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HEP_24281_sm_suppinfofig1.tif2087KSupplementary Fig. 1. NOX1 and NOX2 proteins are expressed in the fibrotic liver. Livers were obtained from WT mice after 12 times of intraperitoneal injection of CCl4 (n=8). (A) NOX1 or (B) NOX2 expression (all red fluorescence) was detected in CCl4-treated liver by means of immunofluorescence staining. Staining with NOX1 or NOX2 isotype antibody was used as controls. Original magnification, x50 (A,B).
HEP_24281_sm_suppinfofig2.tif222KSupplementary Fig. 2. Liver injury is attenuated in NOX1KO and NOX2KO mice after CCl4 treatment. Serum levels of ALT, total bilirubin and liver/body weight ratio were assessed in WT, NOX1KO, and NOX2KO mice after (A) 12 times of intraperitoneal injection of CCl4 (n=8) or (B) 3 weeks of BDL (n=10). Statistical significance relative to WT CCl4-treated mice or WT BDL mice: *P < 0.05, **P < 0.01.
HEP_24281_sm_suppinfofig3.tif273KSupplementary Fig. 3. Hepatic mRNA expressions of M1 and M2 macrophage markers in WT, NOX1KO, and NOX2KO mice after CCl4 or BDL treatment. (A) Hepatic mRNA levels of M1 macrophage markers, including TNF-α, iNOS, and CCL3, were measured in WT, NOX1KO and NOX2KO mice after 12 CCl4 injections (n=8)or 3 weeks of BDL (n=10) by quantitative real-time PCR. (B) Hepatic mRNA levels of M2 macrophage markers, including arginase-1, Mgl-1, and Mrc2, were measured in WT, NOX1KO, and NOX2KO mice after 12 CCl4 injections (n=8) or 3 weeks of BDL (n=10) by quantitative real-time PCR. Ribosomal 18S RNA was used as an internal control.
HEP_24281_sm_suppinfofig4.tif5341KSupplementary Fig. 4. Double immunofluorescent staining of NOX2 and F4/80 in NOX2 chimeric mice after CCl4 treatment. Double Immunofluorescence staining for NOX2 (red fluorescence) and F4/80 (KCs) (green fluorescence). The liver sections were from NOX2 BM chimeric mice treated with CCl4 including WT BM -> WT, WT BM -> NOX2KO, NOX2KO BM -> WT, and NOX2KO BM -> NOX2KO. Arrow head indicates double positive cells. Original magnification, x200.
HEP_24281_sm_suppinfofig5.tif218KSupplementary Fig. 5. Expression of NOX components in quiescent HSCs and in vivo- activated HSCs by CCl4. mRNA levels of NOX1, NOX2, NOX4, p22phox, p40phox, p47phox, p67phox, NOXO1, NOXA1, and Rac1 were evaluated by quantitative real-time PCR in quiescent (1 day in culture) HSCs (n=2), and in vivo activated (isolated from WT mice after 5 days of CCl4) HSCs (n=2). Ribosomal 18S was used as an internal control. Statistical significance compared to quiescent HSCs: *P < 0.05, **P < 0.01.
HEP_24281_sm_suppinfofig6.tif3452KSupplementary Fig. 6. Activated human HSCs express NOX1 and NOX2. LX-2 activated human HSCs were fixed in ice-cold methanol and then permeablized using 0.3M glycine in 0.1% PBS-Tween. For the detection of (A) NOX1 or (B) NOX2, the cells were incubated with rabbit anti-human NOX1 or rabbit anti-human NOX2 primary antibody, respectively. Then the cells were incubated with AlexaFluor 594 goat anti-rabbit secondary antibody (red). Cells stained with NOX1 or NOX2 isotype antibody were used as controls. DAPI was used to stain the cell nuclei (blue). NOX1 was diffusely observed in the cytoplasm, while, NOX2 was predominantly found in the perinuclear area. The cells were visualized on a Zeiss fluorescent microscope. Original magnification, x400 (A,B).
HEP_24281_sm_suppinfofig7.tif3509KSupplementary Fig. 7. Both NOX1 and NOX2 mediate ROS production in HSCs. HSCs were isolated from WT, NOX1KO and NOX2KO mice. Activated HSCs (7 days in culture) were loaded with redox-sensitive dye CM-H2DCFDA (10 μM) for 20 minutes. Cells were then washed twice and subsequently stimulated with angiotensin II (10-6 M). Fluorescent signals from WT HSCs, NOX1KO HSCs, and NOX2KO HSCs were observed and continuously recorded every 10 seconds for 10 minutes under the fluorescent microscope.
HEP_24281_sm_suppinfofig8.tif575KSupplementary Fig. 8. NOX2, but not NOX1 plays a role in superoxide generation in Kupffer cells. (A, B) Kupffer cells were isolated from WT mice. (A) Kupffer cells (2 days in culture) were treated with DMSO (solvent), PMA (1 μg/ml) with or without preincubation of DPI (10 μM), then superoxide generation was continuously monitored by detecting luminol- enhanced chemiluminescence using luminometer for 40 minutes. PMA-induced superoxide generation rapidly dropped to baseline level after adding superoxide dismutase (50 μg/ml) 25 minutes after PMA stimulation (arrow). (B) Kupffer cells were isolated from WT, NOX1KO and NOX2KO mice. Cells were treated with PMA (1 μg/ml), and superoxide generation was monitored by measurement of luminol-enhanced chemiluminescence for 40 minutes.
HEP_24281_sm_suppinfomov1a.avi759KSupporting Information Movie 1
HEP_24281_sm_suppinfomov1b.avi650KSupporting Information Movie 2
HEP_24281_sm_suppinfomov1c.avi542KSupporting Information Movie 3
HEP_24281_sm_suppinfo.doc72KSupporting Information

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