DND41, REC-1 and ALL/SILL cells were resuspended in PBS, spotted on immunofluorescence slides (Thermo Fisher Scientific, Waltham, MA) by a cytospin centrifuge (CR2000, Small Prime Centrifuge, Centurion) fixed for 10 min in 4% paraformaldehyde (#28908, Thermo Fisher Scientific, Waltham MA, USA), permeabilized in 0.2% Triton X-100 for 5 min, and blocked in 5% bovine serum albumin for 1 hour. Then, the cells were incubated with primary antibodies against full length NOTCH1 (#SC-6014 (C-20) Santa Cruz Biotechnology, Santa Cruz, CA, USA or #ab44986 (A6) Abcam, Cambridge, United Kingdom), GOLGA1 (#SAB1409131, Sigma-Aldrich, St. Louis, MO, USA), and ATF6 (#37149, Abcam, Cambridge, United Kingdom).
Alexa Fluor 488 (#A11029, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568 (#A11036, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies and cells were incubated 1 hour at room temperature protected by the light. Nuclei were stained with DAPI (#D9542, Sigma-Aldrich, St. Louis, MO, USA). Coverslips were mounted with Prolong Gold Antifade reagent (#P36934, Thermo Fisher Scientific, Waltham MA, USA). Images were captured using a EVOS FL microscope (Thermo Fisher Scientific, Waltham MA, USA) and analyzed using ImageJ software (http://rsbweb.nih.gov/ij/ ).
Alexa Fluor 488 (#A11029, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568 (#A11036, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies and cells were incubated 1 hour at room temperature protected by the light. Nuclei were stained with DAPI (#D9542, Sigma-Aldrich, St. Louis, MO, USA). Coverslips were mounted with Prolong Gold Antifade reagent (#P36934, Thermo Fisher Scientific, Waltham MA, USA). Images were captured using a EVOS FL microscope (Thermo Fisher Scientific, Waltham MA, USA) and analyzed using ImageJ software (