EMBO/soda-nel huggingface dataset:
Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism (2021)
Figure 2

Figure 2: Drug combinations synergistically induce the integrated stress response

A Relative ATF4 and CHOP mRNA expression levels in HCT116 cells upon the indicated treatments determined by qPCR (NEN (N) 1.2 µM; Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM).

B Immunoblot analysis of CHOP, ATF4, phosphorylated (p-) and t-) eIF2alpha (eIF2a) of HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM; Domperidone (Domp), Amitriptyline (Ami), each 30 µM).

C Caspase activity in HCT116 cells either transfected with control (Ctrl), ATF4- or CHOP-targeting siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM).

D Relative mRNA expression levels of the indicated genes upon drug treatments (Control (Ctrl), NEN (N) 1.2 µM, Domperidone (D) 30 µM) and inhibition of the ISR using ISRIB (1 µM), determined by qPCR.

E Caspase activity in HCT116 cells grown as monolayer (2D) and treated as indicated for 24h (Control (Ctrl), NEN 1.2 µM, Domperidone (D) 30 µM, ISRIB 1 µM).

F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM, ISRIB 1 µM).

Data information: Data are presented as mean (SD) and were analyzed by one-way ANOVA with Tukey post-hoc test (A, upper left and lower left graphs, (N=3), (D (N=3)), Kruskal-Wallis with Dunn´s post-hoc tests (A, upper right and lower right graphs, (N=5, besides NEN+Clomi (N=4) and NEN+Desi (N=4)) or two-way ANOVA with Tukey post-hoc test (C (N=3), E (N=4), F (N=4). In (A), right graphs, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001. Exact p-values for all comparisons are listed in Appendix Table S1.
Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism (2021)
Figure 3

Figure 3: Combinatorial treatments induces CLEAR network associated with a blockage in autophagy

A Relative mRNA expression levels of indicated CLEAR network genes in HCT116 cells treated as indicated ((NEN (N) 1.2 µM; Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM), determined by qPCR.

B Immunofluorescence (IF) of HCT116 cells treated with DMSO (Control (Ctrl)), NEN, Domperidone (Domp) or NEN+Domp as indicated (NEN, 1.2 µM; Domp, 30 µM) for 16 h and stained with anti-Lamp1 (top panel), Alexa-555 LC3 (middle panel) co-stained with Alexa-647 p62 (bottom panel). Nuclei, Blue (dapi). Scale bar 20 µm. Mean (SEM) fluorescence of Lamp1 (Ctrl, NEN, Domp (N=20 cells); NEN+Domp (N=24 cells)), LC3-II (Ctrl, NEN+Domp (N=16 cells)); NEN, Domp (N=16 cells)) or P62 (Ctrl (N=38 cells); NEN (N=27 cells); Domp (N=28 cells); NEN+Domp (N=21 cells)) was quantified and analyzed by one-way ANOVA with Tukey post-hoc test (for LC3-II) or Kruskal-Wallis with Dunn´s post-hoc test (for Lamp1 and p62). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.

C Immunoblot analysis and quantification (mean ± SD) of LC3-II/LC3-I ratios and P62 in HCT116 cells. Cultured cells were treated with DMSO (Control), NEN or NEN and Domperidone (Domp) as indicated (NEN, 1.2 µM; Domp, 30 µM) for 16 h. Protein band signal intensities were normalized to vinculin (loading control) and the ratios LC3-II bands LC3-I (N=3) and p62 levels (N=3) were analyzed by one-way ANOVA with Tukey post-hoc test. * p< 0.05; *** p< 0.001; **** p < 0.0001.

D Immunoblot analysis and quantification of LC3 in HCT116 cells (mean ± SEM). Cultured cells were treated with DMSO (Control (Ctrl)), NEN or NEN and Domperidone (NEN+Domp) as indicated (NEN, 1.2 µM; Domp, 30 µM) for 16 h, followed by control or 20 µM chloroquine treatment for 3 h. Protein band signal intensities were normalized to vinculin (loading control) and the ratios LC3-II bands in presence or absence of chloroquine treatment (N=6) were analyzed by Kruskal-Wallis with Dunn´s post-hoc test. * p< 0.05; *** p<0.001.

Data information: In (A) (first (TFE3) and third (CD68) graph (N=3); second (TFE3) and fourth (CD68) graph (N=5, besides NEN+Clomi (N=4) and NEN+Desi (N=4)) data are presented as mean (SD) and were analyzed by a one-way ANOVA with Tukey post-hoc test. In the second and the fourth graph, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls (Ctrl). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001. Exact p-values for all comparisons are listed in Appendix Table S1.
Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism (2021)
Figure 4

Figure 4: UPP1 induction deregulates pyrimidine metabolism and contributes to cell death

A Heat map for pyrimidine pathway-related metabolites derived from a non-targeted metabolomics analysis, depicting relative levels in HCT116 cells treated as indicated for 18h (NEN (N) 1.2 µM, Domperidone (Dom) 30 µM or in combination (Comb).

B Relative UPP1 mRNA expression levels in HCT116 cells treated as indicated (NEN (N) 1.2 µM, Domperidone (Domp, D) 30 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami) each 30 µM, Clomipramine (Clomi) 20 µM), determined by qPCR.

C Relative UPP1 mRNA expression levels in HCT116 cells either transfected with control (siCtrl) or combined TFE3- and MITF- targeting (siTFE3/siMITF) siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM, ISRIB 1 µM), determined by qPCR.

D Relative UPP1 mRNA expression in HCT116 cells transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM).

E Ratio of toxicity and viability measurements of HCT116 spheroids (3D) and 2D cultures, treated as indicated (NEN 1.2 µM, Domperidone 30 µM). Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs prior to spheroid formation or during cell seeding.

F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN (low) 0.6 µM or NEN (High) 1.2 µM, Domperidone (Domp) 30 µM) Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 48h prior to treatment.

G Immunoblot of HCT116 whole cell lysates using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Amitriptyline (Ami) and Desipramine (Desi), each 30 µM, Clomipramine (Clomi) 20 µM).

H Ratio of toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM). Cells were transfected with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 24h prior to spheroid formation.

Data information: In (B, C, D, E and H), data (N=3) are presented as mean (SD) and were analyzed by one-way ANOVA with Tukey post-hoc test (B) or two -way ANOVA with Tukey post-hoc test (C, D, E,H). In (B), right panel, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls. ** p< 0.01;*** p< 0.001; **** p < 0.0001. Exact p-values for all comparisons are listed in Appendix Table S1.
Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism (2021)
Figure 5

Figure 5: Drug-induced regulation of pyrimidine biosynthesis enzyme DHODH contributes to cell death

A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM, Clomipramine (Clomi) 20 µM).

B Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Domperidone (Domp) 20 µM, A77126 (A77) 50 µM, JNK inhibitor II (Jnki) 1µM).

C Immunoblot analysis of HCT116 whole cell lysates using antibodies against indicated proteins and treated as follows: NEN and Domperidone for 12h, A77126 for an additional 6h (NEN 0.6 µM, Domperidone (Domp) 20 µM, A771726 50 µM).

D Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 20 µM, Clomipramine (Clomi) 10 µM, A77126 (A77) 50 µM).

Data information: In (A, B and D), data are presented as mean (SD) (N=3) and were analyzed by Kruskal-Wallis with Dunn´s post-hoc test (A, left graph), one-way ANOVA with Tukey post-hoc test (A, right graph) or two-way ANOVA with Tukey post-hoc test (B, D). In (A), significance is indicated for the comparison of the different treatments to controls. ** p < 0,01; **** p < 0,0001. Exact p-values for all comparisons are listed in Appendix Table S1.
Combination therapies induce cancer cell death through the integrated stress response and disturbed pyrimidine metabolism (2021)
Figure 6

Figure 6: Cholesterol dysregulation contributes to stress pathway induction and cell death

A Immunofluorescence (IF) images of HCT116 cells treated as indicated for 12h (Imipramine and Domperidone, each 30 µM) and stained for cholesterol content using filipin III.

B Immunofluorescence (IF) images of HCT116 cells treated with Domperidone and co-stained for cholesterol and Lamp1.

C Ratio of toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Domperidone (Domp) 30 µM). Cyclodextrin content in the medium is indicated in percentage (%CD).

D Ratio of toxicity and viability of U87 spheroids (3D) treated as indicated (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Amitriptyline (Ami), Desipramine (Desi), each 30 µM, Clomipramine (Clomi 20 µM, Cyclodextrin (CD) 0.75%).

E Relative mRNA expression levels of the specified genes in HCT116 cells, treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (D) 30 µM, Cyclodextrin (CyD) 0.75%).

F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h (NEN 1.2 µM, Domperidone (Domp) 30 µM, Cyclodextrin (CD) 0.75%).

Data information: In (C, D and E), data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test or a two-way ANOVA with Tukey post-hoc test (C and D).* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001. Exact p-values for all comparisons are listed in Appendix Table S1.
Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing (2022)
Figure 1

Figure 1 - Preparation of TAMEMs from primary murine macrophages.

A Schematic diagram of the generation of TAMEMs.

B Comparison of Wright-Giemsa staining (morphology) of freshly induced (Day 0) TAMEMs and typical macrophage subtypes and their continued cultured for 4 days in RPMI-1640 complete medium (Day 4) (scale bar: 10 μm; n=3, biological replicates).

C The viability curve of TAMEMs and typical macrophages subtypes, with the survival rate presented as a percentage of the initial cell vitality at day 1 (**P < 0.01 and ****P < 0.0001 vs. the M0 group; n=6, biological replicates).

D Immunofluorescent staining for Ki67 in TAMEMs and typical macrophages subtypes (scale bar: 10 μm; n=3, biological replicates).

E Quantification of mean fluorescence intensity (MFI) for CD80, CD86, CD206, CD163, CCR2, and MHC-Ⅱ in TAMEMs and typical macrophages subtypes via flow cytometry (*P < 0.05, ***P < 0.001, and ****P < 0.0001; n=6, biological replicates).

F Representative immunofluorescent (IF) staining images for CD86 and CD206 in TAMEMs and typical macrophages subtypes (scale bar: 10 μm; n=6, biological replicates).

Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (C) and ordinary one-way ANOVA with Tukey's multiple comparison test in (E) in Graph Pad Prism 8.
Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing (2022)
Figure 5

Figure 5 - TAMEMs resolve inflammation and rebuild vasculature in diabetic wounds.

A, B The MFI of pro-inflammatory macrophages (CD86 positive cells) (A) and anti-inflammatory macrophages (CD206 positive cells) (B) gating from F4/80+ macrophages on postinjury days 1, 3 and 7 (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=5, biological replicates).

C Ly6CHi monocytes plotted as a percentage of live cells on postinjury days 1, 3 and 7 (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=5, biological replicates).

D Quantification of the expression of the angiogenesis - associated Vegfa and Pdgfb mRNAs in different wound tissues at day 7 post-wounding (**P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=3, biological replicates).

E IF staining for CD31 (red; endothelial marker, for new capillary formation) at day 7 and α-SMA (green; smooth muscle marker, for vascular maturation) at day 14, with both the entire wound region and magnified yellow-box zone (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium).

F Quantification of the CD31+ vessels at day 7 and CD31+ α-SMA+ vessels and CD31+ α-SMA- vessels at day 14 (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates).

G Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 post-injury, uninjured skin was analyzed as positive control. Up: the images of the entire sampling area (yellow circled) containing the healed wound (green circled); Down: magnified zone (blue boxed in the upper images; scale bar: 100 μm; n=3, biological replicates).

H, I Orientational distribution (H) and quantification of blood vessel alignment (I), based on pixel orientation (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=3, biological replicates).

Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (A, B, C, D) and ordinary one-way ANOVA with Tukey's multiple comparison test in (F, I) in Graph Pad Prism 8.
Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing (2022)
Figure 6

Figure 6 - Preparation of a nine-factor cocktail and transplantation of TAMEMsC-h.

A The absolute concentration of the selected nine proteins by ELISA (n=6, biological replicates).

B Gene expression heatmap of complete transcriptome genes in TAMEMs and TAMEMsC-m, as determined using bulk RNA-seq analysis.

C Correlation of TAMEMs with TAMEMsC-m, each dot represents a gene expression (Pearson's correlation analysis), as determined using bulk RNA-seq analysis.

D The real-time qPCR analysis of pro-inflammatory genes (Il12, Tnfa, Ly6c, and Nos2), anti-inflammatory genes (Il10, Mrc1, Tgfb, and Arg1), and growth factors (Pdgfb, Vegfa, and Ang) to compare TAMEMs and TAMEMsC-m (ns: not significant (P > 0.05); n=6, biological replicates).

E The real-time qPCR analysis of M2 polarization genes (Arg1 and Mrc1) in BMDMs treated with TAMEMs-CM, TAMEMsC-m-CM, and normal medium for 48 h (****P < 0.0001 vs. the normal medium group; n=6, biological replicates).

F Scratch wound assay of L929 cells treated with TAMEMs-CM, TAMEMsC-m-CM, and normal medium for 6 h, and quantification of the remaining scratch area (the area between the two red dotted lines) was performed using an image analysis, the rates are presented as a percentage of the initial scratch area at 0 h (scale bar: 200 μm, *P < 0.05 and ****P < 0.0001 vs. the normal medium group, n=6, biological replicates).

G The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK-8 kit (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates).

H Immunofluorescent staining for α-SMA and type I Collagen in L929 cells after 48 h treatment with different CMs (scale bar: 10 μm; n=6, biological replicates).

I ELISA for α-SMA and type I Collagen in L929 cells after 48 h treatment with different CMs (*P < 0.05, **P < 0.01, and ****P < 0.0001 vs. the Normal Medium group; n=6, biological replicates).

Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (E, F, I), and two-way ANOVA with Tukey's multiple comparison test in (D, G) in Graph Pad Prism 8.
Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing (2022)
Figure 7

Figure 7 - Transplantation of TAMEMsC-h promotes wound healing in immunocompromised diabetic mice.

A Schematic depicting the education and transplantation of TAMEMsC-h.

B Gross view of the wound closure process was captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates).

C The percentage of wound closure determined at each time point (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates).

D Full-thickness skin samples containing entire wound sites were stained with H&E (day 7 and day 14) and Masson (day 14) (scale bar: 500 μm, n=8, biological replicates), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).

E Quantitative analysis of regenerated granulation on day 7 and Masson's trichrome-stained tissues on day 14 (blue for collagen) (**P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates).

F Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 postinjury, uninjured skin was analyzed as positive control (Up: the images of entire sampling sites (the yellow circular region) containing healed wound sites (the green circular region); Down: the images of higher magnification that indicates an area in the blue boxed region in the upper images; scale bar: 100 μm; n=3, biological replicates).

G quantification of blood vessel alignment, based on pixel orientation (**P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=3, biological replicates).

Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (E, G), and two-way ANOVA with Tukey's multiple comparison test in (C) in Graph Pad Prism 8.
The glucocorticoid receptor in brown adipocytes is dispensable for control of energy homeostasis (2019)
Figure 1

Fig 1. Hypothalamic-pituitary-adrenal axis is functional in GRBATKO mice.

(A) Plasma corticosterone levels at baseline, 30 minutes after induction of stress by placing mice in a clean cage, and after 90 minutes upon return in the home cage. DEX mice received an injection of dexamethasone at 0.1 mg/kg body weight 150 minutes before collecting the first blood sample (n = 5-6 animals per group).

(B) Flox and GRBATKO mice were injected with 0.02 mg/kg dexamethasone and the procedure described in (A) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in (A) (n = 6-7 animals per group).

(C) GR mRNA levels in BAT of Flox and GRBATKO mice. Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage. Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure, controls received an injection with saline (n = 5-6 animals per group).

(D) Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice (n = 5-6 animals per group).

(E) Blood glucose levels (n = 5-6 animals per group).

Data information: Data were analyzed with Two-way ANOVA. *, p < 0.05; **, p < 0.01 for genotype effect; #, p < 0.01 for treatment effect. Error bars denote SEM.
The glucocorticoid receptor in brown adipocytes is dispensable for control of energy homeostasis (2019)
Figure 2

Fig 2. Flox and GRBATKO mice respond similarly to cold.

(A) Knockdown of GR in BAT assessed by immunoblotting. Β-Tubulin was used as loading control.

(B) Quantification of immunoblot in (A) using Image Lab (n = 6 animals per group).

(C) Body temperature over the course of the cold exposure, determined daily (n = 6 animals per group).

(D-E) Body weight and BAT weight at the end of the cold exposure (n = 6 animals per group).

(F) Average daily food intake in g/day (n = 6 animals per group).

(G) Representative Hematoxilin and eosin staining image of BAT of cold-exposed Flox and GRBATKO mice. Scale bar 50 µm.

(H) Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group).

(I) Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals, indicating unchanged thermogenic marker gene expression. Color gradient ranges from 2-fold higher expression in GRBATKO over Flox controls (red) to 0.1-fold (lower) expression (blue) (n = 4 animals per group).

(J) Volcano plot from microarray gene expression data as in (I), with cut-off values for the adjusted p-value <0.1 and fold change >1.5.

(K) Serum corticosterone levels (n = 6 animals per group).

Data information: Data were analyzed with two-tailed Student t-test. **, p < 0.01. Error bars denote SEM.
The glucocorticoid receptor in brown adipocytes is dispensable for control of energy homeostasis (2019)
Figure 3

Fig 3. BAT GR does not impact the fasting-refeeding transition.

(A) Body weight after 16 h fasting and 6 h chow refeeding at 22°C (n = 6 animals per group)

(B) Food intake during 6 h refeeding (n = 6 animals per group).

(C) Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h (n = 6 animals per group).

(D) Blood glucose (n = 6 per group).

(E-F) Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group).

(G, H) Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).

Data information: Data were analyzed with Two-way ANOVA. #, p < 0.05; ##, p < 0.01 for treatment effect. Error bars denote SEM.
The glucocorticoid receptor in brown adipocytes is dispensable for control of energy homeostasis (2019)
Figure 4

Fig 4. Knockdown of GR in BAT reduces oxygen consumption.

(A) Body weight gain over a 24-week period of high-fat feeding (n = 13-14 animals per group). Panel shows experimental design for Figures 4-6.

(B) Average daily food intake in g (n = 6 cages per genotype).

(C) Feed efficiency to evaluate weight gain per ingested J (n = 6 cages per group).

(D) Fecal energy loss over a 3-day period determined by combustion of lyophilized feces of a subgroup of HFD-fed mice (n = 9 animals per group).

(E) Oxygen consumption over a 90-hour period. Grays columns indicate dark phase (n = 6-8 mice per group).

(F) Average oxygen consumption specified for light phase, dark phase, and total (n = 6-8 animals per genotype).

(G) Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight (n = 6-8 animals per group).

(H) Locomotor activity over a 90-hour period (n = 6-8 animals per group).

(I) Cumulative food intake over the 90-hour period specified for each phase and as grand total (n = 6-8 animals per group).

Data information: All data shown refer to 10 weeks HFD time point. Data were analyzed with two-tailed Student t-test (F). **, p < 0.01. Analysis of co-variance was applied in (G) with body weight as covariate using SPSS 25. Error bars denote SEM.