Fig 1.
Hepatitis B virus (HBV) inhibits AP-1 activation in LX-2 cells.
(A) LX-2 cells were transfected with pAP-1-luc with pCXN2-HBx or pCXN2. After 24 hours of transfection, LX-2 cells were treated with or without 100 ng/mL LPS (Novus Biologicals, Littleton, CO, USA), 0.1% lipid mixture 1 (Sigma), or 5 ng/mL TGF-β (Wako Pure Chemical, Osaka, Japan). (B) After 24 hours of transfection with pAP-1-luc, the cells were treated with conditioned media from HepG2 or from HepG2.2.15 (indicated as 2.2.15). (C), (D) After 24 hours of transfection with pAP-1-luc, the cells were co-cultured with HepG2 or HepG2.2.15 with (C) or without the transwell system (D). After 48 hours of transfection, AP-1 activity was measured by luciferase assay. Data are expressed as mean ± standard deviations of triplicate determinations. *P < 0.01 (vs. control).
Fig 2.
Conditioned media from HepG2.2.15 (2.2.15-CM) inhibits phosphorylated-c-Jun (p-c-Jun) in LX-2 cells.
(A)-(C) Western blot analyses of phosphorylated-c-Jun, c-Jun and GAPDH expression in LX-2 cells treated with conditioned media from HepG2 (HepG2-CM) or 2.2.15-CM. (B), (C) Densitometric analyses were performed by ImageJ software. (D) Western blot analyses of JNK and GAPDH expression in LX-2 cells treated with HepG2-CM or 2.2.15-CM. Data are expressed as mean ± standard deviations of triplicate determinations.
Fig 3.
Conditioned media from HepG2.2.15 (2.2.15-CM) protects hepatic stellate cells from MG132-induced apoptosis.
(A) LX-2 cells were cultured with mock control, conditioned media from HepG2 (HepG2-CM), or conditioned media from HepG2.2.15 for 24 hours with or without MG132 (20 μM). *P < 0.01, **P < 0.05. (B) HHSteC cells were cultured with mock control, conditioned media from HepG2 (HepG2-CM), or conditioned media from HepG2.2.15 for 24 hours with or without MG132 (20 μM). *P < 0.01, **P < 0.01. Apoptosis was quantified using the APOPercentage Apoptosis Assay. Data are expressed as mean ± standard deviations of triplicate determinations. (C) Caspase-3/-7 activities following 20 μM of MG132 treatment were measured in conditioned media from LX-2 cells incubated with conditioned media from HepG2 or HepG2.2.15 for 24 hours. Caspase-3/-7 activities were determined using the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA). (D) Western blot analysis of PARP and GAPDH expression in LX-2 cells treated with conditioned media from HepG2 (HepG2-CM) or 2.2.15-CM after 24 hours of MG132 treatment.
Fig 4.
c-Jun is important for apoptotic hepatic stellate cell death induced by MG132.
(A) Apoptotic cell deaths were lower in LX-2 cells transfected with siRNAs against c-Jun (si-c-Jun1 and si-c-Jun2) compared with LX-2 cells transfected with siRNA-control (si-C) after incubation with conditioned media from HepG2 in the presence of MG132 (lower panel). Western blot analysis of c-Jun and GAPDH expression in LX-2 cells treated with conditioned media from HepG2 after 24 hours of MG132 treatment (upper panel). (B) Overexpression of c-Jun by the transfection of pMEKK into LX-2 cells enhanced apoptosis in LX-2 cells treated with conditioned media from HepG2.2.15 in the presence of MG132 (lower panel). Western blot analyses of c-Jun and GAPDH expression in LX-2 cells treated with conditioned media from HepG2.2.15 after 24 hours of MG132 treatment (upper panel). Apoptosis was quantified using the APOPercentage Apoptosis Assay. Data are expressed as mean ± standard deviations of triplicate determinations. (C) Conditioned media from HepG2.2.15 (2.2.15-CM) protects hepatic stellate cells from MG132-induced apoptosis and DNA damage. Confocal microscopic findings with a high-power view (x200) of the expression of phosphorylated histone H2AX (γ-H2AX) (red), a DNA damage marker, and Annexin V (green), an apoptosis marker, in LX-2 cells treated with conditioned media from HepG2.2.15 (upper panel) or HepG2 (lower panel) in the presence of MG132.
Fig 5.
Real-time PCR array analysis for ER stress-associated genes in conditioned media associated with or without HBV.
(A) Conditioned media from HepG2.2.15 (2.2.15-CM) and conditioned media from HepG2 (HepG2-CM) were used for the treatment of LX-2 cells for 48 hours. (B) Conditioned media from HBV-infected PXB (HBV-infected) and conditioned media from HBV-uninfected PXB (HBV-uninfected) were used for the treatment of LX-2 cells for 48 hours. Eighty-four ER-stress-associated gene expression profiles using real-time PCR-based focused microarrays (see Materials and Methods section). Three genes are indicated by arrows: solute carrier family 17, member 2 (SLC17A2); inhibin, beta E (INHBE); and cAMP responsive element binding protein 3-like 3 (CREB3L3), are indicated by arrows.
Table 1.
Up-regulated genes (> 4-fold differences) in LX-2 cells treated with conditioned media from HepG2.2.15 compared with in LX-2 cells treated with that from HepG2.
Fig 6.
Possible involvement of hepatitis B virus e antigen (HBeAg) in the attenuation of apoptosis in hepatic stellate cells induced by MG132.
(A) HBeAg inhibits AP-1 activation in LX-2 cells. LX-2 cells were transfected with pAP-1-luc with or without pCXN2-HBeAg(+) or pCXN2-HBeAg(-) [25]. After 48 hours of transfection, AP-1 activity was measured by luciferase assay. Data are expressed as mean ± standard deviations of triplicate determinations. *, **P < 0.01 (vs. mock control). (B) Inhibitory effects of conditioned media from HBeAg-positive HepG2 on MG132-induced apoptosis of LX-2. Apoptosis was quantified using the APOPercentage Apoptosis Assay. Data are expressed as mean ± standard deviations of triplicate determinations.