Neon transfection

Square microfluidic chambers (150 or 450 microgroove) were purchased from Xona microfluidics. The chambers were sterilized under UV for 15 min and soaked in 70% ethanol for 2 min and allowed to air dry under a sterile TC hood. We used MatTek dishes (P50G-1.5, MatTek corp.) that we pre-coated with PLL overnight, rinsed three times with sterile water and air dried overnight in a sterile hood. Using sterile forceps, we carefully place the microfluidic chamber on the glass area of the MatTek dish, and gently apply pressure to ensure the chambers are sitting on the glass. 3 whole brain P1–2 cortices after papain enzymatic digestion and Optiprep gradient were resuspended in 200 µl of NB with full supplements. We then seeded 15 µl of the neuronal suspension on one side of the microgroove and place in a 37°C incubator for 20 min to allow the neurons to adhere. All wells were filled with 150 µl of supplemented NB. Once we observe neurites growing across the microgroove (2–3 days), we then did treatments on the neurite compartment and wait 48 h before we add 4% Paraformaldehyde (PFA) with 4% Sucrose. We used the Neon transfection (Invitrogen) method to overexpress YB-1.

Cells were transfected with hGFP1-10 with Neon Transfection (Invitrogen) and seeded into 96-well plates at 20,000 (CHO-K1) and 30,000 cells/well (HCC827). Stable cell lines were seeded at 10,000 cells/well. The next day recombinant CPP_TRX_S11 proteins were added to final concentrations of 5, 10, 20 or 40 μM. After 24 h, cells were prepared for flow cytometry.

For caspase 8 downregulation experiments, hESC were transfected with siRNA using a Neon transfection instrument (Invitrogen) according to the manufacturer's instructions. 100 pmoles of caspase 8 siRNA (Santa Cruz Biotechnology Inc.) were added to 1 × 10⁵ cells and transfection was performed with one 1100 V pulse for 30 ms. Cells were seeded on Matrigel and cultured in CM without antibiotics. Cells were harvested at 48 and 72 hours after transfection.

NB4 [60 (link)], HL-60 [61 (link)] and COS-7 cells were cultured as described [36 (link), 62 (link), 63 (link)]. We generated NB4 cell populations silenced for PML-RAR and the RARα isoforms infecting cells with the SCRsh, PMRsh, RA1sh and RA2sh retroviral vectors according to standard protocols (System Biosciences). Following infection, NB4 cells were selected in RPMI medium containing 10% bovine serum and puromycin (1.0 μg/ml) for at least 15 days. Only NB4 cell populations characterized by >95% positivity to GFP following FACS analysis were considered. Subsequent passages of the cell populations were performed in complete RPMI medium containing puromycin (0.5 μg/ml). To isolate NB4 populations over-expressing RARα2, cells were electroporated with pCDH-CMV lentiviral vectors (System Biosciences) containing RARα2 (pRA2) using Neon Transfection (Invitrogen, Life Technologies). HL-60 cells were infected as above with the RA2sh, SCRsh or the void (Void) retroviral vector.

To create the TP53- and POLQ-deficient cell lines, we used the Alt-R–CRISPR–Cas9 system (IDT). We performed neon transfection (Invitrogen) and followed the manufacturer’s protocol with Alt-R HiFi Cas9 nuclease, crRNA and tracrRNA purchased from IDT. crRNA was designed using MIT CRISPR (http://crispr.mit.edu) to target exon 2 of the TP53 gene to create the TP53^−/− cell line and the polymerase domain of the POLQ gene to create the TP53^−/−POLQ^−/− cell line. Forty-eight hours after transfection, cells were seeded for single-clone selection. Restriction enzyme screening, western blots, PCR screening and Sanger sequencing confirmed gene targeting, as well as functional assays.