Mgcl2

This study adopted the prototype Fully Automated Nucleic Acid Amplification Testing System (FANAT-1), as well as primers and probes (Sangon Biotech) and nucleic acid extraction kit (QIAGEN). The basic PCR system contains High-Affinity HotStart Taq and TIANSeq M-MLV (defined as N enzyme, TIANGEN Biotech, Cat nos. ET108 and NG212, separately), MgCl₂ (25 mM) (Sangon Biotech, Cat no. B601193), One-Step PrimeScript III (defined as A enzyme, Takara Biotech, Cat no. RR601A), Anstart One-Step RT-PCR Mix (heat-labile UDG) (defined as E enzyme, Fapon Biotech, Cat no. MD013P), and 5 × Neoscript RT Premix-UNG (Probe qRT-PCR) (DG) (defined as F enzyme, Biori Biotech, Cat no. FM5254).

DNA was extracted from 25–30 mg of dried leaf material following the methods from Doyle & Doyle⁴³ with a minor modification. Using Liu et al.^{44 (link)} as a reference, we selected 12 pairs of SSR primers, fluorescently labeled the forward primers with Fam, Rox, and Hex, which were produced by Sangon Biotech (Shanghai, China). PCR was performed in 10 μl volume, which included ca. 20 ng of DNA, 0.2 μM of primers, 2.5 mM of MgCl₂, 0.2 mM of dNTP (Sangon Biotech (Shanghai, China)), 1 × Taq of buffer, and 0.5 U of Taq polymerase. The PCR was carried out in a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA: ABI) and proceeded as follows: 4 min at 94 °C; then 35 cycles for 45 s at 94 °C, 30 s at 55–62 °C, 45 s at 72 °C; then a final extension step of 10 min at 72 °C. Alleles were sized on a Hitachi ABI 3730 (ABI) automated sequencer using LIZ 500 (ABI) as a ladder and analysed in Genemapper 4.0 (ABI).

Cu(NO₃)₂, Na₂HPO₄, sodium ascorbate, boric acid, 1,4-dithiothreitol (DTT), and ethylenediaminetetraacetic acid disodium salt were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Tris(hydroxymethyl) aminomethane, NaCl, and MgCl₂ were bought from Sinopharm Group Co., Ltd. (Shanghai, China). HCl was bought from Hengyang Kaixin Chemical Reagent Co., Ltd. (Hengyang, China). SYBR Green I and DNA Marker were bought from Sangon Biotechnology Co., Ltd. (Shanghai, China). λ-Exo was obtained from New England Biolabs. Ltd. (Beijing, China). UO₂(NO₃)₂, Pb(NO₃)₂, HgCl₂, AgNO₃, CrCl₃, AlCl₃, FeCl₃, and KCl were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the conventional chemical reagents were of analytical grade. The DNA oligonucleotides used were ordered from Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table S1).
The types of buffers used were as follows: buffer A (50 mM Na₂HPO₄ and 500 mM NaCl; pH 7.6), buffer B (50 mM Na₂HPO₄ and 50 mM NaCl; pH 7.6), buffer C (25 mM Tris-HCl, 50 mM NaCl, 2 mM MgCl₂, and 1 mM DTT; pH 9.4), and buffer D (50 mM Na₂HPO₄ and 1 mM EDTA; pH 7.6). Among these, buffer A was employed as the DNA stock solution (10.0 μmol·L⁻¹) of oligonucleotides, while the others were used as DNA reaction buffers. The solvent in each step was deionized water (DI water, 18 MΩ·cm) from a Milli-Q water purification system.

The expression of human placental SEAP in a cell culture medium was quantified using a p-nitrophenyl phosphate-based light absorbance time course assay. Briefly, 120 µL of substrate solution [100 µL of 2 × SEAP buffer (pH 9.8) containing 20 mM L-homoarginine hydrochloride (catalog no. A602842, Sangon Biotech), 1 mM MgCl₂ (catalog no. A610328, Sangon Biotech), and 21% (v/v) diethanolamine (catalog no. A600162, Sangon Biotech), and 20 µL of substrate solution containing 120 mM p-nitrophenyl phosphate (catalog no. 333338-18-4, Sangon Biotech)] were added to 80 µL of heat-inactivated (65 °C, 30 min) cell culture supernatant^{69 (link)}. The time course of absorbance at 405 nm was measured at 37 °C using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments) with Gen5 software (version 2.04).

Brusatol (HY-19543, purity 99.89) and polydatin (HY-N0120A, purity 98.55) were obtained from MedChemExpress (Monmouth Junction, NJ, USA). DMSO was obtained from Beyotime Biotechnology (Shanghai, China). CH₃COOK, MgCl₂, DTT, NaCl were fetched from Sangon Biotech (Shanghai, China). Triton X-100 was purchased from Aladdin (Shanghai, China). DMEM and RPMI-1640 medium were obtained from Biological Industries (Kibbutz Beit Haemek, IL, USA). Fetal bovine serum (FBS) was obtained from MIKX (Shenzhen, China). CCK-8 kit was obtained from Yeasen (Shanghai, China). The commercial antibodies used in the study: Nrf2 and LaminA/C were obtained from Cell Signaling Technology (Danvers, MA, USA). GAPDH is obtained from Proteintech (Wuhan, China). HRP-conjugated affinipure goat anti-rabbit/mouse IgG (H+L) secondary antibodies were obtained from Proteintech.
Human BC MDA-MB-231 cells and SUM159 cells were purchased from Procell Life Science & Technology Co., Ltd. (CL-0150B and CL-0622, Wuhan, China), MDA-MB-231 cells were cultured in DMEM with high glucose (BI) supplemented with 10% FBS and 1% of penicillin/streptomycin (Beyotime) at 37 °C and 5% CO₂. SUM159 cells were cultured in RPMI-1640 medium (BI) supplemented with 10% FBS and 1% of penicillin/streptomycin (Beyotime) at 37 °C and 5% CO₂.

Cells were lysed in NP-40 buffer containing 50 mM Tris-HCl (pH 7.5) (Sigma, St Louis, MO, USA), 150 mM NaCl (Sangon, Shanghai, China), 0.5% Nonidet P-40 (Sigma), 1 μg/ml aprotinin (Sigma), 1 μg/ml leupeptin (Sigma), 1 μg/ml pepstatin (Sigma), 1 mM Na₃VO₄ (Sigma) and 1 mM PMSF (Sigma). For immunoprecipitation, 500 μl of cell lysate was incubated with HA antibody (provided by the Zhao lab of Fudan University) for three hours at 4°C with rotation. Then, 30 μl Protein A Agarose (Millipore) was added for 12 hours at 4°C with rotation, and the beads were washed three times with lysis buffer before proteins were dissolved in loading buffer. Deacetylation assays were carried out in the presence of 5 μg enzyme and 0.3 μg peptide in 30 μl reaction buffer (30 mM HEPES (Sigma), 0.6 mM MgCl₂ (Sangon), 1 mM DTT (Sigma), 1 mM NAD⁺ (Sigma), 10 mM PMSF (Sigma)). The deacetylation reaction was incubated for 3–5 hours at 37°C before the mixture was desalted by passing it through a C18 ZipTip (Millipore). The desalted samples were analyzed using a MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Grand Island, NY, USA). The acetylated peptide used in the assay was GILRRLKK^AcYDNCWL (Glssale, Shanghai, China).

CdCl₂, ZnCl₂, MgCl₂, MnSO₄, CaCl₂, NiSO₄, CuSO₄, EDTA, dithiothreitol (DTT), sodium dodecyl sulfate(SDS), nucleotide triphosphates (NTPs), and deoxynucleotide triphosphates (dNTPs) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Amino acids, ATP Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China).