Ms column

Mouse cardiomyocytes isolation was performed by langendorff perfusion method, which was described in details previously (21 (link)). Cardiac fibroblast and endothelial cell isolation was performed using MACS cell separation (Miltenyi Biotech) following the manufacturer’s instructions. Briefly, the mouse heart was dissected and minced into small pieces. Collagenase type I (Worthington LS004196) was used for digestion in 37°C for 1 h. After 1 h incubation, a strainer was used to remove large particle and undigested tissues. RBC lysis buffer (ab204733) was used to remove red blood cells. Next, CD45 microbeads (Miltenyi Biotec, 130-052-301) and MS column (Miltenyi Biotec, 130-042-201) were used to remove leukocytes. To isolate cardiac fibroblast, CD90.2 microbeads (Miltenyi Biotec, 130-049-101) and MS were used. CD90.2 positive cells were collected from MACS column. CD31 microbeads (Miltenyi Biotec, 130-097-418) and MS column were used to isolate endothelial cells from flow through samples. The enrichment of the target cells was validated by qRT-PCR.

Isolation of lysosomes was conducted as described previously (Eguchi et al., 2018 (link); Komori et al., 2023 (link)). Cells on a 10-cm dish were cultured in DMEM containing 1 mM HEPES-NaOH (pH 7.2) and 10% dextran-coated magnetite (DexoMAG 40; Liquids Research Ltd.) for 24 h, and then chased in normal media for 24 h. Cells were harvested with trypsin, centrifuged at 60 × g for 5 min, washed with ice-cold PBS, lysed in 2 ml of ice-cold Buffer A (1 mM HEPES, 15 mM KCl, 1.5 mM Mg(Ac)₂, 1 mM DTT, protease/phosphatase inhibitors) with a Dounce homogenizer, and passed through a 23G needle for eight times. After homogenization, 500 μl of ice-cold Buffer B (220 mM HEPES, 375 mM KCl, 22.5 mM Mg(Ac)₂, 1 mM DTT, 0.1 mM sucrose, and 50 μg/ml DNase I) was immediately added, and samples were inverted for five times, incubated for 5 min, and then centrifuged at 400 × g for 10 min. The supernatant was then applied to an MS Column (Miltenyi Biotec) set on an OctoMACS separator (Miltenyi Biotec) and equilibrated with 0.5% BSA in PBS and the flow through was collected. 1 ml of DNase I solution (50 μg/ml DNase I, 0.1 mM sucrose in PBS) was added and the column was incubated for 10 min and washed with 1 ml of ice-cold sucrose buffer (0.1 mM sucrose in PBS). After removing the column from the OctoMACS separator, lysosomes were eluted with 250 μl of ice-cold sucrose buffer using a plunger.

Isolation of CD45⁺ leukocytes and α‐SMA⁺ fibroblasts from peritumoral liver and HCC tissues using MicroBeads and an MS Column (Miltenyi Biotec, Bergisch Gladbach, Germany) was performed according to the manufacturer's instructions. Phenotypic characterization of the isolated cells was conducted using flow cytometry as previously described 24. Details are included in the supplementary material, Supplementary materials and methods.