Cf543

Cells grown on coverslips were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 10 min, and blocked in 3% normal donkey serum in PBS for 2 h at room temperature. Primary antibodies were diluted in 3% normal donkey serum in PBS and applied at 4 °C overnight. The primary antibodies used in these experiments were as follow: Nestin (Abcam; 1:3000), Sox2 (Santa Cruz; 1:500), after rinsing in PBS three times and incubating for 2 h with CF488 and CF543 (Biotium; 1:1000), coverslips were washed three times, cell nuclei were stained with DAPI. Images were acquired on a Leica TCS SP2 confocal fluorescence microscope.

Chlorpromazine (C8138), dynasore (D7693), and pitstop 2 (SML1169) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mowiol was obtained from Calbiochem (Merck Chemicals, Darmstadt, Germany), To-Pro-3 from Thermo fisher (Waltham, MA, USA) and human transferrin (Tf) CF^®543 and CF^®555-Labeled Dye Dextran 10,000 MW conjugates were purchased from Biotium (Fremont, CA, USA). X-tremeGENE 360 Transfection Reagent (XTG360-RO) was obtained from Roche (Basel, Switzerland). All drugs in this study were dissolved in 0.1% v/v dimethyl sulfoxide (DMSO), except for chlorpromazine and Tf which were dissolved in sterile distilled water.
Primary antibodies used were mouse monoclonal anti-GFP (11814460001, Roche), mouse monoclonal anti-β-actin peroxidase antibody (A3854, Sigma), rabbit monoclonal anti-AP2M1 (ab75995, abcam, Cambridge, UK), and rabbit anti-IE180 (Gómez-Sebastián and Tabarés, 2004 (link)) (kindly provided by Dr. Enrique Tabarés). Horseradish peroxidase conjugate (HRP) secondary anti-IgG antibodies were purchased from Millipore (Darmstadt, Germany).

Cells grown on coverslips were fixed in 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100 (Sigma), and blocked in 3% normal donkey serum in PBS for 30 minutes at room temperature. Rabbit anti-human γH₂AX (Abcam; 1:1000) antibody was diluted in 3% normal donkey serum in PBS and applied at 4°C overnight. After rinsing with PBS three times and incubating for one hour with donkey anti-rabbit secondary antibody (CF543, Biotium; 1:1000), slides were washed three times in PBS and cell nuclei were stained with DAPI (Invitrogen) for 10 minutes at room temperature. Images were acquired on a Leica TCS SP2 confocal fluorescence microscope.