Doxorubicin

To ensure doxorubicin entry, cells on coverslips were treated with 10 μm doxorubicin (MedchemExpress) for 2 h, then washed with PBS. doxorubicin autofluorescence (Ex/Em = 488/600 nm) was monitored using total internal reflection fluorescence (TIRF)/spinning disk confocal microscopy (Carl Zeiss, TIRF 3/Cell Observer SD; Imaging Core, First Core Labs, NTU College of Medicine). FIJI (ImageJ; NIH, Bethesda, MD, USA) was used to quantify fluorescence intensity in regions of interest.

Male C57BL/6 mice (6–8 weeks old; weighing 18–22 g) were obtained from Shanghai Jiesjie Laboratory Animal Co., LTD. Following a 1‐week acclimation period, the mice were randomly assigned to four groups (n = 5 per group): a control group (sham+Saline), a doxorubicin‐treated group (sham+Dox), a mitochondrial injection control group (mito+Saline) and a mitochondrial injection group with doxorubicin‐treated (mito+Dox). Mice in the Dox and mito+Dox groups received an intraperitoneal injection of doxorubicin (5 mg kg⁻¹; MedChem Express) once a week for 4 weeks, while the Saline group received an equivalent volume of PBS. The mito+Dox group also received mitochondrial transplantation 1 day before each doxorubicin injection, with a total of 50 000 isolated mitochondria suspended in 80 µL (20 µL per injection site), as previously described.^[10 (link)
^]To obtain the cardiomyocyte‐specific Sting knockout mice, sting‐floxed mice were crossed with Myh6‐Cre mice as previously described. Age matched Sting^flox/flox/Cre− and Sting^flox/flox/Cre+ mice were used for the experiment. To overexpress mito‐DNase1, 2 weeks prior to Dox induction, mice received a 1E11 vg single i.v. injection of adeno‐associated serotype 9 (AAV9) virus carrying cardiac troponin T (cTnT) promoter‐driven mito‐DNase1 cDNA (AAV9‐mito‐DNase1). Empty AAV9‐cTnT served as vector control (AAV9‐Vector) (Genomeditech).
Transthoracic echocardiography was performed using the Vevo3100 High‐Resolution Imaging System (FUJIFILM VisualSonics) by an experienced investigator. Anesthesia was induced with 2% isoflurane and maintained with 0.5%–1.0% isoflurane to keep the heart rate between 410 and 450 beats per minute. M‐mode images from the left ventricular long‐axis view were obtained at the mid‐papillary muscle level. Echocardiographic data were analyzed using the VevoLAB Version 3.0 software package (FUJIFILM VisualSonics), and left ventricular ejection fraction and fractional shortening were measured as described.^[20 ,
⁵⁵ (link)
^]At the sixth week (4 weekly Dox injections and 2 weeks rest), echocardiography was performed, and ventricular tissues were collected from euthanized mice. Hearts were subjected to Langendorff perfusion as previously described. Ventricular cardiomyocytes were seeded onto laminin‐coated (1:100 in dH₂O; Sigma) 96‐well confocal glass‐bottom plates and used for MitoTracker Green (Thermo), MitoSox (Thermo), and TMRM (Thermo) live‐cell imaging on an Operetta CLS High Content Imaging System (PerkinElmer). The cells were imaged over time using the Operetta CLS High Content Imaging System (PerkinElmer).

The topoisomerase II inhibitor doxorubicin (MedChemExpress) was used as previously described [70 (link)]. Cytotoxicity was evaluated in GSTC cells by the CCK-8 assay [71 (link)]. Confluent cells in 96-well plates were treated with increasing concentrations of doxorubicin ranging from 0 to 300 nM for 24 h. After incubation, CCK-8 solution (Dojindo, Japan) was added and incubated for another 4 h. Colorimetric measurements were performed on a microplate reader at 450 nm. The cell viability was calculated for each concentration as OD_T/OD₀, where OD_T and OD₀ correspond to the absorbance of drug-treated and non-drug-treated cells, respectively.
For the drug inhibition assay, confluent GSTC cells in 24-well plates were treated with different concentrations of doxorubicin for 1 h, followed by infection with recombinant ADRV expressing the mCherry tag at its TK gene locus (no published data) at 0.5 MOI. The insertion of exogenous genes in the TK locus of ranavirus has been proven to have no effect on virus replication in vitro. The infected cells were incubated for another 24 h with the drug. After incubation, the cells were observed under an Olympus IX73 fluorescence microscope and collected for virus titer and genome copy number determination.
The virus titers were determined using a 50% tissue culture infectious dose (TCID₅₀) assay as described previously [63 (link)]. Total DNA was extracted with the TakaRa MiniBEST Universal Genomic DNA Extraction Kit (TakaRa, Japan). Viral genomes, based on detection of the relative numbers of major capsid protein gene (MCP), were detected by qPCR conducted using a StepOne Real-Time PCR system (Applied Biosystems, USA) as described previously [68 ]. The β-actin gene was used as a loading control, as in a previous study [72 (link)]. MCP levels were normalized to β-actin levels in each sample. The level of the viral genome (MCP level) in the treated group versus that in the control group (no drug) was calculated by the 2^−ΔΔCT method [73 (link)]. All experiments here and above were performed in triplicate. Data were presented as means ± SD. P values were calculated by using Student’s t test.

NOZ cells were obtained from the Health Science Research Resources Bank (Osaka, Japan), GBC-SD, SGC-996, and EH-GB1 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, and human embryonic kidney 293 T (HEK293T) cells were purchased from the American Type Culture Collection. NOZ and GBC-SD both were P53 WT genotype (P53^+/+). GBC-SD, SGC-996, EH-GB1, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco), and NOZ cells were cultured in William’s E medium (Gibco). All cell lines were supplemented with 10% fetal bovine serum (Gibco), penicillin (100 mg ml⁻¹) and streptomycin (100 mg ml⁻¹) and were incubated in a humidified chamber with 5% CO₂ at 37 °C. All cell cultures were ensured to be mycoplasma-negative cultures by monthly mycoplasma tests and were passaged with 0.25% trypsin containing 2.21 mm ethylenediaminetetraacetic acid (EDTA) in PBS when the cells reached 80~90% confluency. Gemcitabine (GEMZAR) was purchased from Eli Lilly. Cisplatin, cycloheximide, doxorubicin, and puromycin were purchased from MedChem Express.