Figure 1.
LPS- and ASA-induced ROS production.
J774.2 macrophage cells were cultured to 80% confluence and treated with LPS and ASA alone or in combination as described in Materials and Methods. ROS production was measured in cell lysate using DCFDA and fluorescence was measured by flow cytometry (1A) using FACSDiva software as described before [13]. Results are expressed as a typical representation of three determinations. Percent change in ROS production from a typical histogram is also presented. Results are expressed as mean +/− SEM of at least three experiments. Asterisks indicate significant difference (** p ≤0.01) from control (C). In some cases, cells were washed twice with PBS and DCFDA-induced ROS fluorescence was immediately analyzed microscopically using an Olympus fluorescence microscope (1B). Typical results from control and LPS alone or in combination with ASA with or without NAC treatment from three experiments are shown.
Figure 2.
A–C: LPS- and ASA-induced apoptosis and alteration in GSH.
Macrophage J774.2 cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA in the presence or absence of NAC as described in Materials and Methods. Apoptosis was measured by flow cytometry as described in Materials and Methods. Bar diagram shows %age apoptotic death from a representative result (2A), expressed as mean +/− SEM of at least three experiments. GSH was measured by enzymatic recycling method (2B–2C) using glutathione reductase and NADPH as described before [13]. Results are expressed as mean +/− SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05; ** p≤0.01 from control (C), ≠p≤0.05 compared to LPS-treated cells and δ p≤0.05 compared to LPS and ASA treated cells).
Figure 3.
A–D: LPS- and ASA-induced alterations in GST and GSH Px activities.
J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described in Materials and Methods. GST-dependent GSH conjugation (3A–3B) and GSH-Px (3C–3D) activities were measured as described before [13]. Results are expressed as mean +/− SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001 from control (C) and δ p≤0.05 compared to LPS and ASA treated cells.
Figure 4.
LPS- and ASA-induced alterations in CYP activities.
J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described in Materials and Methods. CYP2E1 (4A), CYP 1A1 (4B) and CYP 3A4 (4C) activities were measured as described in Materials and Methods. Results are expressed as mean +/− SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C).
Figure 5.
LPS- and ASA-induced alterations in cytokines.
J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described above and cytokines IL6 and TNF-α were measured using standard ELISA kits as described in Materials and Methods. Results are expressed as mean +/-SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C) and δ p≤0.05 compared to LPS and ASA treated cells.
Figure 6.
LPS- and ASA-induced alteration in mitochondrial membrane potential.
J774.2 macrophage cells were cultured to 80% confluence and treated with LPS and ASA alone or in combination as described above. Mitochondrial membrane potential was measured using a cationic fluorescent dye as described before [13]. A typical histogram representative of at least three experiments showing % loss of mitochondrial membrane potential is shown. Results are expressed as mean +/−SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C).
Figure 7.
A–C: LPS- and ASA-induced alterations in mitochondrial respiratory functions.
J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described above. Freshly isolated mitochondria from control and treated cells were used to assay Complex I and Complex IV activities (7A–7B) and ATP content (7C) as described before [13]. Results are expressed as mean +/−SEM of at least three experiments. Asterisks indicate significant difference (*p≤0.05, **p≤0.001) from control (C), ≠p≤0.05 compared to LPS-treated cells and δ p≤0.05 compared to LPS and ASA treated cells.
Figure 8.
LPS- and ASA-induced alterations in protein expression.
J774.2 macrophage cells were cultured to 80% confluence and treated with LPS alone or in combination with ASA with or without NAC as described above. Proteins (50–100 µg) from cell lysates were separated by SDS-PAGE analysis and transferred on to membranes (Western blot). Transferred proteins were incubated with primary antibodies against cytochrome c, Bcl-2, TNF-α, Nrf-2, HO-1, IκB-α and NF-κBp65 and antibody reacting proteins were visualized as described before [13]. Beta-actin was used as loading control. Results from representative SDS-PAGE/Western blot analysis are shown. The quantitation of protein bands is expressed as relative intensity (R.I) of the protein considering the expression of proteins in control untreated cells as 1.0. Molecular weight markers (kDa) are indicated by arrows.