Fig 1.
Inhibition of CTSS activities induces apoptotic hallmarks.
(A) After OEC-M1 cells were incubated with DMSO or 20 μM of the CTSS inhibitor 6r for the indicated time points, the cells were fixed with 4% paraformaldehyde, and stained with DAPI. The cell morphology and nucleus were monitored under a phase-contrast and a fluorescent microscope, respectively. Merged images are shown. (B) Cells were treated with 20 μM of 6r at different times. Nuclei were stained with DAPI, and images were taken using the fluorescent microscope. Relative nuclear sizes were analyzed using the Image Pro Plus software. Nuclear area was defined as the area within the outlined nuclear perimeter. Differences were found to be statistically significant at ***P < 0.001. (C) Time-course analysis of apoptosis during 6r treatment. Cells were exposed to 20 μM of 6r at the indicated time points, harvested, and stained with Annexin V and PI, as described in the materials and methods section. The numbers of apoptotic cells were quantified using flow cytometry. Data shown in the below panel are representative of 3 independent experiments. Differences were found to be statistically significant at **P < 0.01 and ***P < 0.001. (D) Left panel: Cells were treated with 20 μM of 6r for various durations, and cleaved caspase-3 fragments were analyzed through western blotting. The upper panel indicates short-term exposure of the blot. ACTIN was shown as an internal control for semiquantitative loading in each lane. Right panel: After OEC-M1 cells were transfected with pCMV vector or pCMV-CTSS for 24 h, cells were subsequently treated with 20 μM of 6r for additional 24 h. Compared to vector control cells, cells with increased CTSS expression showed reduced caspase-3 activation upon 6r treatment. (E) Representative images of a TUNEL assay in cells treated with 20 μM of 6r or ZFL for 24 h. Apoptotic cells display TUNEL-positive nuclei stained green.
Fig 2.
Autophagy is uninterruptedly up-regulated in CTSS-inhibiting cells.
(A) The OEC-M1 cells were treated with 20 μM of 6r or ZFL for the indicated times. Autophagy activation was evaluated with the detection of Lipidated LC3-II accumulation. The amount of loading proteins was assessed with anti-ACTIN antibodies. (B) Autophagy inhibition by CQ or BAF suppressed 6r-induced p62 degradation. The relative band intensities are shown. (C) The inhibition of CTSS by 6r or ZFL induced the autophagic vacuolization of MDC-labeled vesicles. After treatment with 20 μM of 6r or ZFL for 24 h, the cells were further incubated with MDC for 10 min, and immediately analyzed using fluorescent microscopy. (D) The visualization of autophagosomes by LC3 puncta formation in CTSS-inhibiting cells. Cells were treated with 20 μM of 6r or ZFL for 24 h, and then processed for immunofluorescence by using an antibody against the LC3 protein. Aggregated LC3 proteins appeared as puncta structures in cells.
Fig 3.
Prolonged inhibition of CTSS by 6r causes a second oxidative burst from the mitochondria.
(A) Cells were incubated with 20 μM of 6r for the indicated times, and intracellular ROS levels were detected using DCFH-DA, and were quantified through flow cytometry. The data represent the mean ± SD of 3 independent experiments. (B) After treatment with 20 μM of 6r for the indicated times, elevated mitochondria-generated ROS were identified using MitoSOX, and were quantified using flow cytometry. The data represent the mean ± SD of 3 independent experiments. Differences were found to be statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001. N.S. denotes no significant difference. (C) Cells were treated with 20 μM of 6r for 24 h, and then labelled with CytoPainter MitoGreen and MitoSOX red. Yellow color in the merged figure denotes mitochondria that are actively producing ROS.
Fig 4.
CTSS inhibition by 6r causes mitochondrial damage.
(A) The mitochondrial membrane potential was determined using JC-1, and was quantified using flow cytometry. Cells were treated with 20 μM of 6r for 4 h, and then stained with JC-1 for 30 min. The JC-1 aggregated form exhibited red fluorescence, indicating high membrane potential, whereas the JC-1 monomer form exhibited green fluorescence, indicating the collapse of membrane potential. CCCP was used as a positive control. Differences were found to be statistically significant at **P < 0.01. N.S. denotes no significant difference. (B) The inhibition of CTSS by 6r for 24 h induced the collapse of the mitochondrial membrane potential. Differences were found to be statistically significant at *P < 0.05. (C) Cells were exposed to 6r at the indicated concentrations for 24 h. Mitochondrial and cytosolic fractions were prepared as described in the materials and methods section, and were subjected to western blot analysis for cytochrome c, Bax, ACTIN, and COX-IV. ACTIN and COX-4 were used as the internal control for cytosolic and mitochondrial fractions, respectively. (D) Cells were treated with 6r for the indicated concentrations, and cleaved caspase-9 fragments were analyzed with western blotting. ACTIN was shown as an internal control for semiquantitative loading in each lane.
Fig 5.
CTSS inhibition-induced early ROS is responsible for mitochondrial damage and the second oxidative burst.
(A) For the inhibition of XO-generated early ROS, cells were pretreated with 200 μM of allopurinol for 1 h and cotreated with 20 μM of 6r for an additional 4 h. The intracellular cellular ROS level was determined using DCFH-DA and was quantified by flow cytometry. The data represent the mean ± SD of 3 independent experiments. Differences were found to be statistically significant at *P < 0.05 (B) After pretreatment with 200 μM of allopurinol for 1 h and cotreatment with 20 μM of 6r for an additional 24 h, the mitochondrial membrane potential was determined using JC-1 and quantified by flow cytometry. The data represent the mean ± SD of 3 independent experiments. Differences were found to be statistically significant at *P < 0.05 (C) Reducing the XO-generated early ROS level by allopurinol attenuated mitochondria-specific ROS generation. After pretreatment with 200 μM of allopurinol for 1 h and cotreatment with 20 μM of 6r for 24 h, the mitochondria-specific ROS level was determined using MitoSOX and quantified by flow cytometry. (D) Allopurinol treatment reduces Bax translocation and cytochrome c release in 6r-treated cells. Cells were pretreated with 200 μM of allopurinol for 1 h, and then cotreated with 20 μM of 6r for 24 h. Mitochondrial and cytosolic fractions were prepared and subjected to western blot analysis for cytochrome c, Bax, ACTIN, and COX-IV.
Fig 6.
CTSS inhibition-induced early ROS contributes to mitochondria-dependent apoptotic signaling.
(A) Allopurinol treatment reduced 6r-induced caspase-9/-3 activation. Cells with and without allopurinol pretreatment were incubated with 20 μM of 6r for 24 h, and subjected to western blot analysis for detecting cleaved caspase-9 and caspase-3. (B) Inhibition of XO expression by siRNA silencing approach. Cells transfected only with RNAiMax was used as a negative control (siNC) and transfected with scramble negative siRNA was used as a scramble control (siSC). (C) Reducing the expression of XO by siRNA decreased 6r-induced caspase-9/-3 activation. After 48 h of siRNA knockdown of XO, cells were treated with 20 μM of 6r for 24 h and the levels of cleaved caspase-9/-3 were assessed by western blotting. (D) Allopurinol treatment reduced the number of 6r-induced apoptotic cells. After pretreatment with allopurinol for 1 h and cotreatment with 6r for 24 h, the numbers of apoptotic cells were determined by Annexin V/PI double staining and quantified by flow cytometry. Differences were found to be statistically significant at *P < 0.05.
Fig 7.
Conclusion model of autophagy-regulated early ROS in the connection between death autophagy and apoptosis.
In CTSS-targeting cells, autophagy is quickly activated to regulate XO for the first oxidative burst (early ROS generation). The harmful early ROS can subsequently act as an early effector to for triggering Bax activation and subsequently causing cytochrome c release, and mitochondrial membrane depolarization for the second oxidative burst. Consequently, these factors act as death executors for the triggering of mitochondria-dependent apoptosis. Thus, these dying cells simultaneously display mixed autophagic and apoptotic hallmarks. When upstream autophagy and/or early ROS level are attenuated, mitochondria-dependent apoptotic signaling cascades diminish.