated with siRNA against CBFA2T2 or control siRNA for 36 h. b–c 786-O and A-498 treated with siRNA against CBFA2T2 fill the wound area more slowly than those treated with control siRNA at 20 h. The wound-healing assay was expressed as wound closure rate (20 h average healed width divided by 0 h wound width). d–e CBFA2T2 silencing significantly blocked cell invasion of 786-O and A-498 cells. Images displaying the bottom side of the filter inserts with cells that migrated through the filter pores. The data are presented as the mean ± standard deviation with three independent experiments. **indicates p < 0.01
Fig 2: CBFA2T2 is required for cancer stem cell state of RCC cells. a 786-O and A-498 cells were treated with siRNA against CBFA2T2 or control siRNA for 24 h and subjected to the Aldefluor assay. Proportions of the ALDH-positive cell subpopulation in total cancer cells were indicated in the figure. b, c 786-O and A-498 cells were plated into ultra low-attachment 24-well plates at a density of 1000/well and cultured in tumor sphere medium containing siRNA against CBFA2T2 or control siRNA. Tumor spheres were photographed (b) and tumor sphere numbers were counted (c). The data are presented as the mean ± standard deviation with three independent experiments. **indicates p < 0.01
Fig 3: CBFA2T2 is required for the expression of known cancer stem cell markers in RCC cells. a Quantitative real-time RT-PCR analysis for the expression of ALDH1A3, CD133, OCT4, Sox2, and Nanog mRNAs in 786-O cells after siRNA treatment for 48 h. P < 0.05 was considered as significant. b Quantitative real-time RT-PCR analysis for the expression of ALDH1A3, CD133, OCT4, Sox2, and Nanog mRNAs in A-498 cells after siRNA treatment for 48 h. p < 0.05 was considered as significant. c The interaction between OCT4 and CBFA2T2 in CSCs population showing by pull-down assay
Fig 4: Kaplan-Meier survival curves of CBFA2T2 and stem cells marker expression in RCC. a RCC patients with high CBFA2T2 expression had shorter 5-year overall survival than those with low expression. b RCC patients with high OCT4 expression had shorter 5-year overall survival than those with low expression. c RCC patients with high ALDH1A3 expression had shorter 5-year overall survival than those with low expression. d RCC patients with high OCT4 expression had shorter 5-year overall survival than those with low expression. e RCC patients with high SALL4 expression had shorter 5-year overall survival than those with low expression
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