L glutamine

HPV18-positive HeLa (RRID: CVCL_0030) and HPV16-positive SiHa (RRID: CVCL_0032) cervical cancer cells, HPV-negative RKO (RRID: CVCL_0504) and HCT116 (RRID: CVCL_0291) colon cancer cells, and HPV-negative U2OS (RRID: CVCL_0042) osteosarcoma cells were obtained from the tumor bank of the German Cancer Research Center (DKFZ), Heidelberg. HeLa “p53 null” cervical cancer cells (HeLa A1 and HeLa C2) were described before [31 (link)]. HeLa-mKate2 and SiHa-mKate2 cells stably express a nuclear-restricted mKate2 fluorescent protein and were generated as detailed previously [72 (link)]. Cell lines were cultivated in Dulbecco’s minimal essential medium (DMEM) (HeLa, SiHa, U2OS, HeLa A1, HeLa C2, HeLa-mKate2, SiHa-mKate2), RPMI 1640 (RKO), or McCoy’s 5A medium (HCT116) (Gibco, Thermo Fisher Scientific), containing 4.5 g/L glucose (if not specified otherwise), 10% fetal bovine serum (PAN-Biotech) and 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Sigma-Aldrich) at 37 °C, 5% CO₂, and 21% O₂. Authentication of the cell lines was performed by single-nucleotide polymorphism profiling within the last year (Multiplexion GmbH), and all experiments were performed with cells validated to be negative for mycoplasma.

Live imaging experiments were performed on intact brains. Larvae expressing nuclear membrane and spindle marker (Klaroid::GFP and UAS-Cherry::Jupiter) together with UAS-Dicer, UAS-Sdc RNAi (VDRC 13322) and worniu-GAL4 were dissected seventy-two or ninety-six hours after egg laying, respectively, in imaging medium (Schneider’s insect medium mixed with 10% fetal bovine serum (FBS) (Sigma, F7524), 2% PenStrepNeo (Sigma, P4083), 0.02 mg/mL insulin (Sigma, 11070), 20mM L-glutamine (Sigma, G8540), 0.04 mg/mL L-glutathione (Sigma, G4251) and 5 mg/mL 20-hydroxyecdysone (Sigma, H5142)) warmed up to room temperature before use. Brains were then transferred onto IbiTreat micro-slide 15 well 3D (Ibidi, 81506) and imaged with a confocal spinning disc. Neuroblasts were imaged in the brain lobes, and we did not distinguish between type I and II neuroblasts. All images presented in Fig 4 are single z sections. Anaphase onset is defined as t=0.

A mixture of 0.5 mL heparinized blood and 4.5 mL RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 2 mM L-glutamine (Sigma, St. Louis, MO, USA), 10% fetal bovine serum (Gibco, São Paulo, Brazil), 100 μg/mL antibiotic-antimycotic (Sigma, St. Louis, MO, USA) and 2% phytohemagglutinin (Sigma, St. Louis, MO, USA) was prepared. Aliquots were incubated at 37 °C for 44 h under 5% CO₂. After incubation, 6 μg/mL cytochalasin B (Sigma) was added to inhibit cytokinesis. Lymphocytes were then collected by centrifugation at 1200 rpm for 8 min, fixed in methanol/acetic acid solution (25:1, v/v), mounted on clean slides and stained with Diff-Quik (Medion Diagnostics, Düdingen, Switzerland). To assess DNA damage and chromosome instability indices, we examined 2000 binucleated (BN) cells per blood sample, with 1000 cells evaluated from each duplicate culture slide. Endpoints included micronuclei (MNBN), nucleoplasmic bridges (NPB), and nuclear buds (NBUDs), which were analyzed by light microscopy at 200–1000× magnification [51 (link)]. All slides were coded with unique identifiers to ensure unbiased analysis according to the criteria described by Fenech et al. [52 (link)].

The dual luciferase assay was conducted in cultured HEK293T cells (American Type Culture Collection ATCC) seeded in Corning white-walled 96-well adherent plates (Sigma-Aldrich) in phenol-red-free Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, UK), supplemented with L-glutamine (2 mM) (Sigma-Aldrich, UK), sodium pyruvate (1 mM) (Thermo Fisher Scientific), 10% heat-inactivated foetal bovine serum (HI-FBS) (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Sigma-Aldrich). Cells were transfected with Transit-LT1 (Mirus Bio) at 24 h post-seeding with 50 ng of dual luciferase reporter, 75 ng of RNA editing effector and 150 ng of gRNA. Positive and negative control wells were included on all plates, transfected with identical quantities of luciferase plasmid, RNA editing effector and non-targeting gRNA plasmids as in the experimental plates. All transfected plasmid preparations were made with endotoxin-free mini- (Zymo Research, Germany) or maxi-prep kits (Qiagen).
In the dual luciferase plasmid, a single SV40 promoter drives the production of a transcript encoding Renilla as a normalisation control, a ‘target cassette’ containing the mutation of interest with flanking F2A sequences, Firefly as a reporter of editing activity and a SV40 PolyA site from 5′ to 3′. The two F2A sequences enable co-translational excision of the target cassette peptide, encoding Renilla and Firefly proteins independent of the identity of the intervening target cassette sequence. At baseline, Firefly is turned off as the presence of the termination codon stalls translation after the production of Renilla but before the production of Firefly. Repair of the premature stop codon to a non-stop amino acid codon allows translation to proceed and Firefly is turned on.
The Dual-Glo luciferase assay kit (Promega, Madison, WI, USA) was used to detect luciferase expression at 48 h post-transfection according to the manufacturer’s instructions. Briefly, plates were allowed to come to room temperature, and 50 µL of media was removed. For cell lysis and detection of Firefly activity, 60 µL of Dual-Glo luciferase reagent containing Firefly luciferase substrate was added to each well and left to incubate at room temperature for 15 min. Luminescence was recorded using a FLUOstar Omega microplate reader (BMG Labtech, Germany). For Renilla detection, 60 µL of Stop & Glo reagent was added to quench Firefly activity and provide Renilla luciferase substrate, left to incubate for 15 min and luminescence recorded.
Mean background Firefly and Renilla luminescence was calculated from transfection-reagent-only wells and subtracted from all raw values. The editing efficiency is calculated from a ratio of Firefly to Renilla luciferase activity, determined for each well and normalised between the positive control and negative control ratios. All assays were performed in technical duplicates, and the experimental sample ratio is the mean of the duplicates for that condition.