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. 2018 Oct 12;1(2):84–95. doi: 10.1021/acsptsci.8b00007

Figure 3.

Figure 3

Effects of aglycone polyethers on calcium and mitochondrial function. (A and B) Proteome analysis of the (A) calcium signaling pathway and (B) mitochondrial signaling pathway after J1-001-2 treatment of MDA-MB-231 cells. (See the Supporting Information for experimental details.) The dashed red line means no changes (ratio = 1) between treated cells and untreated cells. (A) CAPN2, calpain-2 catalytic subunit, calcium-regulated nonlysosomal thiol-protease; CAPNS1, calpain small subunit 1, regulatory subunit of the calcium-regulated nonlysosomal thiol-protease; VDAC1–3, voltage-dependent anion-selective channel protein 1–3; ATP2B1, plasma membrane calcium-transporting ATPase 1; SLC25A4–6, ADP/ATP translocase 1–3; EGFR, epidermal growth factor receptor; PPIF, peptidyl-prolyl cis–trans isomerase F, mitochondrial. (B) AIFM1 (apoptosis-inducing factor 1, mitochondrial), CYCS (cytochrome c), UQCRB (Cytochrome b-c1 complex subunit 7), CYC1 (Cytochrome c1, heme protein, mitochondrial), UQCRC1–2 (Cytochrome b-c1 complex subunit 1–2, mitochondrial). (C and D) Effects on calcium and ROS. Flow cytometry analyses of (C) intracellular calcium levels and (D) intracellular ROS levels. MDA-MB-231 cells were seeded in 6-well plates at a density of 1 × 105 cells/well and incubated for 24 h before treatment with J1-001-1 and J1-001-2 at the indicated concentrations. MDA-MB-231 cells were stained with (C) Fluo-3 M after 16 h or (D) DCFH-DA after 24 h. (E–H) Effects on H+ and K+.Permeabilization of the mitochondrial inner membrane to H+ and K+ following exposure to J1-001-1 (E and G) and J-001-2 (F and H). Effects of J1-001-1 (I) and J-001-2 (J) on mitochondrial ROS levels. ROS levels in liver mitochondrial were measured by flow cytometry using the probe DCFH-DA (λex = 495 nm, λem = 520 nm). Isolated mitochondrial fractions were incubated in buffer G (containing 100 mM sucrose, 10 mM Tris-HCl, 50 mM KCl, 10 mM MOPS, 5 mM K2HPO4, 2 μM rotenone, pH ∼7.4) with DCFH-DA (20 μM) at 37 °C for 20 min in the dark. Then, the pellets were incubated with (I) J1-001-1 or (J) J1-001-2 at the indicated concentrations for 30 min, n = 3. The fluorescence in the FL-1 channel was detected for at least 100 000 events with BD AccuriTM C6 (BD, USA) and analyzed with BD AccuriTM C6 System software.