T pJNK, pERK, pPERK, pSTAT-1 (tyrosine 701), pSTAT-1 (serine 727), and a-tubulin. Primary antibodies were diluted in 2 BSA in TBSt. Membranes were incubated with primary antibodies overnight at 4 C, after which they were washed with TBSt followed by the addition of secondary antibody. Goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)conjugated secondary antibody was diluted in 5 BSA in TBSt at a concentration of 1:2500 for pJNK and 1:5000 for all others. Clarity Western ECL substrate (Bio-Rad, Hercules, California) was used to visualize HRP, and the substrate was developed on HyBlot CL film (Denville Scientific, Metuchen, New Jersey). All images were quantified by performing densitometry using Image J software. Statistical analysis. All results are expressed as mean 6 standard error of the mean (SEM). Data were subjected to log transformation as necessary to achieve equal variance and normality. Data were analyzed by either a 1-way or 2-way analysis of variance (ANOVA), as appropriate. For 1-way and 2-way ANOVAs, the Holm-Sidak post hoc test was used for multiple, pair-wisecomparisons between treatment groups. The criterion for significance was set at a ?0.05.RESULTSDCLF Promotes an Increase in Cytosolic Free Ca11 Ca�� levels were measured at 2 times prior to the onset of cytotoxicity. Treatment of cells with TNF did not promote an increase in intracellular calcium at the times examined. Treatment with IFN promoted an increase in intracellular Ca�� at 6 h but not at 12 h. Treatment with DCLF caused an increase in intracellular Ca�� at 6 h that was still apparent at 12 h. Interestingly, the DCLF-induced increase in intracellular Ca�� at 12 h was enhanced by treatment with TNF/IFN (Figure 1). An Intracellular Ca11 Chelator Reduces Cytotoxicity Mediated by DCLF/Cytokine Cotreatment Consistent with previous observations, treatment with DCLF by itself did not AZD0156 site result in cell death (LDH release) (Figure 2A). Similarly, treatment with the cytokines individually or in combination did not result in cytotoxicity. DCLF synergized with TNF to cause LDH release from cells. Although DCLF did not synergize|TOXICOLOGICAL SCIENCES, 2016, Vol. 149, No.FIG. 7. Ca�� AZD0156 site contributes to DCLF/IFN-mediated phosphorylation of STAT-1 at Ser 727. HepG2 cells were treated with VEH (0.1 DMSO), (A) BAPTA/AM (10 lM, 4 h before addition of DCLF/cytokines) or (B) 2-APB (100 lM, simultaneous addition with DCLF/cytokines) and treated with sterile water (Control) or DCLF (250 mM) alone or in combination with TNF and/or IFN. Proteins were collected 18 h after drug treatment. pSTAT-1 (Tyr 701), pSTAT-1 (Ser 727), and a-tubulin levels were detected via western analysis. a, significantly different from corresponding bar in VEH group. b, significantly different from corresponding bar in TNF group. c, significantly different from Control within a cytokine group. d, significantly different from DCLF within a cytokine group. Western analysis of proteins from cells treated with and without BAPTA/ AM or 2-APB was performed simultaneously. Data are represented as mean 6 SEM of at least 3 experiments. Abbreviations: VEH, vehicle; DCLF, diclofenac; pSTAT-1, phosphorylated signal transducer and activator of transcription-1; Tyr, tyrosine; Ser, serine; BAPTA/AM, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid; APB, aminophenoxydiphenyl borate.with IFN alone, IFN enhanced the cytotoxic interaction between DCLF and TNF. Pretreatment of cells with the intra.T pJNK, pERK, pPERK, pSTAT-1 (tyrosine 701), pSTAT-1 (serine 727), and a-tubulin. Primary antibodies were diluted in 2 BSA in TBSt. Membranes were incubated with primary antibodies overnight at 4 C, after which they were washed with TBSt followed by the addition of secondary antibody. Goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)conjugated secondary antibody was diluted in 5 BSA in TBSt at a concentration of 1:2500 for pJNK and 1:5000 for all others. Clarity Western ECL substrate (Bio-Rad, Hercules, California) was used to visualize HRP, and the substrate was developed on HyBlot CL film (Denville Scientific, Metuchen, New Jersey). All images were quantified by performing densitometry using Image J software. Statistical analysis. All results are expressed as mean 6 standard error of the mean (SEM). Data were subjected to log transformation as necessary to achieve equal variance and normality. Data were analyzed by either a 1-way or 2-way analysis of variance (ANOVA), as appropriate. For 1-way and 2-way ANOVAs, the Holm-Sidak post hoc test was used for multiple, pair-wisecomparisons between treatment groups. The criterion for significance was set at a ?0.05.RESULTSDCLF Promotes an Increase in Cytosolic Free Ca11 Ca�� levels were measured at 2 times prior to the onset of cytotoxicity. Treatment of cells with TNF did not promote an increase in intracellular calcium at the times examined. Treatment with IFN promoted an increase in intracellular Ca�� at 6 h but not at 12 h. Treatment with DCLF caused an increase in intracellular Ca�� at 6 h that was still apparent at 12 h. Interestingly, the DCLF-induced increase in intracellular Ca�� at 12 h was enhanced by treatment with TNF/IFN (Figure 1). An Intracellular Ca11 Chelator Reduces Cytotoxicity Mediated by DCLF/Cytokine Cotreatment Consistent with previous observations, treatment with DCLF by itself did not result in cell death (LDH release) (Figure 2A). Similarly, treatment with the cytokines individually or in combination did not result in cytotoxicity. DCLF synergized with TNF to cause LDH release from cells. Although DCLF did not synergize|TOXICOLOGICAL SCIENCES, 2016, Vol. 149, No.FIG. 7. Ca�� contributes to DCLF/IFN-mediated phosphorylation of STAT-1 at Ser 727. HepG2 cells were treated with VEH (0.1 DMSO), (A) BAPTA/AM (10 lM, 4 h before addition of DCLF/cytokines) or (B) 2-APB (100 lM, simultaneous addition with DCLF/cytokines) and treated with sterile water (Control) or DCLF (250 mM) alone or in combination with TNF and/or IFN. Proteins were collected 18 h after drug treatment. pSTAT-1 (Tyr 701), pSTAT-1 (Ser 727), and a-tubulin levels were detected via western analysis. a, significantly different from corresponding bar in VEH group. b, significantly different from corresponding bar in TNF group. c, significantly different from Control within a cytokine group. d, significantly different from DCLF within a cytokine group. Western analysis of proteins from cells treated with and without BAPTA/ AM or 2-APB was performed simultaneously. Data are represented as mean 6 SEM of at least 3 experiments. Abbreviations: VEH, vehicle; DCLF, diclofenac; pSTAT-1, phosphorylated signal transducer and activator of transcription-1; Tyr, tyrosine; Ser, serine; BAPTA/AM, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid; APB, aminophenoxydiphenyl borate.with IFN alone, IFN enhanced the cytotoxic interaction between DCLF and TNF. Pretreatment of cells with the intra.