Lipofectamine p3000

BECN1 isoforms fused with the GFP were transiently expressed in MDA-MB231 cells. Transient transfections were performed with Lipofectamine P-3000 (L3000-15, Invitrogen) following the manufacturer’s instructions. Briefly, confluent cells were incubated for 6 h with Lipofectamine-P3000 solutions containing 6 µg of plasmid and were cultured for 36 h to allow the expression of the exogenous BECN1. When indicated, the cells were treated with 30 µM Chloroquine or Clq (C6628, Sigma-Aldrich) for 4 h for the LC3 or p62 turnover assay, or with 10 µM of the uncoupler cyanide m-chlorophenylhydrazone or CCCP (C2579, Sigma-Aldrich) for 3 h to induce mitophagy.

Mouse embryonic fibroblasts or HEK-293T cells (Takara 632180) were cultured in high-glucose DMEM (Corning) containing 1 mM pyruvate and supplemented with 10% foetal bovine serum (Denville) and penicillin/streptomycin (Corning). Then 50 mM of lactate or pyruvate (Sigma-Aldrich) was supplemented into the cell culture media in the presence or absence of 1 µM trichostatin A for 3 h before collection. Lipofectamine P3000 (Invitrogen) was used for transfection experiments.

Infection with pegRNA library: Cells were infected with the pegRNA library (separate infections for each target site and library set), aiming at a multiplicity of infection of 0.5 and a guide coverage of >1,000×. Each screen was performed in three biological replicates and independently infected. To achieve this, 6 × 10⁶ cells were plated in three wells of a six-well plate and spin-infected for 15–30 min at 2,000 r.p.m. Following infection, cells were resuspended and replated at 2 × 10⁴ cells per cm². Cells were cultured for seven d and selected for pegRNA integration with 10 µg ml⁻¹ blasticidin.
Transfection with prime editors: HEK293T cells were seeded at a concentration of 6.9 × 10⁴ cells per cm² in a 15-cm dish. The next day, the medium was replaced with fresh medium and the cells were transfected using Lipofectamine LTX reagent. Then, 72 µg of PE-Puro or PE-FeLV plasmid was mixed with 8 µg of pCS2-GFP and 40 µl of Lipofectamine P3000 (Invitrogen) in 3.2 ml of Opti-Mem (Gibco). In another tube, 40 µl of Lipofectamine 3000 and 160 µl of Lipofectamine LTX were mixed in 3.2 ml of Opti-Mem. The solutions were combined, incubated for 30 min at room temperature and then added to the cells. For PE3, an additional 6 µg of nicking guide RNA was added. For screens with nuclease overexpression, an additional 30 µg of flap nuclease or eGFP plasmid in the pTwist vectors was added.

Human codon optimized MEDLE2 lacking the N-terminal signal peptide (aa 21–209) was synthesized by Integrated DNA Technologies (IDT, Coralville, IA) and cloned into the mEGFP-Lifeact-7 mammalian expression plasmid (Addgene #54610), replacing Lifeact-GFP and appending a 3× HA tag. Point mutations were engineered by Gibson cloning. HEK293T cells (ATCCCRL-3216; RRID:CVCL_0063) were transfected with 5 µg of each plasmid using Lipofectamine P3000 (Thermo Fisher Scientific). 24 hr post transfection, cells were harvested and processed for western blot analysis. Additionally, a MEDLE2-EGFP plasmid was cloned by Gibson cloning to introduce human codon optimized MEDLE2 lacking the N-terminal signal peptide (aa 21–209) into the same mEGFP-Lifeact-7 mammalian expression plasmid, replacing Lifeact. A GFP-only-expressing plasmid was engineered removing the Lifeact from Addgene plasmid #54610. Similarly, T. gondii GRA16 omitting the sequence encoding the N-terminal signal peptide (aa 24–505) was amplified from gDNA of T. gondii strain ME49 parasites and cloned into the mEGFP-Lifeact-7 mammalian expression plasmid in place of Lifeact.

Adherent hAC were incubated in a digest solution containing 1 mg/mL collagenase, 1 mg/mL protease, and 40 U/mL hyaluronidase to increase siRNA transfection efficacy. Detached cells were counted and seeded at a density of 20,000 cells/cm². Silencer Select siRNA (Thermo Fisher Scientific) against CDKN2A (ID: s218; targeting both p16^INK4a and the related transcript p14^ARF), VIM (ID: s14800), or Silencer Negative Control siRNA was diluted in Lipofectamine P3000 (Thermo Fisher Scientific) and added to the cells at a final concentration of 5 pmol/μL in serum-free Opti-MEM (Thermo Fisher Scientific; 31985062) without antibiotics. After 4 hr at 37°C, transfection medium was removed and serum-containing chondrocyte medium was added. After 72 hr, hAC were trypsinized, counted, and seeded as mentioned above. The next day, transfection was repeated in adherence. This protocol allows a consistent silencing over 7 days.