Df3727

Immunohistochemical reactions were carried out using the labeled streptavidin-biotin method. Tumors dissected from mouse xenograft models were fixed in 10% formalin solution overnight at 4 °C, and sections of 6 μm were cut and embedded in paraffin. Deparaffinized sections were heated for 5 min at 100 °C in a pressure cooker to reactivate the antigens and blocked with hydrogen peroxide. After being washed three times, the samples were incubated with antibodies at 4 °C overnight. The primary antibodies included anti-ACSS1 (Affinity, DF3727, 1:200), anti-VDAC1 (Affinity, DF6140, 1:200), and anti-SIRT3 (Affinity, AF5135, 1:200). After incubation with a secondary antibody at room temperature for 1 h, the samples were washed, and staining was developed by incubation with a DAB substrate kit (Pierce). Immunostained sections were assessed and photographed under a microscope at magnification of 200× (IX71, Olympus, Japan).

HepG2 (3×10⁵) and L02 (3×10⁵) cells were seeded into dishes and co-cultured with or without SWNHs for 48 h. Then, total protein was extracted with a protein extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The concentration of these proteins was monitored by the BCA protein assay (Thermo Fisher Scientific, Inc.). The proteins extracted from each sample were subjected to 10% SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). Next, the membranes were incubated with primary antibodies [including acetyl-CoA synthetase 2 (AceCS2) (DF3727, 1:200), SCNN1A (DF9199, 1:200), SIRT3 (AF5135, 1:200), VDAC1 (DF6140, 1:200) (all from Affinity Biosciences, Cincinnati, OH, USA), Cyt-C (sc-13561, 1:50), Bax (sc-4239, 1:50) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)] and secondary antibodies (S0001, 1:5,000; Affinity Biosciences) at 4°C overnight. The blots were projected onto X-ray film (Carestream, Shanghai, China) following visualization by chemiluminescence using the ECL kit (EMD Millipore, Billerica, MA, USA). Image Pro Plus 6.0 software (Media Cybernetics) was used to measure the intensity of protein bands.