Figure 2

Effect of TEA on tumorigenic potential of TIC. (A) Ishikawa H (upper panels) or Hec50co (lower panels) cells were propagated in the presence of a cell membrane marker PKH26 and either kept untreated (left panels) or treated with TEA (right panels). PKH26(+) cells were isolated, and then ALDH1(+) cells obtained from that population by fluorescent cell sorting. Cells in the box in the right of the plots were collected for the soft agar assay in Figure 2B. N = 3. (B) Ishikawa H cells untreated (Ishikawa H ALDH1/PKH26(+), UN, solid bar) or pre-treated with TEA (Ishikawa H ALDH1/PKH26(+), PRE, shaded bar, p ≤ 0.05) were separated by the fluorescent cell sorting. ALDH1/PKH26 positive fractions were seeded onto the TEA-free soft agar and colonies counted 21 days later. Similarly, Hec50co cells untreated (Hec50co ALDH1/PKH26(+), UN, solid bar) or pre-incubated with TEA (Hec50co ALDH1/PKH26(+), PRE, shaded bar, p ≤ 0.05) were grown in TEA-free agar for 21 days. N = 4. (C) Untreated Ishikawa H cells with high PKH26/ALDH1 levels were seeded onto the unaltered soft agar (Ishikawa H, ALDH1/PKH26(+), UN, solid bar) or on agar supplemented with TEA (Ishikawa H, , ALDH1/PKH26(+), TEA, shaded bar, p ≤ 0.05) and inspected 21 days later. Similarly, PKH26/ALDH1-positive Hec50co cells on untreated agar (Hec50co, ALDH1/PKH26(+), UN, solid bar) or agar supplemented with TEA (Hec50co, ALDH1/PKH26(+), TEA, no bar) were counted 21 days later. N = 3.