Monobromobimane

For the quantification of the anthocyanin content, approximately 40–70 mg of leaf tissue was homogenized in 1 ml of propanol:HCl:water (18:1:81) and further extracted in a boiling-water bath for 3 min. The mixture was centrifuged at 5000 g for 40 min. The absorbance of the supernatant was measured at 535 and 650 nm, and referred to the fresh weight as described (Laureano-Marín et al., 2016 (link)).
To quantify hydrogen peroxide, 50 mg of leaf tissue was ground in liquid nitrogen with 250 μl of 50 mM phosphate buffer, pH 7.4, vortexed and shaken continuously at room temperature for 30 min. Samples were centrifuged at 4 °C at 12 000 g for 10 min. Fluorescence quantification of H₂O₂ was performed in the supernatant after incubation with Amplex Red reagent (Thermo Fisher Scientific) and horseradish peroxidase using 560 nm excitation and 590 nm emission filters. Standard calibration curves were obtained with known H₂O₂ concentrations.
To quantify the total Cys and glutathione contents, thiols were extracted, reduced with NaBH₄, and quantified by reverse-phase HPLC after derivatization with monobromobimane (Thermo Fisher Scientific) as described previously (Dominguez-Solis et al., 2001 (link)).

Buffer A consists of 20 mM Tris and 300 mM NaCl, adjusted to pH 7.4 unless otherwise stated.
Buffer B consists of 300 mM NaCl, 2.7 mM KCl, 5.3 mM Na₂HPO₄ and 1.5 mM KH₂PO₄, adjusted to pH 8 unless otherwise stated. Both buffers were supplemented with 0.02% DDM (Anatrace, Maumee, OH) when specified.
Buffer C consists of 15 mM Na₂HPO₄, 150 mM NaNO₃ adjusted to pH 7.
Pre-mix buffer consists of 10 mM Na₂HPO₄, 140 mM NaNO₃ adjusted to pH 7 unless otherwise stated.
Quenching buffer consists of 10 mM Na2HPO4, 90 mM TlNO3, 50 mM NaNO3 adjusted to pH 7 unless otherwise stated.
MBS buffer consists of 88 mM NaCl, 1 mM KCl, 2.5 mM NaHCO₃, 5 mM HEPES, 0.7 mM CaCl₂ and 1 mM MgSO₄.
MES buffer consists of 100 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 10 mM MES.
CHO labeling buffer consists of 150 mM NaCl, 8.1 mM Na₂HPO₄, 1.9 mM NaH₂PO₄, 0.1 mM CaCl₂ and 1 mM MgCl₂, adjusted to pH 7.4.
CHO conservation buffer consists of 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES and 8 mM glucose, adjusted to pH 7.4.
Monobromo bimane (ThermoFisher Scientific, Pittsburgh, PA) was dissolved in 100% DMSO at 10 mM and stored at −20° C.
Bimane Bunte salt was dissolved at 50 mM in water and stored at −80° C.
Unless otherwise stated, all chemicals were purchased from Sigma Aldrich (St Louis, MO).

BPTES (HY-12683), CBR-5884 (HY-100012), SHIN1 (HY-112066), DS18561882 (HY-130251), and sorafenib (HY-10201) were purchased from MedChemExpress. Trolox (S3665), lenvatinib (S1164) were from selleckchem. Crystal violet (V5265), H₂DCFDA (D399), MitoSOX^TM Red (M36008), monobromobimane (mBBr, M1378), Propidium Iodide (P3566), RNase A (10109169001), (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT, M6494), Hoechst 33342 (H3570), and Lipofectamine^TM RNAiMAX Transfection Reagent (13778150) were obtained from Thermo Fisher Scientific. H₂O₂ (216763) was purchased from Sigma-Aldrich. Triton X-100 (0694) was purchased from AMRESCO. Primary antibodies against SHMT2 (sc-390641), PHGDH (sc-100317), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology. MTHFD2 (98116 S) was acquired from Cell Signaling Technology. Secondary antibodies of the mouse and rabbit were from Cell Signaling Technology and Abcam respectively (7076 S and ab6721). Small interfering RNA (siCTRL and siATF4) oligonucleotides were purchased from Santa Cruz Biotechnology (sc-37007 and sc-35112, respectively).