Fig 1.
3-AT selectively decreased peroxisomal matrix protein abundance in Raw264.7 cells.
(A) RAW246.7 cells were treated with 5 mM, 10 mM, or 20 mM of 3-AT for 24 h and catalase activity was measured. (B) Cells were treated as in (A) and followed by immunoblotting with antibodies as shown. (C) Cells were treated as in (A), followed by subcellular fractionation, and by immunoblotting for Pex5, PMP70, β-actin and UBXD8. (D) Cells were treated as in (A) and mRNA expression levels of Catalase, PMP70, ACOX1 and DBP were measured. (E) Cells were treated as in (A) and immunostaining was performed with anti-catalase (green) and anti-PMP70 (red). (F) The ratio of catalase positive PMP70 puncta to the total PMP70 puncta per cell was determined from (E) was calculated by dividing total number of catalase puncta to total number of PMP70 puncta per cell. At least 30 cells were counted in each experiment. All error bars represent the mean ± S.D. (n = 3, independent experiments), *p < 0.05.
Fig 2.
Scavenging of peroxisomal ROS prevented the 3-AT mediated degradation of peroxisome matrix proteins.
(A) Cells treated with H2O2, 3AT (10 mM), NAC (2 mM) or co-treatment with NAC and 3-AT for 24 h. Cells were then treated with DCFH-DA (1 μM) and examined under fluorescence microscope. (B) Bar graph showing the fluorescent intensity of DCFH-DA from (A). (C) Cells were transiently transfected with HyPer-SKL plasmid for 24 h and then treated with 3AT, for 24 h. (D) Peroxisomal ROS production was measured by fluorescence intensity of HyPer-SKL in the transfected cells from (C). (E) Cells were treated with 3-AT alone, combination of 3-AT and NAC or NAC alone and followed by immunoblotting. (F) Densitometric analysis of band from (E). (G) Cells were treated as in (E) and immunostaining was performed with anti-catalase and anti-PMP70. Representative images are shown. All error bars represent the mean ± S.D. (n = 3, independent experiments), *p < 0.05, ns: non significance.
Fig 3.
3-AT induced peroxisome leakage and promoted the ubiquitin mediated proteasomal degradation of peroxisomal matrix proteins.
(A) Cells were treated with 3-AT (10 mM), MG132 (10 μM), chloroquine (5 μM), combination of 3-AT and MG132, 3-AT or chloroquine for 24 h and followed by immunoblotting. (B) Cells were transfected with HA-Ub plasmid for 12 h. Cells were then treated with 3-AT, MG132 or combination of 3-AT and MG132 for additional 24 h. Cell lysates were then subjected for immunoprecipitation and immunoblotting. (C) Cells were treated with 3-AT alone, or combination of 3-AT and MG132 for additional 24 h and immunostaining was performed with anti-PMP70 (red) and anti-ACOX1 (green). (D) Cells were treated as in (C), followed by Proteinase K protection assay, and immunoblotting for PMP70, Catalase, DBP and ACOX1.
Fig 4.
3-AT promotes the formation of 4HNE-IκBα adduct.
(A) Cells were treated with LPS (100 ng/ml) for different time points and mRNA expression levels of TNF-α, IL-1β, and IL-6 were measured. (B) Cells were treated with LPS for different time points and expression of TNF-α, IL-1β, and IL-6 was measured from the culture medium at each time points. (C) Cells were treated with LPS or 3-AT (10 mM) for different time points, added with DCFH-DA (1 μM), and examined under fluorescence microscope. Bar graph shows the fluorescent intensity of DCFH-DA. (D) Cells were transfected with HyPer-SKL and then treated with LPS or 3-AT for different time points. Peroxisomal ROS production was measured by fluorescence intensity of HyPer-SKL from the transfected cells. (E) Cells were treated with LPS or 3-AT for different time points and immunoblotting was performed with anti-4HNE. (F) Cells pre-treated with 3-AT for 12 h were further incubated with or without LPS for additional 12 h. Cells were then subjected to immunoprecipitation with anti-IκBα and followed by immunoblotting. (G) Cells treated with 3-AT for 12 h were subjected to immunoprecipitation with anti-4HNE and followed by immunoblotting. All error bars represent the mean ± S.D. (n = 3, independent experiments), *p < 0.05, ns: non-significance.
Fig 5.
3-AT suppresses inflammatory response through promoting the formation of 4HNE-IκBα adduct.
(A) Cells pre-treated with 3-AT for 12 h were further incubated with or without LPS for additional 12 h. Cells were then subjected to immunoblotting with anti-IκBα and anti-phospho-IκBα. (B) Cells were treated as in (A), followed by subcellular fractionation, and immunoblotting for NF-κB, β-actin and CREB. Cells were treated as in (A) and mRNA expression levels pro-inflammatory genes were measured by qPCR. All error bars represent the mean ± S.D. (n = 3, independent experiments), *p < 0.05.