Fig. 2. The expressions of TSPYL5 and CD44 are elevated in ALDH1^high CSC-like cells isolated from NSCLC cells.

a, d Flow cytometric analysis of CSC-like cells using Aldefluor staining. A549 or H460 cells were stained with Aldefluor substrate and sorted into ALDH1^low (AL^low) and ALDH1^high (AL^high) cells. Sorted cells were re-examined by Aldefluor assay to confirm their ALDH1 activity. b, e Sphere formation, migration/invasion assay, and colony formation assay of AL^low and AL^high cells sorted from A549 or H460 cells. Scale bar: 20 μm. c, f Western blot analysis of CSC markers (ALDH1A1, ALDH1A3, CD44, OCT-4, and SOX-2) and TSPYL5 in AL^low and AL^high populations sorted from A549 or H460 cells. g Flow cytometric analysis of CSC-like cells using anti-CD44 immunostaining. A549 cells were stained with APC-conjugated anti-human CD44 antibody and sorted into CD44^low and CD44^high cells. Sorted cells were re-examined by CD44 staining to confirm their CD44 expression. h Sphere formation, migration/invasion assay, and colony-formation assay of CD44^low and CD44^high cells sorted from A549 cells. i Western blot analysis of CSC markers (ALDH1A1, ALDH1A3, and CD44) and TSPYL5 in CD44^high and CD44^low cells sorted from A549 cells. Data represent mean ± s.d. using two-tailed t-test. n = 5 independent experiments for each group. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.