Cd44v6

The miR-193b-5p mimic, pCMV-CD44v6 plasmid and their respective control RNAs were transfected into Hs-578t and BT-549 cells using Lipofectamine 3000 transfection reagent (Invitrogen, USA) according to the manufacturer's protocol. Additionally, 50 nM miRNA and 0.5 μg of pCMV-CD44v6 plasmid were used in a 6-well plate. Protein and RNA were collected 48 h after transfection. RIPA buffer (Beyotime, China) was used for protein extraction. After the total protein concentration was determined by a bicinchoninic acid protein assay kit (Sigma, USA), 30 μg protein samples were separated by 8% SDS polyacrylamide gels and transferred onto PVDF membranes(Millipore, Billerica, USA). The membrane was blocked with 5% nonfat milk in TBST for 1 h and incubated with the indicated antibody (CD44v6, R&D Systems. BBA13, 1:1000; CD44s, CST #5640, 1:1000; GAPDH, 1:5000) at 4 °C overnight. Then HRP-conjugated secondary antibodies (1: 5000) were added. Bands were subsequently visualized using the enhanced plus chemiluminescence assay (Pierce, USA). Measurement of the bands was conducted on an ImageQuant LAS 4000 mini.

Flow cytometric analyses and sorting were performed on single-cell suspensions derived from each cell line. ALDH1 enzymatic activity was detected using the ALDEFLUOR assay kit (StemCell Tehnologies, Durham, NC). The basis for this assay is that uncharged ALDH substrate (BODIPY-aminoacetaldehyde [BAAA]) is taken up by living cells via passive diffusion. Once inside the cell, BAAA is converted into negatively charged BODIPY-aminoacetate (BAA) by intracellular ALDH. The negatively charged BAA is then retained inside the cell, causing the cell to become highly fluorescent. Diethylaminobenzaldehyde (DEAB) was used to inhibit the ALDEFLUOR reagent, providing a negative control. Antibodies (FITC-conjugated anti-CD44s, PE-conjugated anti-CD24, and anti-EpCAM were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). PE-conjugated-CD44s, -CD44v3, and -CD44v6 were purchased from R&D Systems (Minneapolis, MN). Fluorescence activated cell analyses and sorting were done by FACS Caliber and FACS Aria III (BD Bioscience, San Jose, CA). Hoechst 33342 was used for specifically staining the nuclei of the cells.