Pan cytokeratin

Total cell lysate was prepared in RIPA (Boston Bioproducts) containing protease and phosphatase inhibitor cocktail (Thermo). The cell lysate was quantified and proteins separated on SDS-PAGE and transferred to the PVDF (Bio-Rad) membrane. Membranes were blocked in 5% nonfat milk in TBS with 0.1% Tween-20 (TBST) for 1 h at room temperature followed by incubation with primary antibodies in TBST with 1% BSA overnight. The following primary antibodies were used – p Smad-2, p Smad-3, Smad-2, Smad-3, Snail, Vimentin, EPCAM, c-Myc, NFAT-1, Cyclin B, Cyclin D1, (Cell signaling Technology), Fibronectin, N-Cad (BD Biosciences), Pancytokeratin, GAPDH, α tubulin and β actin (Sigma), Acidic and basic Cytokeratin AE1/AE3 (Millipore), EMA (Dako), p27 (Santa Cruz Biotechnology), and cytokeratin-8 (Developmental Studies Hybridoma Bank, University of Iowa). Secondary antibodies (from sigma) were used at a concentration of 1:10,000. Equal loading was verified by immunoblotting with GAPDH, αTubulin or β Actin [44 (link)].

Monolayers and spheroids were washed in PBS before being fixed for 30 min at 4 °C with 2% paraformaldehyde, followed by blocking with 2% bovine serum albumin. Cells were reacted with primary antibodies for EpCAM (Sigma, Burlington, MA, USA), Pancytokeratin (Sigma, Burlington, MA, USA), and CK8/18/19 (Sigma, Burlington, MA, USA) and secondary antibodies conjugated to Alexa 594 and 488 (Invitrogen, Waltham, MA, USA) and were counterstained with 4’,6-diamidino-2-phenylindole for nucleus. The cell images were acquired using a confocal microscope (Leica, Wetzlar, Germany).

For tissue immunofluorescence analysis, brains were carefully removed, post‐fixed overnight in 4% paraformaldehyde, and placed in 30% sucrose for 48 h. They were then embedded in OCT compound (Thermo Fisher Scientific, Waltham, MA, USA) and frozen at −80 °C before cryostat sectioning (10 μm). Immunofluorescence was performed by blocking sections in 5% normal donkey serum and 0.1% Triton‐X in PBS, and incubating them overnight at 4 °C with primary antibodies against CD19 and pan‐cytokeratin (Sigma, St Louis, MO, USA) (supplementary material, Table S3). After washing with PBS, sections were incubated with the appropriate Alexa Fluor‐conjugated secondary antibodies (Life Technologies, Grand Island, NY, USA). Hoechst 33258 (Molecular Probes, Eugene, OR, USA) was used for nuclear counterstaining. Slides were mounted with Prolong Gold (Life Technologies) and examined using a Nikon Eclipse Ci fluorescence microscope. Image acquisition was performed using Nikon Digital sight DS‐U3 and Nis‐Elements D viewer software. Finally, Fiji software was used for image processing (http://fiji.sc/; last accessed 3 May 2018).