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. 2023 May 19;26(6):106899. doi: 10.1016/j.isci.2023.106899

Figure 3.

Figure 3

The combination of gemcitabine with perhexiline enhances the energetic and oxidative stress induced by perhexiline in primary pancreatic cancer cells

(A) Mitochondrial respiration (oxygen consumption rate, OCR) was measured on a Seahorse oxygraph in PDAC084T cells 6 h after the start of treatment (no cell death was observed at this time point) with gemcitabine (1 μM), perhexiline (10 μM), or combination treatment at same concentrations (DMSO 0.05% was used as vehicle for the controls). The basal and maximal respiration, ATP production by mitochondria, and spare respiratory capacity were calculated (graphics on the right). Values are presented as percentage of the vehicle-treated. Data are mean ± SEM of three independent experiments performed in triplicates. To calculate p values, one-way ANOVA test was used; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

(B) PDAC084T cells were treated with gemcitabine (1 μM), perhexiline (7 μM), or the combination for 24 h. Then, total ROS and mitochondrial superoxide anion (O2.-) levels were measured by flow cytometry with the CellROX orange and MitoSOX red probes, respectively. Data are expressed as mean ± SEM and are representative of three independent experiments performed in triplicates (CellROX) or four independent assays done in duplicates (MitoSOX). To calculate p values, one-way ANOVA test was used; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

(C) PDAC084T cells were treated with gemcitabine (1 μM), perhexiline (7 μM), or the combination for 24 h; then cells were fixed and stained with BODIPY 493/503 for lipid droplet detection and DAPI. Representative fluorescence microscopy images from three independent experiments performed in duplicates are shown. Scale bar: 50 μM. The graph on the right shows the quantification of BODIPY staining mean area (±SEM) of the three independent experiments. p values calculated from t test; ∗p < 0.05, ∗∗p < 0.01.

(D) PDAC084T cells were treated with gemcitabine (1 μM), perhexiline (7 μM), or the combination for 24 h; then cells were fixed and stained with BODIPY 581/591 C11 for lipid peroxidation detection and DAPI. Representative fluorescence microscopy images from three independent experiments done in duplicates are shown. Scale bar: 50 μM. The graph on the right shows the quantification of BODIPY staining mean area (±SEM) of the three independent experiments. p values calculated from t test; ∗∗p < 0.01, ∗∗∗p < 0.001.