Skip to main content
. 2017 Jul 19;4(3):339–363. doi: 10.1016/j.jcmgh.2017.07.003

Figure 7.

Figure 7

Inhibition of signal transducer and activator of transcription 3 (STAT3) by AG-490 and its consequences on apoptosis signaling in hepatocytes. (A) Hepatitis B virus (HBV)–infected differentiated HepaRG cells were either treated with 0.1% dimethyl sulfoxide (DMSO) or with different amounts of AG-490 (0.2 μM, 1 μM, 5 μM, 20 μM, and 100 μM); 24 hours after administration of AG-490, cell viability was analyzed by CellTiter-Blue Cell Viability Assay. Values are given as median ± SD (n = 3; statistical significance relative to DMSO-control; Student's t test). (B) Mock- or HBV-infected primary human hepatocytes (PHHs) were either incubated with 0.1% DMSO or with 100 μM of AG-490 for 24 hours. Nuclear or cytosolic proteins were prepared using the NE-PER Nuclear and Cytoplasmic extraction Reagents. Inhibition of STAT3 phosphorylation (upper panel) was analyzed by Western blotting using nuclear proteins and anti-pSTAT3 antibodies. Purity of nuclear protein preparations was controlled by Western blotting using antialbumin antibodies. Cleavage of poly(ADP-ribose) polymerase (PARP) (lower panel) was analyzed by Western blotting using nuclear proteins and anti–poly(ADP-ribose) polymerase (PARP) antibodies. Positions of 116 kDa uncleaved (upper band) and 85 kDa cleaved PARP (lower band) are indicated. Membranes were reprobed with anti-lamin antibodies to control equal protein loading. (C) Caspase 3/7 activity in mock- or HBV-infected PHHs treated with either DMSO or with 100 μM AG-490 was measured by luminescent caspase 3/7 assay. Mean ± SD is given (n = 3; ***P < .001; Student's t test). (D) Expression levels of cIAP2, Mn-SOD, IER3, and TNFAIP8 genes in HBV-infected PHHs treated with either DMSO or with AG-490 (20 or 100 μM) were determined by real-time reverse transcription polymerase chain reaction. Gene expression levels in HBV-infected cells treated with DMSO were set to 100%. Values are shown as mean ± SD, statistical significance was calculated relative to the respective DMSO-control sample (n = 3; *P < .05; **P < .01; ***P < .001; Student's t test). ns, not significant.