Catalase

Dried ripe fruits of Gardenia jasminoides J.Ellis (Fructus Gardeniae) (No. 180525) and the fermented ripe seeds of Glycine max (L.) Merr. (Semen sojae praeparatum) (No. 180716-1) were purchased from Tong Han Chun Tang Chinese Herbal Factory (Shanghai, Chinese). Authenticated by Professor Lu-Ping Qin, Fructus Gardeniae (Voucher number 2018082001) and Semen sojae praeparatum (Voucher number 2018082002) were deposited at the herbarium of pharmaceutical analysis, School of Pharmacy, Second Military Medical University, Shanghai, China. Fluoxetine was purchased from Eli Lilly and Company (China). Sodium carboxymethyl cellulose (CMC-Na) and sucrose were obtained from Sangon Biotech Co., Ltd. (Shanghai). LC-MS grade formic acid, acetonitrile, and methanol were supplied by Honeywell (United States). Urethane was purchased from meilunbio (China). Deionized water was collected by a laboratory water purification system (HITECH Instruments CO., Ltd.). Reactive Oxygen Species, Glutathione and Glutathione disulfide, catalase, glutathione reductase, glutathione peroxidase, and SOD assay kit were purchased from Beyotime (China).

4T1 cells were seeded into a 96-well culture plate (1 × 10⁴/well) or a 6-well culture plate (4 × 10⁵/well) followed by 12 h starvation. Cells were pretreated with Yoda1 (Selleck, China), GSK2795039 (Selleck, China), or catalase (Beyotime, China) for 1 h and then incubated at 37 or 43°C for 5 h. After the heat treatment, the plates were returned to a 37°C incubator for 24 h as recovery. Subsequently, cells were harvested for the following experiments. All incubation temperatures were maintained within ±0.01°C.
As for the experiments of photothermal nanomaterials (TiCN), cells were incubated in a medium with TiCN (100 μg/mL) after 12 h starvation. Then, the cells were given local irradiation with a 1,064 nm laser (0.6 W/cm²) for 7 min/well. After the heat treatment, the plates were returned to a 37°C incubator for 24 h and harvested for the following tests.
In the tumor models, 4T1 cells were used to establish the model, 7 days later, 200 µl of photothermal TiCN (500 µg/mL) were intravenously injected into the 4T1 tumor-bearing mice. One day later, the breast tumors of mice were exposed to a 1,064 nm laser (0.6 W/cm²) for 10 min, and then, the volumes of the tumors were measured daily. Yoda1 was administrated 1 h before laser exposure (1.5 mg/kg, intraperitoneal injection). Ten days later, the mice were sacrificed and the tumors were harvested for measurement.

FAC, calcein acetoxymethyl ester (CA-AM), deferiprone (L1) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (USA). DFO was from Novartis Pharma (Switzerland). NAC, Catalase, Beclin-1, ATG5 and ATG7 were from Beyotime Biotechnology (Shanghai, China). AICAR was from Med Chem Express (USA). AMPK, p-AMPK (Thr172), p-Drp1 (Ser616), p-Drp1 (Ser637), p-ACC (ser79), LC3, GAPDH and VDAC1 were from Cell Signalling Biotechnology (Danvers, MA, USA). MFF was from Abcam (Cambridge, MA, USA). p-MFF (ser155) was generated by YenZym Antibodies (South San Francisco, CA, USA). TOM20 and Drp1 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) secondary antibody and MitSOX^TM Red Mitochondrial Superoxide Indicator were from Thermo Scientific (Rockford, IL, USA).