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. 2021 Jun 16;14(9):101154. doi: 10.1016/j.tranon.2021.101154

Fig. 5.

Fig 5

Fig 5

Endoplasmic reticulum stress induction is also an important mechanism of the combination's action. (A) Cells were treated for 48 h with 5 μM simvastatin and 20 nM romidepsin with or without 5 μg/ml cycloheximide (CHX). Apoptotic cells were detected by annexin-V assay using flow cytometry. 10,000 cells were counted. Bar graphs show the increase in annexin-V positive cells. Data are expressed as mean ± SD from three independent experiments. FITC, fluorescein isothiocyanate; 7-AAD, 7-amino-actinomycin D. *p = 0.0495. (B) Western blotting for glucose-regulated protein (GRP) 78, endoplasmic reticulum resident protein (ERp) 44, acetylated histone, and peroxisome proliferator-activated receptor (PPAR) γ. Cells were treated for 48 h with 5 μM simvastatin and 20 nM romidepsin with or without 5 μg/ml CHX. Actin was used for the loading control. Representative blots are shown. (C) Cells were treated for 48 h with 5 μM simvastatin and 20 nM romidepsin with or without 5 μg/ml CHX and reactive oxygen species production was measured by dihydroethidium (DHE) staining using flow cytometry. 10,000 cells were counted. Bar graphs show the increase in relative DHE fluorescence intensity. Data are expressed as mean ± SD from three independent experiments. *p = 0.0495. (D) Cells were treated for 48 h with 0.1–2 μM tunicamycin and cell viability was measured using CCK-8 assay. Mean ± SD, n = 6. (E) Western blotting for GRP78, ERp44, AMP-activated protein kinase (AMPK), acetylated histone, and PPARγ. Cells were treated for 48 h with 0.5–2 μM tunicamycin. Actin was used for the loading control. Representative blots are shown.