Journal: International Journal of Molecular Medicine

Article Title: The marine natural product, dicitrinone B, induces apoptosis through autophagy blockade in breast cancer

doi: 10.3892/ijmm.2022.5186

Figure Lengend Snippet: DB disrupts the autophagic flux. (A) Representative western blots and corresponding protein quantification plots of LC3-I/II protein expression in MCF7 and MDA-MB-231 cells following treatment with DB in the presence or absence of 5 µ M RAPA or 60 µ M CQ for 6 h. β-actin was used as the loading control. * P<0.05, ** P<0.01 and *** P<0.001. (B) Representative western blots and corresponding protein quantification plots of SQSTM1/p62 protein expression in MCF7 and MDA-MB-231 cells following treatment with increasing concentrations of DB for 6 h or for different time periods (0-24 h) with 20 µ M DB. β-actin was used as a loading control. (C) Representative images of MCF7 and MDA-MB-231 cells treated with 20 µ M DB in the presence or absence of 5 µ M RAPA or 60 µ M CQ for 6 h and subjected to anti-LC3-II (red), anti-SQSTM1/p62 (green) and 4,6-DAPI (blue) staining. Scale bars, 10 µ m. LC3, microtubule associated protein 1 light chain 3; RAPA, rapamycin; CQ, chloroquine; DB, dicitrinone B; SQSTM1, sequestosome 1; DAPI, diamidino-2-phenylindole.

Article Snippet: To analyze autophagic flux, ~10,000 MCF7 and MDA-MB-231 cells were plated in 96-well plates, incubated at 37°C in a humidified atmosphere with 5% CO 2 for 24 h, then transfected with the lentivirus pGMLV-CMV-RFP-GFP-hLC3-Puro (cat. no. GM-3394LV, Genomeditech Co., Ltd.) at a MOI of 30 for 72 h at 37°C and then treated with 20 μ M DB in the presence or absence of 5 μ M RAPA (MedChemExpress, cat. no. HY-10219) or 60 μ M CQ (Aladdin, cat. no. C129284) for a further 6 h. The MCF7 and MDA-MB-231 cells were pre-treated with 10 mM NAC (Beyotime Instittue of Biotechnology, cat. no. S0077) for 1 h and incubated with 20 μ M DB for 6 h at 37°C.

Techniques: Western Blot, Expressing, Control, Staining