Upregulation of Nestin immunoreactivity in differentiating P19 and ES lines. (A–D) Fluorescent photomicrographs of mTERT-expressing P19 and ES lines 3 d after RA treatment reacted with anti-Nestin mAb. Nuclei were counterstained with DAPI. (A and B) mTERT-expressing P19 lines A4 and A5. (C and D) mTERT-expressing ES lines M1 and M3. Arrows point to “perinuclear rosette” staining characteristic of differentiating cells. Bar, 40 μm. (E) The percentage of total (DAPI-labeled) cells expressing Nestin was counted after RA treatment in P19 lines (E) and ES cells (F). Parent cells (white bars) the control lines P2 and P3 and E1 and E2 (gray bars) and the mTERT-expressing lines A4 and A5 and M1, M2, and M3 (dark gray bars). Both mTERT expressing P19s and ES cells up-regulated Nestin in a timely manner. Parent P19s up-regulated Nestin earlier, at 2 d, and by day 4 there was no significant difference in the percent of P19 cells expressing Nestin. MTERT expressing ES cells up-regulated nestin more efficiently than parent or control lines.