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) Dual immunofluorescence for GFP (green) and SRC-2 (red) in the ARH of POMC-EGFP mice (controls, upper panels) and pomcSRC-2-KO/POMC-EGFP mice (lower panels). Arrowheads point to double-labeled neurons. Scale bar represents 100 μm. 3V, 3 rd ventricle; ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial hypothalamic nucleus. (B and C) Changes in body weight (B), fat mass, or lean mass (C) of chow-fed male control and pomcSRC-2-KO littermates (3 to 4 months old) after a 24-h fasting. Data are presented as mean ± SEM (% baseline before fasting). n = 6 or 10 mice per group. **p < 0.01 in t tests. (D) Daily energy expenditure measured by CLAMS in male control and pomcSRC-2-KO littermates (3 months old) during ad libitum fed, fasted, and refed periods. Data are presented as mean ± SEM. n = 8 or 9 mice per group. *p < 0.05 and ***p < 0.001 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (E) Representative microscopic images showing a recorded POMC neuron (GFP-labeled in POMC-EGFP mice). Scale bar represents 10 μm. (F) Representative action potential traces in POMC neurons from fasted control versus pomcSRC-2-KO mice. (G and H) Quantifications of firing rate (G) and resting membrane potential (H) in POMC neurons from fasted control versus pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 17 or 21 neurons from 3 mice per group. *p < 0.05 in t tests. (I) Representative traces for mIPSC recorded in POMC neurons from fasted control versus pomcSRC-2-KO mice. (J and K) Quantifications of frequency (J) and amplitude (K) of mIPSC. Data are presented as mean ± SEM. n = 15 or 17 neurons from 3 mice per group. *p < 0.05 in t tests. (L) Relative mRNA levels of indicated genes measured in POMC neurons isolated from control versus pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 8 samples per group. *p < 0.05 in t tests.

Article Snippet: The blot was probed with anti-Flag-HRP (1:3,000–10,000, A8592, Sigma), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Immunofluorescence, Labeling, Isolation

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: The blot was probed with anti-Flag-HRP (1:3,000–10,000, A8592, Sigma), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Luciferase, Activity Assay

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: The blot was probed with anti-Flag-HRP (1:3,000–10,000, A8592, Sigma), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

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