L histidinol dihydrochloride

To culture hTERT RPE-1 cells (ATCC, CRL-4000), DMEM/F-12 (Gibco, #31331-028) containing 0.5% sodium bicarbonate, supplemented with 10% foetal bovine serum (Gibco, #10500-064), 1% (v/v) Penicillin-Streptomycin (Gibco, #15140-122) and 10 µg/ml Hygromycin B Gold (InvivoGen, #ant-hg-1) was routinely used. Cells were cultured at 37°C, 5% CO₂. Transfection was conducted using Lipofectamine 3000 Reagent (Invitrogen, #L3000001) following the manufacturer's instruction. To select clones with ER^T2-Cre-ER^T2 integration at AAVS1 locus, puromycin (InvivoGen, #ant-pr-1) was added to the media at a final concentration of 6–8 µg/ml. To select clones with a LoxP cassette integration at KRAS gene locus, L-histidinol dihydrochloride (Sigma-Aldrich, #H6647) was added to the media at a final concentration of 1.35–1.5 mg/ml. For doxycycline treatment, doxycycline (Alfa Aesar, #J60422) was dissolved in H₂O at 1 mg/ml and used at a final concentration of 1 μg/ml in the medium. For 4-OHT treatment, 4-OHT (Merck, #H6278) was dissolved in ethanol at 1 mM and used at a final concentration of 0.5 μM in the culturing medium.

2 × 10⁶ CHO(DYS‐HAC2) cells were co‐transfected with p17 (8 μg) and Cre‐expressing plasmids (1 μg) using Lipofectamine 2000 (Invitrogen) to induce insertion of p17 into DYS‐HAC2 via Cre/LoxP system and generate DYS‐HAC3. 24 h after transfection, CHO cells were expanded with a hypoxanthine–aminopterin–thymidine (HAT)‐supplemented medium (Sigma‐Aldrich) to select only CHO cells containing DYS‐HAC3. CHO(DYS‐HAC3) cells were further transfected with pP‐ΔHR and Bxb1‐expressing plasmid following the same protocol detailed above. Selection was performed according to the following: 8 μg of blasticidin S (Wako), 800 μg of G418 (Promega), 7 mM of L‐histidinol dihydrochloride (Sigma‐Aldrich) and HAT medium.