Hoechst staining kit

Radiotherapy, 8 μg/mL of BJOE, or both radiotherapy and 8 μg/mL of BJOE were used to treat EC109 cells, respectively. Hoechst Staining Kit (C0003, Beyotime, Shanghai, China) was used to detect apoptosis according to the manufacturer’s instructions.

Sodium hydrosulfide (NaHS) and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Specific monoclonal anti-PCSK9 (cat. no. 55206), anti-LDLR (cat. no. 66414), anti-SREBP-2 (cat. no. 557037) and anti-SREBP-1-c (cat. no. 66875) antibodies were purchased from Proteintech (Rosemont, IL, USA). Specific monoclonal anti-protein kinase B (Akt) antibody (cat. no. BM4400) was purchased from Wuhan Boshide Bioengineering Co. (Wuhan, China). Specific monoclonal anti-phosphorylated Akt antibody (cat. no. YM3621) was purchased from ImmunoWay Biotechnology (Plano, TX, USA). Anti-β-actin antibody (cat. no. LCA01) was purchased from Aijia Biological Technology Co., Ltd. (Changsha, China). Cy3-conjugated goat anti-rabbit IgG secondary antibody (cat. no. E031620) was purchased from EarthOx Life Sciences (Millbrae, CA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biocompany (Hangzhou, China). The Hoechst Staining kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled LDL (DiI-LDL) was purchased from Haoyuan Biological Technology Co., Ltd. (Guangzhou, China). siRNAs were synthesized by Ruibo Biotechnology Co., Ltd. (Guangzhou, China).

NCI-H1299 cells were inoculated onto coverslips at a density of 5 ×10⁴ per well in 12-well plates. When reached to 80% confluences, the suspension was decanted and cells were treated with 10 µM TBMS1 for 48 h. Hoechst staining assay was performed with the Hoechst Staining kit (Beyotime Institute of Biotechnology) following the manufacturer instructions. Briefly, the cells on coverslips were fixed for 20 min using indicated stationary liquid and stained at room temperature for 5 min using a total of 0.5 ml Hoechst 33258 solution with dropwise addition. Then the coverslips were mounted inversely onto slides with indicated anti-fluorescein quencher and observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

The Hoechst staining kit (Beyotime Institute of Biotechnology, Haimen, China) was used to detect the state of nucleus condensation. Cells were seeded onto cover slides in 6-well plates overnight at 37°C and then treated with 12.5 µg/ml of cisplatin for 48 h followed by adding 0.5 ml of fixation fluid for 30 min. After washing with phosphate-buffered saline (PBS) twice, 0.5 ml of Hoechst 33258 were added to the plate and incubated for 5 min. The stained cells were washed with PBS twice and then the nuclear morphology was observed under a fluorescence microscope (Zeiss AG, Oberkochen, Germany). The apoptotic cells showed condensed and fragmented nuclei.