From cells treated with and without SP600125 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 BLU-554 price transcription-1; Tyr, tyrosine; Ser, serine; pERK, phosphorylated extracellular signal-regulated kinase.Ca11 Contributes to DCLF-Mediated JNK Activation DCLF/TNF-mediated cytotoxicity and the purchase Quinagolide (hydrochloride) IFN-Mediated enhancement of that cytotoxicity are JNK-dependent processes (Fredriksson et al., 2011; Maiuri et al., 2015). DCLF caused activation of JNK at 18 hours, consistent with previous findings, and this effect was unaltered by cytokine treatment (Figures 5A and B). DCLF/cytokinemediated JNK activation was reduced by pretreatment with BAPTA/AM (Figure 5A) and by treatment with 2-APB (Figure 5B). Ca11 Contributes to DCLF-Mediated ERK Activation The IFN-mediated enhancement of DCLF/TNF-induced cytotoxicity depends on ERK (Maiuri et al., 2015). DCLF treatment promoted strong activation of ERK that was unaffected by cytokine treatment (Figure 6), confirming our earlier observation. Pretreatment of cells with BAPTA/AM significantly reduced ERK activation induced by DCLF (Figure 6A). Similarly, treatment of HepG2 cells with 2-APB markedly reduced DCLF-mediated activation of ERK (Figure 6B). Ca11 Contributes to DCLF/IFN-Mediated Phosphorylation of STAT-1 at Ser 727 Janus kinase (JAK)-mediated phosphorylation of STAT-1 at Tyr 701 and ERK-mediated phosphorylation of STAT-1 at Ser 727 arerequired for maximal activation of STAT-1 and STAT-1-mediated apoptosis (Varinou et al., 2003). We demonstrated previously that DCLF-mediated ERK activation promotes phosphorylation of STAT-1 at Ser 727 in the presence of IFN and that the IFN-mediated enhancement of DCLF/TNF-induced cytotoxicity is driven by ERK (Maiuri et al., 2015). Since Ca�� contributed to DCLF-mediated ERK activation, we evaluated whether Ca�� also contributes to DCLF/IFN-induced phosphorylation of STAT-1 at Ser 727. As reported previously by Maiuri et al. (2015), treatment with IFN led to phosphorylation of Tyr 701 of STAT-1 in the absence and presence of DCLF, but Ser 727 of STAT-1 was only phosphorylated in the presence of both IFN and DCLF (Figure 7). Treatment of HepG2 cells with either BAPTA/AM or 2-APB significantly reduced DCLF/IFN-mediated phosphorylation of STAT-1 at Ser 727 without affecting phosphorylation of STAT-1 at Tyr 701 (Figure 7). JNK Promotes DCLF/IFN-Mediated Phosphorylation of STAT-1 at Ser 727 Via Activation of ERK Activation of JNK and ERK both contributed to the IFN-mediated enhancement of DCLF/TNF-induced cytotoxicity (Maiuri et al., 2015). ERK contributed to the phosphorylation of STAT-1 at Ser 727 (Maiuri et al., 2015), but the role of JNK in this response is|TOXICOLOGICAL SCIENCES, 2016, Vol. 149, No.FIG. 9. Aspirin does not promote activation of JNK, ERK, or PERK. HepG2 cells were treated with VEH (0.1 DMSO), ASA (2 mM), or DCLF (250 mM). Protein was collected 18 h after treatment. (A) pJNK, (B) pERK, and (C) pPERK levels were measured via western analysis. a, significantly different from all treatment groups. Data are represented as mean 6 SEM of at least 3 experiments. Abbreviations: VEH, vehicle; ASA, aspirin; pJNK, phosphorylated c-Jun N-terminal kinase; pERK, phosphorylated extracellular signal-regulated kinase; pPERK, phosphorylated protein kinase RNA-like endoplasmic reticulum kinase.unknown, as is whether there is interde.From cells treated with and without SP600125 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; pERK, phosphorylated extracellular signal-regulated kinase.Ca11 Contributes to DCLF-Mediated JNK Activation DCLF/TNF-mediated cytotoxicity and the IFN-mediated enhancement of that cytotoxicity are JNK-dependent processes (Fredriksson et al., 2011; Maiuri et al., 2015). DCLF caused activation of JNK at 18 hours, consistent with previous findings, and this effect was unaltered by cytokine treatment (Figures 5A and B). DCLF/cytokinemediated JNK activation was reduced by pretreatment with BAPTA/AM (Figure 5A) and by treatment with 2-APB (Figure 5B). Ca11 Contributes to DCLF-Mediated ERK Activation The IFN-mediated enhancement of DCLF/TNF-induced cytotoxicity depends on ERK (Maiuri et al., 2015). DCLF treatment promoted strong activation of ERK that was unaffected by cytokine treatment (Figure 6), confirming our earlier observation. Pretreatment of cells with BAPTA/AM significantly reduced ERK activation induced by DCLF (Figure 6A). Similarly, treatment of HepG2 cells with 2-APB markedly reduced DCLF-mediated activation of ERK (Figure 6B). Ca11 Contributes to DCLF/IFN-Mediated Phosphorylation of STAT-1 at Ser 727 Janus kinase (JAK)-mediated phosphorylation of STAT-1 at Tyr 701 and ERK-mediated phosphorylation of STAT-1 at Ser 727 arerequired for maximal activation of STAT-1 and STAT-1-mediated apoptosis (Varinou et al., 2003). We demonstrated previously that DCLF-mediated ERK activation promotes phosphorylation of STAT-1 at Ser 727 in the presence of IFN and that the IFN-mediated enhancement of DCLF/TNF-induced cytotoxicity is driven by ERK (Maiuri et al., 2015). Since Ca�� contributed to DCLF-mediated ERK activation, we evaluated whether Ca�� also contributes to DCLF/IFN-induced phosphorylation of STAT-1 at Ser 727. As reported previously by Maiuri et al. (2015), treatment with IFN led to phosphorylation of Tyr 701 of STAT-1 in the absence and presence of DCLF, but Ser 727 of STAT-1 was only phosphorylated in the presence of both IFN and DCLF (Figure 7). Treatment of HepG2 cells with either BAPTA/AM or 2-APB significantly reduced DCLF/IFN-mediated phosphorylation of STAT-1 at Ser 727 without affecting phosphorylation of STAT-1 at Tyr 701 (Figure 7). JNK Promotes DCLF/IFN-Mediated Phosphorylation of STAT-1 at Ser 727 Via Activation of ERK Activation of JNK and ERK both contributed to the IFN-mediated enhancement of DCLF/TNF-induced cytotoxicity (Maiuri et al., 2015). ERK contributed to the phosphorylation of STAT-1 at Ser 727 (Maiuri et al., 2015), but the role of JNK in this response is|TOXICOLOGICAL SCIENCES, 2016, Vol. 149, No.FIG. 9. Aspirin does not promote activation of JNK, ERK, or PERK. HepG2 cells were treated with VEH (0.1 DMSO), ASA (2 mM), or DCLF (250 mM). Protein was collected 18 h after treatment. (A) pJNK, (B) pERK, and (C) pPERK levels were measured via western analysis. a, significantly different from all treatment groups. Data are represented as mean 6 SEM of at least 3 experiments. Abbreviations: VEH, vehicle; ASA, aspirin; pJNK, phosphorylated c-Jun N-terminal kinase; pERK, phosphorylated extracellular signal-regulated kinase; pPERK, phosphorylated protein kinase RNA-like endoplasmic reticulum kinase.unknown, as is whether there is interde.