Geneticin

Agrobacterium-mediated transformation of rice was performed as described previously (Toki et al., 2006 (link)). Rice (Nipponbare) was used for GT transformation following a method described previously (Saika et al., 2011 (link)). Calli transformed with Agrobacterium harboring pKOD4/mALS were selected on callus induction (N6D) medium solidified with 0.4% gelrite (Wako Pure Chemical Industries) containing 50 mg/L hygromycin and 25 mg/L meropenem (Wako Pure Chemical Industries, http://www.wako-chem.co.jp/). GT candidate calli were transferred to N6D medium without hygromycin and meropenem and cultured for 1 month. For marker excision, GT candidate calli were transformed with Agrobacterium to introduce an expression vector encoding hyPBase, and were selected on N6D medium with 35 mg/L geneticin (Nacalai tesque, https://www.nacalai.co.jp) and 25 mg/L meropenem. For regeneration, transgenic calli were transferred to regeneration medium with 25 mg/L meropenem, and shoots arising from callus were transferred to Murashige and Skoog (MS) medium (Murashige and Skoog, 1962 ) without phytohormones. The hyPBase-expressing regenerated plants were subjected to marker excision analysis. The T₁ progeny plants were obtained from self-pollinating marker-free T₀ plants containing two point mutations, W548L and S627I, in the ALS locus and were subjected to further analysis.

ZE cells were cultured in Leibovitz's L-15 medium supplemented with 2% FBS. To obtain stable transfectants, ZE cells were transfected with 4 μg of either JNK1 wild-type, JNK1-K55R mutant (DN), nSMase1 wild-type,^{16 (link)} or nSMase1-S270A mutant (substitution of serine-270 to alanine) constructs using FuGENE 6 Transfection Reagent (Roche) following the manufacturer's instructions. Transfected cells were selected with 0.8-mg/ml geneticin (Nakalai Tesque, Kyoto, Japan). Cell lines that overexpressed the JNK1 wild-type, JNK1-K55R mutant, nSMase1 wild-type,^{16 (link)} and nSMase1-S270A mutant stably and at high levels were established.

Briefly, 293F and 293T cells were transduced with retrovirus expressing mouse NRF1 (mNRF1) or its mutant molecule (WT, S-AA, A-SS, and A-AA) with the 3×FLAG tag at the C terminus in a suspension with 12 μg/ml Polybrene. H460 cells were transduced with retrovirus expressing doxycycline-inducible control or OGT shRNA (V3SH11252-227734651; Dharmacon) in a suspension with 12 μg/ml Polybrene. One day after infection, the infected cells were replated and incubated in a selection medium that contained 2 μg/ml puromycin (Sigma). To establish stable cell lines that expressed 3×FLAG-tagged mNRF1-FL, mNRF1-ΔbZip, mNRF1-M1, and mNRF1-M2, 293F cells were transfected with pCMV-3xFLAG-mNRF1-FL, pCMV-3xFLAG-mNRF1-ΔbZip, pCMV-3xFLAG-mNRF1-M1, and pCMV-3xFLAG-mNRF1-M2, respectively. After transfection, the cells were replated and incubated in a selection medium that contained 1.5 mg/ml Geneticin (Nacalai Tesque).