We found that epi-1-triggered mitochondrial hyperpolarization occurs within 30 min and is sustained HIV-1 integrase inhibitor 2 to 3 h (Figure 4ACC). Epi-1 Induces Mitochondrial Hyperpolarization Next, we analyzed the effect of epi-1 on mitochondrial function by TMRE. (F) Cells were pretreated with BA (10 M) for 1 h, followed by epi-1 for an additional 15 min. Cytotoxicity was determined by the trypan blue exclusion assay. (E) Cells were preincubated with PD151746 (PD) for 1 h, followed by epi-1 for an additional 5 h. (D) Cells were preloaded with fluorogenic calpain substrate t-BOC (10 M) for 30 min, followed by epi-1 for the indicated times. Cytotoxicity was assessed by the trypan blue exclusion assay. (C) Cells Itga1 were preincubated with BAPTA (BA 10 M) for 1 h, followed by epi-1 for an additional 5 h. Fluorescence of Fluo-4 was observed HIV-1 integrase inhibitor 2 by fluorescence microscopy (A) and flow cytometry (B).

Cells were preloaded with Fluo-4 (5 M) for 15 min, then treated with epi-1 at different points as indicated. Open HIV-1 integrase inhibitor 2 in a separate window Figure 3 Calcium-dependent calpain activation is required for epi-1-mediated cytotoxicity.

Since BAPTA attenuated epi-1-mediated upregulation of calpain activity (Figure 3F), calcium seems to be required for epi-1-mediated activation of calpain. Calpain activity was also quickly induced within 15 min (Figure 3D), and suppression of calpain activity by PD151746 inhibited epi-1-mediated cytotoxicity (Figure 3E). Calcium chelator BAPTA blocked cell death, suggesting that calcium is necessary for epi-1-mediated cytotoxicity (Figure 3C). Epi-1 treatment elevated the intracellular calcium level within 15 min, and the elevation was sustained to 60 min (Figure 3A,B). Calcium and Calpain are Required for Epi-1-Induced Cell Death Necrosis often involves intracellular calcium overload, which subsequently activates cell death-inducing molecules, such as calpain. Cells were pretreated with Necrostatin-1 (Nec-1) (10 M) (G) or Z-VAD-FMK (Z-VAD) (100 M) (H) for 1 h, followed by epi-1 (6.125 M) treatment for 24 h. After rinsing cells with PBS, PI incorporation was observed by fluorescence microscopy. After stimulation, cells were loaded with propidium iodide (PI 1 g/mL) for 10 min. (F) Cells were treated with epi-1 or stau as described in (A). (D) Cells were treated with epi-1 for different times, and cell lysates and supernatants were collected and immunoblotted with indicated antibodies. (A,B) Band intensities were quantified by ImageJ. Cell lysates were collected and immunoblotted for caspase-3 and -actin. Supernatants were collected and immunoblotted for HIV-1 integrase inhibitor 2 cyclophilin A. (A,C) Cells were treated with epi-1 (6.125 M) or stau (1 M) for 3 h. Open in a separate window Figure 2 Epi-1 triggers caspase-independent cell death. Furthermore, the necrosis inhibitor, Necrostatin-1 (Nec-1), suppressed epi-1-induced toxicity (Figure 2G), but apoptosis inhibitor Z-VAD-FMK (Z-VAD) did not (Figure 2H). Epi-1-treated cells also exhibited propidium iodide incorporation, while stau-treated cells did not (Figure 2F). In contrast, extracellular cyclophilin A was not increased by stau (Figure 2A,C). HIV-1 integrase inhibitor 2 Extracellular cyclophilin A is considered to be a marker of necrosis, and epi-1 effectively increased the levels of cyclophilin A in the culture supernatant (Figure 2A,CCE). Next, the involvement of necrosis in epi-1-mediated cell death was examined. Consistently, epi-1 did not induce activation of caspase-3 at any tested time point (Figure 2D). To monitor whether apoptosis may occur at an earlier time, cells were tested at various time points after treatment (0.5, 1, 3, and 5 h). Activation of caspase-3 was induced by stau but not epi-1 (Figure 2A,B). п»ї(F) Band intensity was measured with ImageJ.

Imagej software band intensity