(A) Quantitative PCR for Plk3. RNA was collected at designated timepoints for control and Lrh-1LKO mice treated with vehicle or 1 mg/kg tunicamycin (TM) (n = 3–6). Data were normalized to Tbp expression. Significance at p<0.01. (B) Immunoblot for PLK3 protein in control and Lrh-1LKO mice treated with vehicle or 1 mg/kg TM for designated timepoints. Liver tissue was boiled in Laemmli buffer to extract insoluble protein and samples were pooled (n = 2) prior to gel loading. β-actin was used as a loading control. (C) Quantitative PCR for Plk3. RNA was collected at designated timepoints for primary hepatocytes isolated from control and Lrh-1LKO mice treated with vehicle or 0.5 µg/ml TM (n = 3). Data were normalized to Tbp expression. Significance at p<0.01. (D) A LRH-1 binding site in the Plk3 promoter was identified 285 bases upstream of the TSS. Chromatin immunoprecipitation was performed from livers of control mice treated with vehicle or TM (1 mg/kg) for 6 hr (n = 3). Immunoprecipation was done with an anti-LRH-1 antibody or mouse IgG as a control. qPCR was used to determine binding by use of primers flanking binding site. Significance at p<0.05 between antibodies with error bars representing SEM. (E) TLR-3 cells were transfected with nonsilencing siRNA or siRNA against Lrh-1 and nuclear protein was prepared at designated timepoints. Samples were immunoblotted for total ATF2 and phospho-ATF2 (mT51/53; hT69/T71) following treatment with vehicle or 1 ug/ml TM. Results representative of three independent experiments. (F) Relative luciferase activity for TLR-3 cells transfected with a cAMP response element (CRE)-luciferase reporter, Atf2, and Lrh-1. 48 hr after transfection, cells were treated with vehicle or 5 µg/ml TM and the following inhibitors: 10 µM D-JNKi for JNKs, 1 µM SB202190 for p38, 10 µM GW84362X for PLK1/PKL3, or 1 µM GSK650394A for SGK. 24 hr after treatment, cells were lysed, and luciferase activity was measured and normalized to β-galactosidase activity. Significance at p<0.01 as compared with TM treated cells (n = 3). Results representative of three independent experiments. (G) Atf3 expression by qPCR in TLR-3 cells transfected with control or siRNA targeting Lrh-1 (knockdown efficiency same as 5E), along with overexpression of constitutively active Atf2 (C2/Atf2), Plk3, or an empty vector. 48 hr post transfection, cells were treated with 1 µg/ml TM for 24 hr. Data were normalized to Tbp expression. Significance at p<0.01 for TM treated vs vehicle treated samples (n = 3). Results representative of three independent experiments. (H) Atf3 expression by qPCR from primary hepatocytes from control and Lrh-1LKO mice treated with vehicle or 5 µg/ml TM and 10 µM PLK3 inhibitor GW843682X for 24 hr. Data were normalized to Tbp expression. Significance at p<0.01 between genotypes (n = cells from 2–3 mice/group). (I) Wild-type mice were i.p. injected with PLK3 inhibitor GW843682X (1 mg/kg BW) or vehicle (DMSO). Mice were also i.p. injected with TM (1 mg/kg BW) or vehicle (DMSO). 48-hr post injection, liver tissue was collected and nuclear protein was isolated to assess accumulation of UPR transcription factors spliced XBP-1, cleaved ATF6, and ATF4. TBP was used as a loading control. Results representative of results from three mice. (J) Wild-type and Plk3−/− mice were treated with TM (0.5 mg/kg) or vehicle. Nuclear UPR proteins were assessed by immunoblot at designated timepoints . TBP was used as a loading control. Results representative of four independent samples.