Hoechst33342 counterstain

Cell lines were plated onto #1.5 thickness, 12 mm round glass coverslips (Thermo Fisher Scientific) coated with Matrigel at ∼20% confluency and allowed to adhere overnight. Coverslips were washed with PBS and subsequently fixed in 4% paraformaldehyde in PBS at room temperature for 15 min. Coverslips were washed with PBS and incubated with 1:200 TOM20 (ProteinTech Group, 11802-1-AP) antibody prepared in PBS and 1% BSA for 2.5 h. Coverslips were then washed three times with PBS and incubated with 10 μg/ml Hoechst33342 counterstain (Invitrogen, H3570) and 1:200 Donkey Anti-Rabbit IgG (H + L) Cy5 (Jackson ImmunoResearch, 711–175–152) for 2 h. Coverslips were mounted onto Fisherbrand Superfrost Plus Microscope Slides (Thermo Fisher Scientific) using Shandon Immu-Mount solution (Thermo Fisher Scientific). Images were acquired through a Zeiss LSM880 AxioObserver Z1 confocal microscope with AiryScan using a 63x 1.4 NA oil objective. Images were processed using ZenBlue 3.2 software (Zeiss; https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html).

Primary antibodies and dilutions used were: rat antibody to CTIP2 (1:500, Abcam); mouse antibody to SATB2 (1:200, Abcam); rabbit antibody to SIRT1 (1:250, Millipore); rabbit antibody to CTIP1 (1:500, Abcam); rabbit antibody to GFP (1:500, Invitrogen); rabbit antibody to Ki67 (1:500, Abcam); chicken antibody to NESTIN (1:500, Novus Biologicals); mouse antibody to TuJ1 (1:500, Covance); and mouse antibody to MAP2 (1:500, Sigma). Alexa fluorophore conjugated secondary antibodies from Invitrogen were used at a dilution of 1:1000. Hoechst 33342 counterstain was used to visualize nuclei (1:3,000, Invitrogen).