Journal: Molecular metabolism

Article Title: The Mediator complex kinase module is necessary for fructose regulation of liver glycogen levels through induction of glucose-6-phosphatase catalytic subunit (G6pc).

doi: 10.1016/j.molmet.2021.101227

Figure Lengend Snippet: Figure 6: Binding of ChREBP and FOXO1 transfactors on G6pc promoter in MED13-LKO mice. ChIP-qPCR was performed to analyze the enrichment for transfactors in the G6pc promoter region. A) ChREBP-specific antibody was used to pull down sonicated liver chromatin of the Med13fl/fland MED13-LKO mice (n ¼ 6). Precipitated DNA enrichment is shown as a percentage of input DNA, with average Ct values of 31.2 (Fasted WT), 29.65 (Fasted KO), 30.38 (Sucrose WT), 29.3 (Sucrose KO), and 30.0 (Fructose WT). B) FOXO1- specific antibody was used to pull down sonicated liver chromatin of the Med13fl/fland MED13-LKO mice (n ¼ 6). Precipitated DNA enrichment is shown as a percentage of input DNA, with average Ct values of 32.4 (Fasted WT), 31.5 (Fasted KO), 33.8 (Sucrose WT), 31.9 (Sucrose KO), 34.1 (Fructose WT), and 31.8 (Fructose KO). Non-specific IgG was used as a control, which displayed Ct values > 38 or were undetectable. A promoter set of the non-specific upstream region was also used as a control, which showed Ct value > 38 or was undetectable. P values were determined by one-way ANOVA followed by the Turkey-Kramer multiple comparisons test. Error bars indicate the mean SEM. Bars with different letters are significantly different from each other (p < 0.05).

Article Snippet: Antibodies and immunoblotting Antibodies against the following proteins were used in this study: MED13 (1:1000, Fisher PA5-35924 or 1:1000 Bethyl A301-278A), MED12 (1:1000, Abcam ab70842), CDK8 (1:1000, Abcam ab54561 or 1:1000, Bethyl A302-501A-M), CCNC (1:1000, Bethyl A301-989A), MED15 (1:1000, Bethyl A302-422A), nucleolin (1:1000, Cell Signaling 14574S), tubulin (1:1000, Cell Signaling 2144S), ChREBP (1:1000, Novus Biologicals NB400e135SS), phospho-S6 ribosomal protein (1:1000, Cell Signaling 4858P), S6 ribosomal protein (1:1000, Cell Signaling 2317S), phospho-AKT S473 (1:1000, Cell Signaling 4060), T308 (1:1000, Cell Signaling 4056), T450 (1:1000, Cell Signaling 12,178), total AKT (1:1000, Cell Signaling 9272), phospho GS (S641, 1:1000 Cell Signaling 3891), GS (1:1000 Cell Signaling 3893), phospho FOXO1 (S256, 1:1000 Cell Signaling 84,192), and FOXO1 (1:1000 Cell Signaling 2880).

Techniques: Binding Assay, ChIP-qPCR, Sonication, Control

Journal: Molecular metabolism

Article Title: The Mediator complex kinase module is necessary for fructose regulation of liver glycogen levels through induction of glucose-6-phosphatase catalytic subunit (G6pc).

doi: 10.1016/j.molmet.2021.101227

Figure Lengend Snippet: Figure 7: Luciferase reporter assay of ChREBP and FOXO1 transfactors in MED13-LKO mice. Isolated primary hepatocytes from adenovirus (Ad)-GFP (Ad-WT) or Ad-Cre (Ad- KO) transduced Med13fl/fl (WT) or Med13fl/flAlb-Cre (KO) mice were used. A) Luciferase reporter assays for the effects of ChREBP-b on ChoRE activity in either Med13fl/fl(WT and Ad-WT) or Med13-LKO (KO and Ad-KO) as indicated. B) Luciferase reporter assays for the effects of FOXO1 on G6pc promoter activity in indicated cells, either from Med13fl/fl(WT and Ad-WT) or Med13-LKO (KO and Ad-KO). P values were determined by Student’s t-test (p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.001, and n ¼ 3. The values above each of the error bars indicate the fold stimulation.

Article Snippet: Antibodies and immunoblotting Antibodies against the following proteins were used in this study: MED13 (1:1000, Fisher PA5-35924 or 1:1000 Bethyl A301-278A), MED12 (1:1000, Abcam ab70842), CDK8 (1:1000, Abcam ab54561 or 1:1000, Bethyl A302-501A-M), CCNC (1:1000, Bethyl A301-989A), MED15 (1:1000, Bethyl A302-422A), nucleolin (1:1000, Cell Signaling 14574S), tubulin (1:1000, Cell Signaling 2144S), ChREBP (1:1000, Novus Biologicals NB400e135SS), phospho-S6 ribosomal protein (1:1000, Cell Signaling 4858P), S6 ribosomal protein (1:1000, Cell Signaling 2317S), phospho-AKT S473 (1:1000, Cell Signaling 4060), T308 (1:1000, Cell Signaling 4056), T450 (1:1000, Cell Signaling 12,178), total AKT (1:1000, Cell Signaling 9272), phospho GS (S641, 1:1000 Cell Signaling 3891), GS (1:1000 Cell Signaling 3893), phospho FOXO1 (S256, 1:1000 Cell Signaling 84,192), and FOXO1 (1:1000 Cell Signaling 2880).

Techniques: Luciferase, Reporter Assay, Isolation, Activity Assay

Journal: Cell reports

Article Title: FOXO1 constrains activation and regulates senescence in CD8 T cells.

doi: 10.1016/j.celrep.2020.108674

Figure Lengend Snippet: Figure 1. FOXO1 limits naive CD8+ T cell AP-1 family member expression (A) mRNA abundance (RNA-seq) of WT and Foxo1-KO naive CD8+ T cells. Selected genes are labeled, including members of the AP-1 family. The gray point color indicates no FOXO1 genomic binding detected at day 0; green indicates R1 peaks detected; the size of points indicates the number of FOXO1 genomic binding sites nearest the gene TSS. (B) Intracellular immunostaining determination of indicated protein abundance in naive P14 cells of the indicated genotypes and CD44 expression level. (C) FOXO1 genomic binding (ChIP-seq) and chromatin accessibility (ATAC-seq) for select AP-1 subunits in naive and post-infection (p.i.) (LCMV-ARM) P14 T cells. The y-axis maximum for all ATAC-seq is 75. For (B), data are averaged from 3 experiments with n = 3 mice per group per experiment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; unpaired Student’s t test was used, and error bars represent means ± SEMs. Informatics experiments are from 1 biological sample per condition.

Article Snippet: Phospho-FoxO1 (CST; 1:1000; Ser256; cat#9461; note antibody is named for human phospho-site) and FoxO1 (CST; 1:1000; C29H4; cat #2880). beta-tubulin (cat# 05-661; 1:1000; Millipore).

Techniques: Expressing, RNA Sequencing, Labeling, Binding Assay, Immunostaining, Quantitative Proteomics, ChIP-sequencing, Infection

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet: (A) Effects of SRC-2 on POMC-luciferase activity in “fed” and fasted cells. Data are presented as mean ± SEM. n = 4 or 5 repeated experiments with 6 biological replicates per group in each experiment. *p < 0.05 and ***p < 0.001 in two-way ANOVA analyses followed by Sidak tests. (B) Effects of SRC-2 on POMC-luciferase activity in fasted cells with or without FoxO1 knockdown. Data are presented as mean ± SEM. n = 4–7 repeated experiments with 6 biological replicates per group in each experiment. ***p < 0.001 in two-way ANOVA analyses followed by Sidak tests. (C) Hypothalamic mRNAs in fasted control and pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 14 or 15 mice per group. *p < 0.05 in t tests. (D) Interaction of endogenous SRC-2 and FoxO1 in the hypothalamus from chow- or HFD-fed male mice. (E) Body weight curves in male control and pomcSRC-2-KO littermates fed HFD ad libitum since 7 weeks of age. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (F) Fat or lean mass in 14-week-old male control and pomcSRC-2-KO littermates after 7 weeks HFD feeding. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in t tests. (G) Cumulative HFD intake in male control and pomcSRC-2-KO littermates fed HFD ad libitum since 7 weeks of age. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (H) Changes in body weight during a 3-week HFD feeding in mice receiving stereotaxic injections of AAV-FLEX-FoxO1 AAA or AAV-GFP into the ARH. Data are presented as mean ± SEM. n = 6–10 mice per group. * or #, p < 0.05 between POMC-Cre+ AAV-FLEX-FoxO1 AAA versus WT+ AAV-FLEX-FoxO1 AAA or versus pomcSRC-2-KO+AAV-FLEX-FoxO1 AAA in two-way ANOVA analyses repeated measurement followed by Sidak tests. (I and J) Changes in body weight (I) and fat mass (J) at the end of the 3-week HFD feeding in mice described in (H). Data are presented as mean ± SEM. n = 6–10 mice per group. *p < 0.05 in two-way ANOVA analyses followed by Sidak tests; p = 0.09 in t tests. (K) Cumulative HFD intake in mice described in (H). Data are presented as mean ± SEM. n = 6–10 mice per group. * or #, p < 0.05 between POMC-Cre+ AAV-FLEX-FoxO1 AAA versus WT+ AAV-FLEX-FoxO1 AAA or versus pomcSRC-2-KO+AAV-FLEX-FoxO1 AAA in two-way ANOVA analyses repeated measurement followed by Sidak tests.

Article Snippet: FoxO1 siRNA (mouse-specific) , Cell Signaling , #6468.

Techniques: Luciferase, Activity Assay, Knockdown, Control

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet:

Article Snippet: FoxO1 siRNA (mouse-specific) , Cell Signaling , #6468.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Transgenic Assay, Control