Figure 7. Squamous transdifferentiation of mammary epithelial cell lines in vitro.

(A) Immunoblot showing K10 and FLG expression in attached cells and induction upon suspension at 24h and 72h in MCF-10A and HMEC^hTert cells. Note induction of BIM (Stressgen antibody AAP-330) in MCF-10A cells upon anoikis as described previously (Reginato et al., 2003). TUB was used as loading control. (B–C) K10 (green) and β1-INT (red) immunofluorescence on cytospin samples of attached cells (B) and cells in suspension for 24h (C). (D) Quantification of Keratin 10-positive MCF-10A cells for attached (7.24% ± 1.99) condition and after 24h (33.8% ± 3.46) or 48h (55% ± 2, p<0.001) in suspension. (E–F) FLG immunofluorescence on cytospin samples of attached cells (E) and cells in suspension for 24h (F). (G) Up-regulation of p38 MAPK pathway activity upon anoikis. Immunoblot showing up-regulation of phosphorylation of p38 MAPK and its upstream MAPKs, MKK3 and MKK6, upon suspension at 24h and 48h in MCF10A cells. Note that total level of MKK3 and MKK6 increased upon anoikis, while p38 MAPK protein did not change. TUB was used as a loading control. (H) Pharmacological inhibition of p38 MAPK activation suppressed K10 expression. SB203580 treatment (10μM in DMSO) decreased phosphorylation of p38 MAPK and prevented K10 up-regulation in suspension at 24h, while treatment with the carrier alone (DMSO) did not inhibit up-regulation of p38 MAPK phosphorylation and K10 protein expression. Note that total level of p38 MAPK protein does not change upon anoikis. TUB was used as a loading control. (I) Microphotography of HMEC^hTert acini after 20 days of culture. Note the “onion skin” shape of the inner cells (black arrow in low magnification panel). Lower panel: high magnification of the upper panel. Note the lumen (black asterisk). (J) Immunostaining for K10 (green) and LOR (red) in HMEC^hTert acini with a hollow lumen (white asterisk) at day 20. The dashed white line displays HMEC^hTert acinus borders. Insert: LOR staining alone with DAPI of the dashed box shown in the main panel. (K) Immunolocalization of K1 in HMEC^hTert acini at day 20. (L) Colocalization of β1-INT (green) and β-CAT (red) by immunostaining in HMEC^hTert acini at day 20. Note the absence of β1-INT expression in the squamous inner cells (double head arrow) and the faint DAPI staining in the center of the acinus (white arrows). [Scale bar: 15 (B-C-E-F), 430 (I), 180 (I insert), 35 (J–K), 45 (L) μm]. Abbreviation: att. and Attach. (attached), ano. (anoikis), sus. (suspension), sq. (squamous), β1-INT (β1-integrin), β-Cat (β-catenin), FLG (Filaggrin), LOR Lloricrin), K1 (keratin 1), K10 (keratin 10), P-MMK3/6 (phospho-MKK3/6), P-p38 (Phospho-p38 MAPK), TUB (tubulin).