Figure 1.
Schematic of APRTP-Js used in the HepG2 cell treatment.
(A) Schematic of the APRTP-Js device and (B) photograph of APRTP-Js acting on the HepG2 cells.
Figure 2.
Typical emission spectra of the plasma emitted by APRTP-Js.
(A) 275-500 nm (B) 500-800 nm.
Figure 3.
APRTP-Js treatment induced apoptosis in HepG2 cells.
Cells were treated by APRTP-Js for 240 s, 480 s and 720 s, and then cultured for 24 h. (A) Cells were double stained with annexin V-FITC and PI and analyzed by flow cytometry. Cells that stained positive for annexin V-FITC and negative for PI were undergoing early stage of apoptosis; Cells that stained positive for both annexin V-FITC and PI were in the end stage of apoptosis; Cells that stained negative for both annexin V-FITC and PI were alive and not undergoing measurable apoptosis. Percentage of apoptotic cells (annexin V-FITC positive) was shown by histogram. (B) Observation of Hoechst 33342 apoptosis staining by fluorescence microscopy. Examples of typical apoptotic nuclei were indicated by white arrows.
Figure 4.
APRTP-Js treatment facilitated ROS and RNS accumulation in HepG2 cells.
(A) Measurement of ROS generation in HepG2 cells 24 h after APRTP-Js treatment. Cells were stained with DCFH-DA probe and DCF fluorescence was measured using a flow cytometer. (B) Effect of the APRTP-Js treatment on the intracellular NO concentration in HepG2 cells after 24 h culture. (C) The influence of APRTP-Js treatment on NOS enzymatic activities in HepG2 cells after 24 h culture. The statistical significance was carried out in comparison with the corresponding control value (*P < 0.05 and **P < 0.01 versus iNOS control, # P < 0.05 versus cNOS control). (D) Effect of plasma on the expression of iNOS. Detection of β-actin was carried out to confirm the equal loading of proteins. (E) The corresponding densitometric analysis of iNOS. Data were normalized using the β-actin signal. Values are means ± SEM obtained from three independent experiments. The asterisk represents statistical significance in comparison with control (*p < 0.05 and **p < 0.01).
Figure 5.
APRTP-Js Treatment compromised cellular antioxidant defense system in HepG2 cells.
Cells were treated without or with APRTP-Js for 240 s, 480 s and 720 s, respectively. 24 h later, the cells were harvested and lysed using cell lysis buffer. The samples were centrifuged and the supernatants were used for antioxidant activities assay. (A) The effect of APRTP-Js treatment on total GSH content in HepG2 cells. (B) The effect of APRTP-Js treatment on the SOD activity in HepG2 cells. (C) The effect of APRTP-Js treatment on the catalase activity in HepG2 cells. Values are means ± SEM obtained from three independent experiments. The asterisk represents statistical significance in comparison with control value (*p < 0.05 and **p < 0.01).
Figure 6.
APRTP-Js treatment induced oxidative and nitrative damage in HepG2 cells.
(A) Status of total protein nitration. The total nitration statuses of proteins were studied using a nitrotyrosine antibody by Western blot in cells with or without APRTP-Js treatment. The deepened nitrotyrosine epitopes were indicated by arrows. (B) Status of protein carbonyl content. The protein carbonyl content was measured using a commercial assay kit. Values are means ± SEM obtained from three independent experiments. The asterisk represents statistical significance in comparison with control (*p < 0.05 and **p < 0.01).
Figure 7.
APRTP-Js treatment disturbed intracellular calcium homeostasis.
Cells were treated without or with APRTP-Js for 240 s, 480 s and 720 s, respectively. After continuous culturing for 24 h, the cells were loaded with calcium probe Fluo-3/AM and the intracellular free Ca2+ was measured using a FACScan flow cytometer.
Figure 8.
APRTP-Js treatment triggered ER stress in HepG2 cells.
(A) Representative autoradiographs of pro-caspase12, GRP78, and CHOP. Detection of β-actin was carried out to confirm the equal loading of proteins. (B–D) The corresponding densitometric analysis of pro-caspase12, GRP78, and CHOP, respectively, and data were normalized using the β-actin signal. The asterisk represents statistical significance in comparison with control (*p < 0.05 and **p < 0.01).
Figure 9.
NAC attenuated the apoptosis induced by APRTP-Js treatment.
(A) HepG2 cells were incubated for 1h with 10 mM NAC (N+) or cell culture medium (N-), followed by treatment with the indicated dose of plasma. Apoptosis was measured 24 h after APRTP-Js treatment by collecting and staining the cells with annexin V-FITC/PI. Samples were run on the flow cytometry. P- N-: control group without any treatment; P+ 240s N-: cells with 240 s plasma exposure only; P+ 480s N-: cells with 480 s plasma exposure only; P-N+: cells with NAC pre-treatment only; P+ 240s N+: cells with NAC pre-treatment followed by 240 s plasma exposure; P+ 480s N+: cells with NAC pre-treatment followed by 480 s plasma exposure. (B) Percentage of apoptotic cells was determined from flow cytometry results.