Journal: Nutrients

Article Title: Wogonin, a Compound in Scutellaria baicalensis , Activates ATF4–FGF21 Signaling in Mouse Hepatocyte AML12 Cells

doi: 10.3390/nu14193920

Figure Lengend Snippet: Primers used for real-time PCR analysis.

Article Snippet: pGL3-FGF21 contained –2 kbp to –40 bp of the mouse Fgf21 promoter [ ]. pGL3-ATF4 contained –0.5 kbp to –100 bp of the mouse Atf4 promoter. pRK-ATF4, the human ATF4 expression vector, was a gift from Yihong Ye (Addgene plasmid #26114) [ ]. siRNAs against luciferase (siLuc) (Invitrogen, Waltham, MA, USA, 12935-146) and ATF4 (Santa Cruz, Dallas, TX, USA, sc-35113) were purchased.

Techniques: Real-time Polymerase Chain Reaction

Journal: Nutrients

Article Title: Wogonin, a Compound in Scutellaria baicalensis , Activates ATF4–FGF21 Signaling in Mouse Hepatocyte AML12 Cells

doi: 10.3390/nu14193920

Figure Lengend Snippet: Wogonin induces Atf4 and Fgf21 expression. ( A ) Wogonin increased Fgf21 expression in AML12 cells. Cells were treated with 10 and 20 µM of baicalin, baicalein, and wogonin for 48 h. n = 4 per group. ( B ) Gene expression of FGF21-regulating transcription factors in AML12 cells. Cells were treated with 10 and 20 µM of wogonin for 48 h. n = 4 per group. ( C ) Wogonin increased the protein levels of ATF4 in AML12 cells. Cells were treated with 10 and 20 µM of wogonin for 48 h. The protein bands were quantified. n = 4 per group. ( D ) Wogonin increased the expression of genes regulated by ATF4 in AML12 cells. Cells were treated with 10 and 20 µM of wogonin for 48 h. n = 4 per group. Data are represented as mean ± SD. * p < 0.05; ** p < 0.01. Comparisons among multiple groups were assessed using one-way ANOVA, followed by Tukey’s post hoc test.

Article Snippet: pGL3-FGF21 contained –2 kbp to –40 bp of the mouse Fgf21 promoter [ ]. pGL3-ATF4 contained –0.5 kbp to –100 bp of the mouse Atf4 promoter. pRK-ATF4, the human ATF4 expression vector, was a gift from Yihong Ye (Addgene plasmid #26114) [ ]. siRNAs against luciferase (siLuc) (Invitrogen, Waltham, MA, USA, 12935-146) and ATF4 (Santa Cruz, Dallas, TX, USA, sc-35113) were purchased.

Techniques: Expressing, Gene Expression

Journal: Nutrients

Article Title: Wogonin, a Compound in Scutellaria baicalensis , Activates ATF4–FGF21 Signaling in Mouse Hepatocyte AML12 Cells

doi: 10.3390/nu14193920

Figure Lengend Snippet: Wogonin affects the promoter activity of Atf4 . ( A ) The protein levels of phospho- and total-eIF2α were not changed in AML12 cells. Cells were treated with 10 and 20 µM of wogonin for 48 h. The protein bands were quantified. n = 4 per group. ( B ) Wogonin increased ATF4-luciferase activity. AML12 cells were co-transfected with the pGL3-ATF4 and pRL-SV40 vectors. After 24 h of transfection, cells were treated with 20 μM of wogonin for 24 h. n = 5 per group. Data are represented as mean ± SD. ( C ) The gene expression of ATF4-regulating transcription factors in AML12 cells. Cells were treated with 10 and 20 µM of wogonin for 48 h. n = 4 per group. Data are represented as mean ± SD. ** p < 0.01. Comparisons between two groups were assessed using unpaired two-tailed t tests and those among multiple groups were assessed using one-way ANOVA, followed by Tukey’s post hoc test.

Article Snippet: pGL3-FGF21 contained –2 kbp to –40 bp of the mouse Fgf21 promoter [ ]. pGL3-ATF4 contained –0.5 kbp to –100 bp of the mouse Atf4 promoter. pRK-ATF4, the human ATF4 expression vector, was a gift from Yihong Ye (Addgene plasmid #26114) [ ]. siRNAs against luciferase (siLuc) (Invitrogen, Waltham, MA, USA, 12935-146) and ATF4 (Santa Cruz, Dallas, TX, USA, sc-35113) were purchased.

Techniques: Activity Assay, Luciferase, Transfection, Gene Expression, Two Tailed Test

Journal: Nutrients

Article Title: Wogonin, a Compound in Scutellaria baicalensis , Activates ATF4–FGF21 Signaling in Mouse Hepatocyte AML12 Cells

doi: 10.3390/nu14193920

Figure Lengend Snippet: Deficiency of ATF4 suppresses wogonin-induced Fgf21 expression. Fgf21 expression was suppressed by transfecting AML12 cells with siRNA against ATF4. After 24 h of transfection, cells were treated with 20 µM of wogonin for 48 h. n = 4 per group. Data are represented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Comparisons among multiple groups were assessed using one-way ANOVA, followed by Tukey’s post hoc test.

Article Snippet: pGL3-FGF21 contained –2 kbp to –40 bp of the mouse Fgf21 promoter [ ]. pGL3-ATF4 contained –0.5 kbp to –100 bp of the mouse Atf4 promoter. pRK-ATF4, the human ATF4 expression vector, was a gift from Yihong Ye (Addgene plasmid #26114) [ ]. siRNAs against luciferase (siLuc) (Invitrogen, Waltham, MA, USA, 12935-146) and ATF4 (Santa Cruz, Dallas, TX, USA, sc-35113) were purchased.

Techniques: Expressing, Transfection

Journal: Cell metabolism

Article Title: Trimethylamine N-Oxide Binds and Activates PERK to Promote Metabolic Dysfunction.

doi: 10.1016/j.cmet.2019.08.021

Figure Lengend Snippet: Figure 3. TMAO Binds Directly to PERK to Induce FoxO1 (A) FLAG-PERK or Myc-PERK was expressed in HEK293T cells, immunoprecipitated using a control immunoglobulin (IgG) or an anti-FLAG antibody (aFLAG), incubated with 3H-TMAO, and subjected to scintillation counting; n = 3. (B) Immunoaffinity-purified FLAG-PERK was incubated with 3H-TMAO in the presence of increasing concentrations of non-radioactive TMAO, choline, or betaine and then subjected to scintillation counting; n = 3. (C) Recombinant proteins encoding the lumenal domain (LD) or cytosolic domain (CD) of PERK (illustrated in schematic) were purified from bacteria, incubated with 3H-TMAO, and subjected to scintillation counting; n = 3. (D) Recombinant protein encoding the LD of PERK was incubated with 3H-TMAO in the presence of increasing amounts of non-radioactive TMAO, choline, betaine, carnitine, or g-butyrobetaine and subjected to scintillation counting; n = 3. (E) Primary mouse hepatocytes were isolated from PERKflox/flox male mice, infected with adenovirus encoding either GFP (Ad-GFP) or Cre (Ad-Cre), treated with 50 mM TMAO for 24 h, and analyzed by immunoblotting. (F and G) Male C57BL/6J mice were placed on a standard chow diet with or without supplementation of 0.12% TMAO at 4 weeks, injected intraperitoneally with the PERK inhibitor GSK2656157 (PERKi, 5 mg/kg BW/day) beginning at 6 weeks, and sacrificed at 8 weeks. Livers were subjected to immunoblotting (F). GTT was performed at 8 weeks (G); n = 6.

Article Snippet: Mouse: ob/ob Jackson Labs Cat. 000632 Mouse: PERK Flox Jackson Labs Cat. 023066 Mouse: C57BL/6 XBP1 Flox Kaser et al., 2008 N. A. Oligonucleotides See Table S2 for oligos used in this study N. A. N. A. Recombinant DNA pMXs Flag-PERK Addgene Cat. #101793 pBabe Flag-PERK cytosolic domain Addgene Cat. #58423 pCDNA Myc-PERK Addgene Cat. #21814 pcDNA Myc-PERK lumenal domain Addgene Cat. #21816 pGL3 ATF4 Addgene Cat. #21850 pTriEx-4 Flag-XBP1u Addgene Cat. #31239 pOFluc-GL3 ATF6 Addgene Cat. #11976 Cas9 sgRNA vector Addgene Cat. #68463 pTK-Gaussia luciferase vector Thermo Fisher Cat. #16192 Software and Algorithms Morpheus https://software.broadinstitute.org/morpheus/ N. A. Enrichr http://amp.pharm.mssm.edu/Enrichr/ N. A. Gene Set Enrichment Analysis http://software.broadinstitute.org/gsea/ index.jsp N. A. ImageJ NIH N. A.

Techniques: Immunoprecipitation, Control, Incubation, Recombinant, Bacteria, Isolation, Infection, Western Blot, Injection