Journal: eLife

Article Title: Autophagy compensates for Lkb1 loss to maintain adult mice homeostasis and survival

doi: 10.7554/elife.62377

Figure Lengend Snippet: Figure 4. p53 deficiency extends the life span of Atg7-/-;Stk11-/- mice. (a) Left: Representative IHC for p53 in different tissues of WT control, Atg7-/-, Stk11-/-, and Atg7-/-;Stk11-/- adult mice shows an increase of nuclear p53 in Atg7-ablated tissues. Right: Bar graphs represent the quantification of nuclear p53 in different tissues. Data are mean ± s.e.m. *p<0.05, **p<0.01, and ***p<0.001. (b) Quantitative real-time PCR of Cdkn1a (p21) and PTEN for intestine tissues of WT control, Atg7-/-, Stk11-/- and Atg7-/-;Stk11-/- adult mice. Data are mean ± s.e.m. *p<0.05, ***p<0.001, and ****p<0.0001. (c) Figure 4 continued on next page

Article Snippet: Antibodies utilized for IHC were Atg7 (Sigma Aldrich, A2856, RRID:AB_ 1078239), Lkb1 (Santa Cruz Biotechnology, sc-32245, RRID:AB_627890), p62 (Enzo Life Sciences, PW9860-0100, RRID:AB_2877676), p-AMPKTh172 (Cell Signaling, 2535S, RRID:AB_331250), p-ACCS79 (Cell Signaling, 3661, RRID:AB_330337), p-S6S235/236 (Cell Signaling, 4858, RRID:AB_916156), p-ULK1S555 (Cell Signaling, 5869, RRID:AB_10707365), p-ULK1S757 (Cell Signaling, 14202, RRID:AB_ 2665508), LC3 (Nano Tools, LC3-5F10, RRID:AB_2722733), Ki67 (Abcam, ab-15580, RRID:AB_ 443209), cleaved caspase-3 (Cell Signaling, 9661S), OLFM4 (Cell Signaling, 39141, RRID:AB_ 2650511), lysozyme (Agilent, A0099, RRID:AB_2341230), and p53 (Novus Biologicals, NB200-103SS, RRID:AB_2877680).

Techniques: Control, Real-time Polymerase Chain Reaction

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Schematic representation of the liver-specific knockout strategy used for the Ncoa6 gene, based on the Cre-LoxP system. Blue arrows denote the specific primer binding sites for genotyping. (B-D) The liver specific knockout of Ncoa6 was confirmed at the level of genomic DNA (gDNA), mRNA and protein. gDNA and mRNA were isolated from the liver, muscle, kidney and white adipose tissue (WAT) of Ncoa6 fl/fl /Cre +/- (LKO) and Ncoa6 fl/fl /Cre -/- (F/F) mice. The Ncoa6 gDNA levels were determined by conventional PCR (B). The Ncoa6 mRNA and NCOA6 protein levels were determined by qRT-PCR (C; n = 4) and western blotting (D; n = 3), respectively. mRNA data are presented as the mean ± SEM; *** P < 0.001 by t -test comparisons of the genotypes.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Knock-Out, Binding Assay, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) The blood glucose levels were measured in Ncoa6 LKO (n = 8) and WT (F/F, n = 9) mice after 16 h fasting. (B) Improved glucose tolerance in the Ncoa6 LKO mice. Intraperitoneal glucose tolerance tests were performed in 10-week-old mice (n = 9 for F/F, n = 8 for LKO) after a 16 h fast. (C) The serum insulin levels were measured in Ncoa6 LKO (n = 6 for fed, n = 7 for fasted) and WT mice (n = 7 for both fed and fasted) before and after a 16 h fast. (D) Enhanced insulin sensitivity in Ncoa6 LKO mice. Intraperitoneal insulin tolerance tests were performed in 10-week-old Ncoa6 LKO (n = 13) and WT mice (n = 10) after a 6 h fast. (E) Pyruvate tolerance was determined in 10-week-old Ncoa6 LKO (n = 11) and WT (n = 10) mice via an intraperitoneal injection with pyruvate (2 g/kg body weight) after overnight fasting for 16 h. AUC values for GTT, ITT, and PTT were calculated and are presented on the right of each graph. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01 by t -test comparisons of the genotypes. WT, wild type; GTT, glucose tolerance test; ITT, insulin tolerance test; PTT, pyruvate tolerance test.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Injection

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Decreased induction of gluconeogenic gene transcripts in the Ncoa6 LKO liver following fasting. Liver RNAs were isolated from Ncoa6 LKO and WT mice after a 24 h fast (n = 6 for each group). The mRNA levels of Pc , Pck1 , and G6pc were determined by real-time qRT-PCR. (B) Attenuated induction of PCK protein in the Ncoa6 LKO liver after fasting. Protein expression was analyzed in the liver of ad libitum fed mice and 16 h fasted mice (n = 7 for each group) by western blot analysis. Intensities of the protein bands were measured using the ImageJ program. (C) Glucagon tolerance of Ncoa6 LKO mice (n = 10 for F/F, n = 14 for LKO). Ten-week-old mice were injected intraperitoneally with glucagon (15 µg/kg) after 6 h of fasting. Blood samples were prepared at 0, 30, 60, 90, and 120 min after the glucagon injection. AUC values are shown on the right. (D) Effects of NCOA6 on the glucose production level in primary hepatocytes. Glucose production was compared between Ncoa6 LKO and WT mouse primary hepatocytes after a 4 h forskolin treatment using glucose free media supplemented with lactate and sodium pyruvate (n = 4 independent experiments). (E) Alteration of Pck1 and G6pc transcript induction in Ncoa6 LKO primary hepatocytes following a 4 h treatment of these cells with forskolin (n = 5 independent experiments). Transcript levels were analyzed by real-time qRT-PCR. Data are presented as the mean ± SEM; ** P < 0.01, *** P < 0.001, determined using one-way ANOVA. Fsk, forskolin.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot, Injection

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Serum glucagon levels in the Ncoa6 LKO (n = 6 for fed, n = 7 for fasted) and WT mice (n = 7 for both fed and fasted) before and after 16 h of fasting. (B) mRNA levels of gluconeogenic transcriptional factors in the liver of Ncoa6 LKO and WT mice in a fed or 24 h fasted state (n = 6 for each group). Hepatic mRNAs were analyzed by real-time qRT-PCR. (C) Transcript levels of the CREB target gene Nr4a1 and FOXO1 target gene Igfbp1 in the primary hepatocytes of Ncoa6 LKO (n = 4) and WT mice (n = 3). (D) Induced phosphorylation of CREB by 16 h of fasting in the livers of Ncoa6 LKO and WT mice (n = 3 for each group). (E) Forskolin-induced phosphorylation of CREB in primary hepatocytes of Ncoa6 LKO and WT mice. Protein levels were determined by western blot analyses using anti-CREB, anti-pCREB (Ser133) or anti-tubulin antibodies (n = 5 independent experiments). The intensities of the protein bands were measured using the ImageJ program. Data are presented as the mean ± SEM; *** P < 0.001, determined using one-way ANOVA. Fsk, forskolin.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Quantitative RT-PCR, Phospho-proteomics, Western Blot

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A) Interactions between CREB and NCOA6. 293T cells were transfected with HA-CREB and FLAG-NCOA6 and lysates were immunoprecipitated from these cells using anti-HA or anti-FLAG antibody. The coimmunoprecipitates were then analyzed by western blotting with anti-HA or anti-FLAG antibody. (B) Sequence alignment of the CRE-containing promoter regions of the Pck1 or G6pc gene among human, mouse, and rat. (C) Schematic diagram of the r Pck1 or h G6PC promoter-Luc reporter containing WT or MT CRE. The red bars indicate the loci of the ChIP primers used in and . (D and E) Effects of NCOA6 on the CREB-mediated transcriptional activation of the r Pck1 (D) and h G6PC (E) promoter (n = 5 independent experiments). WT or MT CRE-containing Pck1 or G6PC promoter-Luc reporters were cotransfected into HepG2 cells with or without HA-CREB, FLAG-NCOA6 and actin-β-galactosidase. Luciferase activities were then measured with a luminometer and normalized using β-galactosidase activities. Data are presented as the mean ± SEM; *** P < 0.001, determined using one-way ANOVA. IP, immunoprecipitation; WT, wild type; MT, mutant; Fsk, forskolin.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Transfection, Immunoprecipitation, Western Blot, Sequencing, Activation Assay, Luciferase, Mutagenesis

Journal: Molecules and Cells

Article Title: Regulation of Hepatic Gluconeogenesis by Nuclear Receptor Coactivator 6

doi: 10.14348/molcells.2022.2222

Figure Lengend Snippet: (A and B) 293T cells were cotransfected with a Pck1 (A) or G6PC (B) promoter-Luc reporter, HA-CREB and FLAG-NCOA6. Modified ChIP assays were then performed using anti-HA, anti-FLAG or IgG antibodies, followed by analysis using conventional PCR (n = 4 independent experiments). The intensities of the PCR bands were measured using the ImageJ program. Data are presented as the mean ± SEM; * P < 0.05, ** P < 0.01, determined by t -test. WT, wild type; MT, mutant; IP, immunoprecipitation. (C) Hypothetical model for the gluconeogenic transcription of the Pck1 and G6pc genes mediated via NCOA6.

Article Snippet: After membrane blocking with 5% skim milk, the membrane was probed with primary antibodies against NCOA6 (Cat. No. NB200-335; Novus Biologicals, USA), PCK1 (Cat. No. sc-32879; Santa Cruz Biotechnology, USA), CREB (Cat. No. 9197; Cell Signaling Technology, USA), p-CREB (Cat. No. 9198S; Cell Signaling Technology), or α-tubulin (Cat. No. T9026; Sigma-Aldrich).

Techniques: Modification, Mutagenesis, Immunoprecipitation