Hoechst 33342

Fluorescent Dye C11 BODIPY 581/591 (D3861, Invitrogen) was used as lipid peroxidation sensor as per the manufacturer's instructions. Briefly, 50,000 cells were seeded on 35‐mm glass bottom dishes and then subjected to the indicated treatments upon attachment. After treatments, cells were stained with 10 μM lipid peroxidation sensor and Hoechst 33342 (Meilunbio, China) for 30 minutes. The cells were then washed for three times with PBS and then imaged on an Olympus IX81 inverted microscope using a 40× objective using filters for Hoechst, FITC, and Texas Red channels. The fluorescent signal was quantified by SlideBook™ 5.0 software, which set the ratio of signals from 510 and 590 channels to quantify lipid peroxidation in cells. Tert‐butyl hydroperoxide (TBH, 200‐500 μM for 1‐3 hours) treatment was as administered a positive control.
Lipid Peroxidation Assay Kit (ab118970, Abcam) was used to evaluate the relative concentration of malondialdehyde (MDA) in cell lysates following the manufacturer's instructions. Data were normalized by corresponding protein concentration.

Cellular proliferation was detected according to the instructions of a Click-iT EdU (5-Ethynyl-2′-deoxyuridine) Cell Proliferation Kit (Meilunbio, Dalian, China). pMSCs at P5, P10, and P15 were plated in a 24-well plate and cultured overnight. For labeling cells with EdU, an equal volume of 2× EdU solution was added to the cells, and the cells were incubated at 37 °C for 2 h. The samples were then fixed and permeabilized. The nuclei were stained using the Hoechst 33342 (Meilunbio, Dalian, China) fluorescent stain. Digital images were acquired using a laser confocal microscope (OLYMPUS, Tokyo, Japan), and the number of EdU-positive cells were calculated using Image-Pro Plus 5.1 software (MEDIA CYBERNETICS, Silver Spring, MD, USA). EdU incorporation (the ratio of EdU-labeled cells to total cells) indicated the cellular proliferation rate.

Internalization of the ADC drugs and colocalization of ADC with lysosomes were observed by confocal fluorescence microscopy. The ADC drug was labeled with AF488 using the Alexa Fluor® 488 Protein Labeling Kit (Thermo Fisher, A10235). BxPC-3 cells were cultured in confocal dishes and incubated with 1 μg/mL labeled ADC for a specific time. Then, lysosomes were stained with 50 nM LysoTracker™ Red DND-99 (Thermo Fisher, L7528) and nuclei were stained with Hoechst 33,342 (Meilunbio, MA0126). The cells were observed by confocal microscopy (Carl Zeiss, Carl Zeiss LSM710).

CTX, Dithiothreitol (DTT), coumarin-6, Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell culture media were purchased from Meilun Biotech Co. Ltd. (Dalian, China). Stearyl alcohol (SAT), dithiodiglycolic acid, 3,3′-dithiodibutyric acid, 4,4′-dithiodibutyric acid, 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI), hydroxybenzotriazole (HOBt) and 4-dimethylaminopyrideine (DMAP) were obtained from Aladdin, (Shanghai, China). Sterile cell culture dishes and centrifuge tubes were bought from NEST Biotechnology (Wuxi, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG_2K) was purchased from AVT (Shanghai) Pharmaceutical Tech Co. Ltd. (Shanghai, China). All other reagents and solvents mentioned in this article were of analytical grade.

Twenty natural pentacyclic triterpenes, quercetin and epicatechin were purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China), and their purities more than 98%. Dithiothreitol (DTT, ≥99%), S-adenosyl-L-methionine (SAM, ≥80%), magnesium chloride hexahydrate (MgCl₂^.6H₂O, ≥99%), entacapone (C₁₄H₁₅N₃O₅, ≥98%) and tolcapone (C₁₄H₁₁NO₅, ≥98%) were supplied by Sigma-Aldrich Co. LLC. (Shanghai, China). 3-(Benzo[d]thiazol-2-yl)-7,8-dihydroxy-2H-chromen-2-one (3-BTD, ≥99%), 3-(Benzo[d]thiazol-2-yl)-7-dihydroxy-8-methoxy-2H-chromen-2-one (3-BTMD, ≥99%) and recombinant human S-COMT were made in our laboratory [22]. Phosphate buffer saline (50 mM, pH 7.4), Dulbecco's modified eagle medium (DMEM, with high glucose, pyruvate and L-glutamine), penicillin/streptomycin (PS, sterile), cell counting kit-8 (CCK-8), Hoechst 33342 and JC-10 dye were obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Foetal bovine serum (FBS, Gbico^®) was purchase from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). Acetonitrile (ACN, ≥99.9%), dimethyl sulfoxide (DMSO, ≥99.7%) and formic acid (CH₃COOH, ≥98%) were of HPLC grade and purchased from Sigma-Aldrich Co. LLC. (Shanghai, China). Ultrapure water (18.2 MΩ cm) was produced using a Millipore water purification system (Milford, MA, U.S.A.).

Cells were seeded and grown on Ψ20mm glass-bottom cell culture dishes and treated as indicated. Next, the cells were stained with Cell Meter™ Autophagy Assay Kit (AAT Bioquest, CA, US) and Hoechst 33342 (Meilunbio, China) based on the manufacturer’s instructions. The images were visualized by a confocal scanning microscope and analyzed by Image J.

To observe the subcellular uptake of CAMNSN@PSN38, CT26 cells were seeded at a density of 1 × 10⁵ in glass-bottom dishes in a complete culture medium (1 mL, RPMI 1640, supplemented with 10% FBS, GIBCO) overnight. The cells were then incubated with fresh medium containing FITC-labeled CAMNSN@PSN38 ([SN38] = 5 µg mL^-1) for 2, 4, and 6 h at 37 °C. Subsequently, the cells were washed and stained with Hoechst 33342 and Lysotracker green (Meilunbio, China) for 15 min and were evaluated by confocal laser scanning microscopy (CLSM, Leica, TCS SP8, US).