Fig 1.
Expression of transcription factors cJun and cFos in GCDCA-treated HepG2.
Effect of the administration of MnTBAP or MitoQ. (A) Representative western blots for cJun (n = 3), phospho-cJun (Ser63) (n = 4), cFos (n = 5) and phospho-cFos (Ser374) (n = 4). Β-Actin was used as loading control. (B) Densitometry analysis of blots included in panel A. In the densitometry analysis, the + sign indicates GCDCA administration plus the showed treatment. Data as mean ± SE versus control group. Statistically significant difference versus control group* or versus GCDCA group# are marked.
Table 1.
Proposed transcription factor binding sites in the pNOS-3 region (1.6 Kb) for AP-1 and GATA-1.
TFSEARCH, TESS and TF SITE SCAN are free online software tools for the prediction of transcription factor binding sites. ‘Position’ makes reference to the pNOS-3 sequence (NCBI accession number GI: 14669582). W is A/T, M is A/C, S is C/G, K is G/T, R is A/G.
Fig 2.
Transcription factors cJun and cFos bind to the NOS-3 promoter after GCDCA administration.
(A) Gene expression of GAPDH and NOS-3 as positive controls. (B) Enrichment of cJun and cFos binding to the NOS-3 promoter (position -666) after GCDCA administration. (C) Evaluation of the binding of GATA-1 and GATA-4 to the NOS-3 promoter (position -235) after GCDCA administration. Data expressed as mean ± SE of three independents experiments. Statistically significant difference versus control group (no GCDCA) is marked*.
Fig 3.
AP-1 downregulates NOS-3 expression during cytotoxicity by GCDCA.
Cells not exposed (control group) / exposed to GCDCA (GCDCA group) were treated with Curcumin, Quercetin or the synthetic retinoid SR 11302. (A) Representative western blot for phospho-SP1 (Thr453) (Curcumin and Quercetin, n = 3; SR 11302, n = 7). (B) NOS-3 promoter activity (Curcumin and Quercetin, n = 6; SR 11302, n = 3). (C) NOS-3 protein expression (n = 3). (D) Dose-dependent NOS-3 expression recovery by SR 11302 during GCDCA cytotoxicity. SR 11302 was assayed at 50, 10 and 1 μM. When no indicated, SR 11302 was used at 50 μM. Densitometry analysis for panels (A), (C) and (D) are shown. Data expressed as mean ± SE versus control group. Statistically significant difference versus Control group* or versus GCDCA group# are marked.
Fig 4.
NOS-3 activity recovery by AP-1 inhibition is associated with cell survival during experimental cholestasis.
Effects of AP-1 inhibition on (A) accumulation of NO-end products (n = 3), (B) caspase-3-associated activity (n = 4), (C) cell proliferation and (D) dead cell count (n = 3). In panel (B), SR 11302 was assayed at 50 and 10 μM. When no indicated, SR 11302 was used at 50 μM. Data expressed as mean ± SE. Statistically significant difference versus control group* or versus GCDCA group# are marked.
Fig 5.
NOS inhibition by L-NAME suppresses cell protection by SR 11302 during cholestasis.
Effect of inhibition of NOS activity on (A) cell doubling time (n = 6) and cell metabolic activity (n = 8); (B) caspase-3-associated activity (n = 3); and (C) cyclin D1 expression (n = 4). SR 11302 was used at 50 μM. In panel (A), only positive data for cell doubling time were used (n = 4 for group ‘GCDCA + SR 11302’, and n = 3 for group ‘GCDCA + SR 11302 + L-NAME’). Data expressed as mean ± SE. Statistically significant difference versus control group* or versus GCDCA group# or between groups ‘GCDCA + SR 11302’ and ‘GCDCA + SR 11302 + L-NAME’ (S) are marked.