Bmal1 integrates mitochondrial metabolism and macrophage activation

  1. Ryan K Alexander
  2. Yae-Huei Liou
  3. Nelson H Knudsen
  4. Kyle A Starost
  5. Chuanrui Xu
  6. Alexander L Hyde
  7. Sihao Liu
  8. David Jacobi
  9. Nan-Shih Liao
  10. Chih-Hao Lee  Is a corresponding author
  1. Department of Molecular Metabolism, Division of Biological Sciences, Harvard TH Chan School of Public Health, United States
  2. School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, China
  3. Institute of Molecular Biology, Academia Sinica, China
8 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Macrophage Bmal1 is induced by M1 activation.

(A–C) Bmal1 protein levels (top) and relative gene expression determined by qPCR (bottom) in bone-marrow-derived macrophages (BMDM) during a 24-hr time course of M1 activation (10 ng/ml Ifn-γ overnight priming + 10 ng/ml LPS) (A), treatment with LPS alone (100 ng/ml) (B), or acute LPS treatment for 1 hr (100 ng/mL) (C). For M1- and LPS-only treatments, LPS was spiked in at time zero without medium change. For acute LPS treatment, cells were grown with LPS for one hour followed by culture in DMEM, 2% FBS without LPS (time zero indicates medium change). N = 3 biological replicates were used for qPCR. (D) Relative expression of circadian clock and inflammatory transcriptional regulators in M1-activated macrophages, as determined by qPCR. N = 3 biological replicates, statistical analysis performed using two-way ANOVA for WT vs M-BKO across the time course. Data are presented as mean ± S.E.M. *, p<0.05. Experiments were repeated at least twice.

Figure 1—figure supplement 1
The circadian clock is a transcriptional module that is induced by M1 activation.

(A) Annotated protein–protein interaction maps generated by STRING of transcriptional regulators that were significantly induced (left) or repressed (right) in WT macrophages by 8 hours M1 activation (Ifn-γ priming followed by stimulation with 10 ng/mL LPS) compared to time-matched controls (Ifn-γ priming without LPS), determined by RNA-seq (p<0.05, FDR <0.05, |F.C.|>1.5). N = 3 biological replicates. F.C., fold change. See Table 1 for the complete gene list. (B) Bmal1 protein levels (top panels) and relative gene expression determined by qPCR (bottom panels) in mouse embryonic fibroblasts during a 24-hr time course of M1 activation (10 ng/ml Ifn-γ overnight priming + 10 ng/ml LPS, left panels), treatment with LPS only (100 ng/ml, middle panels), or 1 hr acute LPS treatment (100 ng/mL, right panels). For M1- and LPS-only treatments, LPS was spiked in at time zero without medium change. For acute LPS treatment, cells were grown with LPS for one hour followed by culture in DMEM, 2% FBS without LPS (time zero indicates medium change). N = 3 biological replicates for qPCR. (C) Gene expression during a 24-hr time course of Il-4 treatment in WT and M-BKO macrophages determined by qPCR. N = 3 biological replicates. Data are presented as mean ± S.E.M. *, p<0.05. Cell culture experiments were repeated at least twice.

Figure 2 with 1 supplement
Bmal1 is required to maintain mitochondrial metabolism.

(A) Assessment of mitochondrial mass in macrophages throughout a time course of M1 activation using Mitotracker Green (mean fluorescence intensity, MFI) determined by flow cytometry. N = 3 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. (B) Basal oxygen consumption rate (OCR) in Ifn-γ-primed macrophages pretreated without or with LPS (10 ng/mL) for 2 or 6 hr before the assay. See Figure 2—figure supplement 1B for the full assay results. The assay medium contained minimal DMEM with 5 mM glucose and 1 mM sodium pyruvate, pH 7.4. N = 5 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. (C) Activities of ETC complexes in isolated mitochondria from WT and M-BKO macrophages after 6 hours M1 stimulation. N = 3 biological replicates, statistical analysis was performed using Student’s T test. (D) Extracellular flux analysis in Ifn-γ-primed macrophages measuring the changes in extracellular acidification rate (ECAR, left panel) and oxygen consumption rate (OCR, right panel) following LPS injection (100 ng/mL). The assay medium contained 5 mM glucose and 1 mM pyruvate in minimal DMEM with 2% dialyzed FBS, pH 7.4. N = 5 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. (E) Glycolytic stress test in Ifn-γ-primed macrophages measuring ECAR following glucose (25 mM) injection, with or without LPS (100 ng/mL). Maximal glycolytic rate was determined by injection of oligomycin (OM, 2 μM), and glycolysis-dependent ECAR was determined by injection of 2-deoxyglucose (2-DG, 50 mM). The assay medium contained minimal DMEM with 2% dialyzed FBS, pH 7.4. N = 5 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. Data are presented as mean ± S.E.M. *, p<0.05. Experiments were repeated at least twice.

Figure 2—figure supplement 1
Effects of Bmal1 gene deletion and overexpression on glycolytic versus oxidative metabolism.

(A) Immunoblot of the mitophagy regulator Bnip3 in M1-activated WT and M-BKO BMDMs. (B) Mitostress test measuring oxygen consumption rate (OCR) in Ifn-γ-primed macrophages pretreated without or with LPS (10 ng/mL) for 2 or 6 hr before the assay. Following three measurements of basal respiration, sequential injections of oligomycin (OM, 2 μM), carbonyl cyanide-4-phenylhydrazone (FCCP, 0.5, μM) and rotenone and antimycin A (rot/AA, 1 μM) were carried out to determine uncoupled respiration, spare respiratory capacity, and non-mitochondrial respiration, respectively. The assay medium contained minimal DMEM with 5 mM glucose and 1 mM sodium pyruvate, pH 7.4. N = 5 biological replicates. (C) Mitostress test measuring OCR in macrophages without (control, Ctl) or with serum shock resulting from treatment with 50% FBS for 2 hr to synchronize the circadian clock (Synchronized). Cells were recovered in fresh growth medium containing 10% FBS and the assay was performed 16 hr after the serum shock treatment. N = 5 biological replicates. (D) Mitostress test measuring OCR in macrophages without or with acute LPS treatment. Cells were cultured in growth medium with 2% FBS without (Ctl) or with 100 ng/mL LPS for 2 hr (LPS recovery), followed by recovery in fresh growth medium with 2% FBS for 24 hr prior to the assay. N = 5 biological replicates, statistical analysis was performed using two-way ANOVA for Ctl vs LPS recovery across the time course. (E) Extracellular flux analysis of thioglycollate-elicited peritoneal macrophages measuring the changes in ECAR (left panel) and OCR (right panel) following LPS injection (final concentration 1 μg/mL). The assay medium contained 5 mM glucose and 1 mM pyruvate in minimal DMEM with 2% dialyzed FBS, pH 7.4. N = 5 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. (F) Bmal1 mRNA expression determined by qPCR (left) and protein levels (right) in RAW264.7 macrophage stable lines. Bmal1-OE, Bmal1 overexpressing stable line. Cells that were transduced with empty vector were used as the control. N = 3 biological replicates for qPCR, statistical analysis was performed using Student’s T test. V, vector control; OE, Bmal1 overexpression. (G) Measurement of the changes in ECAR (left) and OCR (right) in Ifn-γ-primed RAW264.7 stable lines after injection with LPS (final concentration 100 ng/mL). The assay medium contained 25 mM glucose and 1 mM pyruvate in minimal DMEM with 2% dialyzed FBS, pH 7.4. N = 5 biological replicates, statistical analysis was performed using two-way ANOVA for control vs Bmal1 OE across the time course. (H) Blood glucose levels in 4-month-old WT and M-BKO male mice before and 6 hr after i.p. injection with 10 μg LPS per g body weight. N = 10 mice, statistical analysis was performed using Student’s T test. (I) Glycolytic stress test in splenic macrophages isolated from mice in panel (D) that were sacrificed 6 hr after LPS injection. ECAR was measured before and after injection with glucose (25 mM). Maximal glycolytic rate was determined by injection of oligomycin (OM, 2 μM). Glycolysis-dependent ECAR was determined by injection of 2-deoxyglucose (2-DG, 50 mM). The assay medium contained minimal DMEM, pH 7.4. N = 10 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. Data are presented as mean ± S.E.M. *, p<0.05. Experiments were repeated at least twice.

Bmal1 deletion induces a metabolic shift for glycolytic and amino-acid metabolism.

(A) Summary of steady-state metabolomics data for differentially regulated metabolites from WT and M-BKO macrophages throughout a 12-hour M1 activation time course. Data are presented as heat maps (normalized to WT control for each metabolite, each panel is the average of four biological replicates). Statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. (B) Box plots of the relative abundances of select metabolites in panel (A). (C, D) Uptake of [3H]−2-deoxyglucose (C) and lactate secretion (D) in control or M1-activated macrophages. N = 3 biological replicates, statistical analysis was performed using Student’s T test. Cell culture assays were repeated at least twice.

Figure 3—source data 1

Metabolomics data for M1-activated WT and M-BKO macrophages.

https://cdn.elifesciences.org/articles/54090/elife-54090-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
Bmal1 loss-of-function increases oxidative stress and Hif-1α protein accumulation.

(A) Immunoblots of Hif-1α and Bmal1 protein levels in WT and M-BKO macrophages during a 24-hr time course of M1 activation. (B) Relative expression of Hif-1α target genes in WT, M-BKO and M-HKO macrophages determined by qPCR. N = 3 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO or WT vs M-HKO across the time course. (C) Measurement of mROS using MitoSox Red (mean fluorescence intensity, MFI) in mitochondria isolated from control or M1-activated macrophages. Succinate (Suc, 10 mM) was included during MitoSox Red staining where indicated. N = 3 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. (D) Hif-1α protein levels in control or 8-hr M1-activated macrophages co-treated with or without N-acetylcysteine (NAC) or 10 mM dimethylmalonate (DMM). Data are presented as mean ± S.E.M. *, p<0.05 for WT vs M-BKO and p<0.05 for WT vs M-HKO. Experiments were repeated at least twice.

Figure 4—figure supplement 1
Increased oxidative stress and Hif-1α activity in M1 activated M-BKO macrophages.

(A) Hif-1α protein levels in M1-activated (primed with 10 ng/mL Ifn-γ and stimulated with 50 ng/mL LPS) control and Bmal1-OE RAW264.7 stable lines. (B, C) Uptake of [3H]−2-deoxyglucose and lactate secretion in control or M1-activated macrophages. N = 3 biological replicates, statistical analysis was performed using Student’s T test. M-BHdko: myeloid-specific Bmal1 and Hif1a double knockout. (D) Glycolytic stress test in Ifn-γ-primed macrophages measuring ECAR following glucose (25 mM) injection without or with LPS (100 ng/mL). Maximal glycolytic rate was determined by injection of oligomycin (OM, 2 μM), and glycolysis-dependent ECAR was determined by injection of 2-deoxyglucose (2-DG, 50 mM). The assay medium contained minimal DMEM with 2% dialyzed FBS, pH 7.4. N = 5 biological replicates. The difference between LPS-treated WT and M-BHdKO macrophages was determined by two-way ANOVA across the time course. (E) Expression of Nrf2 target genes determined by qPCR throughout a 24-hr time course following M1 stimulation. N = 3 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. Data are presented as mean ± S.E.M. *, p<0.05. Experiments were repeated at least twice.

Figure 5 with 1 supplement
Genes involved in amino-acid uptake and catabolism are upregulated in M-BKO macrophages.

(A) Schematic representation of M1-regulated genes involved in amino-acid and TCA metabolism determined by RNA-seq. Genes in blue are downregulated whereas genes in red are upregulated by 8 hours M1 activation in both WT and M-BKO macrophages. Genes that are differentially regulated in these two genotypes are displayed in heat maps on the right. F.C., fold change. N = 3 biological replicates. BCAAs, branch chain amino acids; Keto AAs, ketogenic amino acids; Cic, mitochondrial citrate carrier. (B) Relative expression of differentially regulated genes identified by RNA-seq and validated by qPCR in a 24-hr time course of M1 activation. N = 3 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO across the time course. (C) Hif-1α protein levels in control or 6-hr M1-activated macrophages with or without co-treatment of the neutral amino-acid transport inhibitor 2-amino-2-norbornanecarboxylic acid (BCH, 10 mM). (D) Gene expression in control or 6-hr M1-activated macrophages with or without co-treatment of 10 mM BCH and/or the complex II inhibitor dimethyl malonate (DMM, 10 mM) determined by qPCR. N = 3 biological replicates, statistical analysis was performed using Student’s T test. Data are presented as mean ± S.E.M. *, p<0.05. Cell culture experiments were repeated at least twice.

Figure 5—source data 1

Functional annotation clustering genes commonly and differentially regulated between WT and M-BKO BMDMs by 8 h M1 activation.

https://cdn.elifesciences.org/articles/54090/elife-54090-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Transcriptome analysis of genes that are regulated by M1 stimulation in WT and M-BKO macrophages.

(A) Functional annotation clustering by biological process and Venn diagram of genes identified by RNA-seq that are mutually induced (2379 genes, left) or suppressed (2644 genes, right) by 8-h M1 activation in WT and M-BKO macrophages. N = 3 biological replicates. F.C., fold change. (B) Enriched biological processes associated with genes that are differentially expressed in M1-activated WT and M-BKO macrophages. (C) Relative expression of the neutral amino-acid transporter Slc7a8 (Lat2) in WT and myeloid Hif1a knockout (M-HKO) macrophages as determined by qPCR. WT samples were from Figure 5B. N = 3 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-HKO across the time course. (D) Determination of glutamine utilization by extracellular flux analysis. OCR was measured before and after LPS injection (final concentration 1 μg/mL), and the assay medium contained 5 mM glutamine in minimal DMEM with 2% dialyzed FBS, pH 7.4. N = 5 biological replicates, statistical analysis was performed using two-way ANOVA for WT vs M-BKO and for WT or M-BKO vs M-BKO cells co-treated with the neutral amino-acid transport inhibitor 2-amino-2-norbornanecarboxylic acid (BCH, 10 mM) across the time course. Data are presented as mean ± S.E.M. *, p<0.05. Cell culture experiments were repeated at least twice.

Figure 6 with 1 supplement
Bmal1 regulates tumor-associated macrophage polarization.

(A) Bmal1 gene expression (left panel) and protein levels (right panel) in WT macrophages treated with control medium or increasing doses of B16-F10 tumor-conditioned medium (T-CM, diluted 1:3 or 1:1 with control medium) for 8 hr. N = 3 biological replicates for qPCR, statistical analysis was performed using Student’s T test. (B) Measurement of mROS using MitoSox Red (mean fluorescent intensity, MFI) in mitochondria from macrophages treated with control medium or T-CM diluted 1:1 with control medium for 1 hr. N = 3 biological replicates, statistical analysis was performed using Student’s T test. (C) Hif-1α protein levels in WT and M-BKO macrophages treated with control medium, T-CM diluted 1:1 with control medium, or undiluted T-CM for 4 hr. (D) Glycolytic stress test in macrophages pretreated with control medium or T-CM diluted 1:1 with control medium for 4 hr. N = 5 biological replicates. Statistical analysis was performed using two-way ANOVA comparing T-CM-treated M-BKO with WT cells across the time course. (E) Relative expression of genes involved in amino-acid metabolism and oxidative stress response in macrophages treated with control medium or T-CM diluted 1:3 with control medium for 8 hr, as determined by qPCR. N = 3 biological replicates, statistical analysis was performed using Student’s T test. (F) Tumor volume in male (left) and female (right) WT and M-BKO mice. 300,000 B16-F10 cells were injected subcutaneously into the right flank. N = 18 (male) and 8 (female) mice, statistical analysis was performed using two-way ANOVA for WT vs M-BKO mice across the time course. (G) Gene expression for F4/80+ cells isolated from B16-F10 tumors or spleens of female mice 14 days after injection. Tissues from six mice per genotype were pooled into three groups for leukocyte isolation. Statistical analysis was performed using Student’s T test. Data are presented as mean ± S.E.M. *, p<0.05. Experiments were repeated at least twice.

Figure 6—figure supplement 1
Increased mROS levels and glucose uptake in M-BKO TAMs.

(A) Tumor volumes of B16-F10 subcutaneous allografts in 3-month-old female WT and M-BKO mice 14 days after tumor cell injection. Each mouse was injected with 500,000 B16-F10 cells in the right and left flanks, and tumors from each mouse were pooled for immune cell isolation and flow cytometry. N = 4 mice, with two tumors per mouse. (B, C) mROS production and glucose uptake by tumor-associated CD45+F4/80+ cells determined by flow cytometry (mean fluorescent intensities) of Mitosox Red and 2-NBDG staining, respectively. N = 4. Statistical analysis was performed using Student’s T test. Data are presented as mean ± S.E.M. *, p<0.05.

Figure 7 with 1 supplement
Macrophage Bmal1 modulates the antitumor activity.

(A) Tumor volume in WT male mice co-injected with 500,000 B16-F10 cells and either 500,000 WT or M-BKO macrophages as indicated. N = 22 mice, statistical analysis was performed using two-way ANOVA to compare WT vs M-BKO macrophage co-injection across the time course. (B) Flow cytometric analysis of tumor-infiltrating CD8+ T cells (CD45+CD3+CD8a+ cells, left panel) and NK cells (CD45+CD3NK1.1+ cells, right panel) stimulated ex vivo with phorbol 12-myristate 13-acetate and ionomycin for Ifn-γ co-staining. Tumors represented in panel (A) were pooled into three groups prior to isolation of infiltrating leukocytes for flow cytometry. Statistical analysis was performed using Student’s T test. (C) Tumor volume in WT male mice co-injected with 500,000 B16-F10 cells and either 500,000 WT or M-BKO macrophages, supplemented without or with dimethylmalonate (DMM, approximately 150 mg/kg body weight per day in mouse diet). N = 8 mice, statistical analysis was performed using two-way ANOVA to compare WT vs M-BKO macrophage co-injection on control diet, or to compare WT macrophage co-injection on the control diet vs WT or M-BKO macrophage co-injection on the DMM-supplemented diet across the time course. Data are presented as mean ± S.E.M. *, p<0.05. Experiments were repeated at least twice.

Figure 7—figure supplement 1
Macrophage Bmal1 regulates tumor growth in a cell-autonomous manner.

(A) Tumor volume in M-BKO male mice co-injected with 500,000 B16-F10 cells and either 500,000 WT or M-BKO macrophages as indicated. N = 6 mice, statistical analysis was performed using two-way ANOVA to compare WT vs M-BKO macrophage co-injection across the time course. (B) Flow cytometric analyses of tumor-infiltrating CD8+ T cells (CD45+CD3+CD8a+ cells, left panel) and NK cells (CD45+CD3NK1.1+ cells, right panel) stimulated ex vivo with phorbol 12-myristate 13-acetate and ionomycin for Ifn-γ co-staining. Tumors that are represented in panel (A) were pooled into three groups prior to isolation of infiltrating leukocytes for flow cytometric analyses. Statistical analysis was performed using Student’s T test. Data are presented as mean ± S.E.M. *, p<0.05. (C) Hif-1α protein levels in WT macrophages treated with (from left to right) control medium or B16-F10 tumor-conditioned medium (T–CM) diluted 1:1 with control medium without or with co-treatment with 10 mM dimethyl malonate (DMM) for 4 hr. (D) Arg1 gene expression in WT macrophages treated as in panel (C) for 8 hr. N = 3 biological replicates. Statistical analysis was performed using Student’s T test. Experiments were repeated at least twice. (E) Schematic showing the working model for Bmal1–Hif-1α crosstalk in the regulation of macrophage bioenergetics and effector functions. Both M1 and tumor-associated macrophages share similar energetically stressed states. Bmal1 preserves mitochondrial oxidative metabolism while reducing oxidative stress to modulate Hif-1α activity and support proper effector functions. Deletion of Bmal1 in the macrophage leads to reliance on glycolytic metabolism and alternative fuel utilization, notably amino-acid metabolism, which further promotes mROS production and Hif-1α protein stabilization. Increased amino-acid utilization by M-BKO macrophages may lead to depletion of amino acids that are critical for lymphocyte activation and cytotoxic function, thereby suppressing anti-tumor immunity in the tumor microenvironment.

Author response image 1

Tables

Table 1
Transcriptional regulators that are differentially regulated in M1-activated macrophages.

Genes encoding transcriptional regulators that were significantly induced or repressed by 8h M1 stimulation (p<0.05, false discovery rate [FDR] <0.05, |F.C.| >1.5) in WT bone-marrow-derived macrophages (BMDM) were identified by gene ontology analysis using the DAVID platform. F.C., fold change. Differentially regulated genes that matched the Transcription GO term in the Biological Processes GO database (accession GO:0006350) were used to generate a protein–protein interaction map using String (Figure 1—figure supplement 1). Uncharacterized zinc-finger proteins (ZFPs) were omitted from analyses by String.

Induced (278 genes)
ADARCRY1GTF2A1KDM4BMNTPPP1R10Snapc1Zkscan17
AFF1CRY2GTF2E2KDM5BMXD1PPP1R13LSNAPC2ZMIZ1
AFF4CSRNP1GTF2F1KDM5CMXI1PTOV1SOX5ZSCAN2
AHRCSRNP2HBP1KEAP1MYBPTRFSPENZSCAN29
AKNADAXXHDAC1KLF11NAB2PURASPICZXDB
ANP32ADDIT3HES1KLF16NACC1RBPJSREBF1
ARHGAP22DDX54HES7KLF4NCOA5RCOR2SRF
ARID3ADEDD2HEXIM1KLF7NCOA7RELST18
ARID5ADNMT3AHIC1KLF9NCOR2RELASTAT2
ARNT2DPF1HIC2LCORNFAT5RELBSTAT3
ASF1ADRAP1HIF1ALCORLNFE2L2RESTSTAT4
ATF3E2F5HIF3ALHX2NFIL3RFX1STAT5A
ATF4E4F1HINFPLIN54NFKB1RING1TAF1C
ATF6BEAF1HIVEP1LITAFNFKB2RNF2TAF7
ATXN7L3EDF1HIVEP2LMO4NFKBIZRORATAL1
BANPEGR2HIVEP3MAFNOTCH1RREB1TBL1X
BATFEID3HLXMAFFNPTXRRSLCAN18TCEB2
BCL3EIF2C1HMG20BMAFGNR1D1RUNX2TCF4
BCL6ELK1HMGA1MAFKNR1D2RUNX3TGIF1
BCORL1ELLHMGA1-RS1MAML1NR1H2RUVBL2THAP7
BHLHE40ELL2HMGN5MAXNR1H3RXRATLE2
BHLHE41ELL3HOPXMBD2NR2F6RYBPTLE3
BRWD1EPAS1HSF4MDFICNR4A1SAFB2TRERF1
BTG2ERFIFI205MECP2NR4A2SAP130TRIB3
CAMTA2ERN1IFT57MED13NR4A3SAP30TRRAP
CASZ1ESRRAILF3MED13LPAF1SBNO2TSC22D4
CBX4ETS1ING2MED15PAX4SCAF1TSHZ1
CCDC85BETV3IRF2BP1MED25PCGF3SEC14L2USP49
CDKN2AFIZ1IRF4MED26PCGF5SERTAD1VPS72
CEBPBFLIIIRF7MED28PER1SETD8WHSC1L1
CEBPDFOXP1IRF8MED31PER2SFPI1ZBTB17
CITED4FOXP4JARID2MEF2DPHF1SIN3BZBTB24
CREB5GATA2JDP2MIER2PHF12SIX1ZBTB46
CREBBPGATAD2AJMJD6MIER3PIAS4SIX5ZBTB7A
CREBL2GATAD2BJUNMITFPMLSLC30A9ZBTB7B
CREBZFGFI1JUNBMIXL1POU2F2SMAD3ZEB1
CREMGLIS3JUNDMKL1POU3F1SMAD4ZFHX4
CRTC2GPBP1KDM3AMLL1POU6F1SMAD7ZGPAT
CRTC3GRHL1KDM4AMNDAPPARDSMYD1ZHX2
Repressed (195 genes)
ACTL6AELK3IRF2NAA15SAP18
AHRRELP2ITGB3BPNCOA1SAP25
AI987944ELP3KDM2BNCOA3SETD7
ANGELP4KLF10NFATC1SETDB1
ASCC1ENY2KLF13NFATC2SNAPC5
ASF1BERCC8KLF2NFIASP3
ATAD2ESR1KLF8NKRFSSBP2
AW146154ETOHI1L3MBTL2NPATSSRP1
BCL9LETV1LBHNPM3STAT1
CBFA2T3EYA1LRPPRCNR2C1SUV39H1
CBX3EYA4LYL1NRIP1SUV39H2
CBX6EZH2MAFBOVOL2SUV420H2
CBX8FLI1MARSPA2G4TADA1
CCNHFNTBMBTPS2PHF19TADA2A
CDCA7FOXM1MCM2PHTF2TAF4B
CDCA7LGTF2H2MCM3PNRC2TAF9B
CEBPAGTF2IMCM4POLR1BTBX6
CEBPGGTF2IRD1MCM5POLR2GTCEA3
CEBPZGTF3AMCM6POLR2ITCEAL8
CHAF1AGTF3C5MCM7POLR3BTCF7L2
CHAF1BHABP4MCM8POLR3HTFB1M
CHD9HDAC10MCTS1POLR3KTFB2M
CHURC1HDAC11MED14PPARGTFDP2
CIITAHDAC2MED18PPARGC1BTHOC1
CIR1HDAC6MED22PRIM1TLE1
CREB3HDAC7MED27PRIM2TRAPPC2
CREB3L1HDAC8MEF2APRMT7TRIM24
CREB3L2HDAC9MEF2CPROX2TWISTNB
CTNND1HELLSMEIS1PSPC1TXNIP
CUX1HHEXMLF1IPRAD54BUHRF1
DDI2HIP1MLL3RB1USF1
DNMT1HIRAMLLT3RBAKVGLL4
DR1HMBOX1MNAT1RCBTB1VPS36
E2F1HMGA2MPV17RCOR3WTIP
E2F2HOXA1MXD3REREZBTB3
E2F6HTATSF1MXD4RFC1ZBTB8A
E2F7IKBKAPMYBL2RPAP1ZHX1
E2F8IKZF2MYCRSC1A1ZIK1
EGR3IL16MYCBP2RSL1ZKSCAN4
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Mus musculus)B6.129S4(CG)-Arntltm1Weit/JThe Jackson LaboratoryJAX: 007668Carry loxp sites flanking exon 8 of the Bmal1 gene
Strain, strain background (Mus musculus)B6.129-Hif1atm3Rsjo/JThe Jackson LaboratoryJAX: 007561Carry loxp sites flanking exon 2 of the Hif1a gene
Strain, strain background (Mus musculus)B6.129P2-Lyz2tm1(cre)lfo/JThe Jackson LaboratoryJAX: 004781Myeloid-specific Cre recombinase expression
AntibodyMouse monoclonal anti-Bmal1 (clone B-1)Santa Cruz BiotechnologyCat# sc365645; RRID:AB_10841724WB (1:1000)
AntibodyRabbit polyclonal anti-Hif-1αNovus BiologicalsCat# NB100-449;
RRID:AB_10001045
WB (1:1000)
AntibodyRabbit monoclonal anti-Bnip3
(clone EPR4034)
AbcamCat# ab109362;
RRID:AB_10864714
WB (1:1000)
AntibodyRabbit polyclonal anti-β-tubulinCell Signaling TechnologyCat# 2146;
RRID:AB_2210545
WB (1:1000)
AntibodyRabbit monoclonal anti-β-Actin (clone 13E5)Cell Signaling TechnologyCat# 4970;
RRID:AB_2223172
WB (1:1000)
AntibodyRat monoclonal anti-CD45 (clone 30-F11), PerCP/Cy5.5-conjugatedBiolegendCat# 103132;
RRID:AB_893340
Flow cytometry (1 μL per test)
AntibodyArmenian hamster monoclonal anti-CD3ε (clone 145–2 C11), PE/Cy7-conjugatedBiolegendCat# 100320;
RRID:AB_312685
Flow cytometry (2.5 μL per test)
AntibodyRat monoclonal anti-CD8a (clone 53–6.7), Alexa Fluor 700-conjugatedBiolegendCat# 100730;
RRID:AB_493703
Flow cytometry (0.5 μL per test)
AntibodyMouse monoclonal anti-NK1.1 (clone PK136), APC-conjugatedBiolegendCat# 108710Flow cytometry (5 μL per test)
AntibodyRat monoclonal anti-F4/80 (clone BM8), Alexa Fluor 488-conjugatedBiolegendCat# 123120;
RRID:AB_893479
Flow cytometry (5 μL per test)
AntibodyRat monoclonal anti-F4/80 (clone BM8), APC-conjugatedBiolegendCat# 123115;
RRID:AB_893493
Flow cytometry (2.5 μL per test)
AntibodyRat monoclonal anti-Ifn-γ (clone XMG1.2), PE-conjugatedThermoFisher ScientificCat# 12-7311-81, RRID:AB_466192Flow cytometry (1 μL per test)
Peptide, recombinant proteinRecombinant murine interferon-γPeprotechCat# 315–05Used 10 ng/mL final concentration
Peptide, recombinant proteinRecombinant murine interleukin-4PeprotechCat# 214–14Used 10 ng/mL final concentration
Sequence-based reagent36b4_FThis paperqPCR primerAGATGCAGCAGATCCGCAT
Sequence-based reagent36b4_RThis paperqPCR primerGTTCTTGCCCATCAGCACC
Sequence-based reagentArg1_FThis paperqPCR primerCGTAGACCCTGGGGAACACTAT
Sequence-based reagentArg1_RThis paperqPCR primerTCCATCACCTTGCCAATCCC
Sequence-based reagentBckdhb_FThis paperqPCR primerTGGGGCTCTCTACCATTCTCA
Sequence-based reagentBckdhb_RThis paperqPCR primerGGGGTATTACCACCTTGATCCC
Sequence-based reagentBmal1_FThis paperqPCR primerAGGATCAAGAATGCAAGGGAGG
Sequence-based reagentBmal1_RThis paperqPCR primerTGAAACTGTTCATTTTGTCCCGA
Sequence-based reagentcMyc_FThis paperqPCR primerCAGCGACTCTGAAGAAGAGCA
Sequence-based reagentcMyc_RThis paperqPCR primerGACCTCTTGGCAGGGGTTTG
Sequence-based reagentCry1_FThis paperqPCR primerCACTGGTTCCGAAAGGGACTC
Sequence-based reagentCry1_RThis paperqPCR primerCTGAAGCAAAAATCGCCACCT
Sequence-based reagentGclc_FThis paperqPCR primerCATCCTCCAGTTCCTGCACA
Sequence-based reagentGclc_RThis paperqPCR primerATGTACTCCACCTCGTCACC
Sequence-based reagentHif1a_FThis paperqPCR primerGAACGAGAAGAAAAATAGGATGAGT
Sequence-based reagentHif1a_RThis paperqPCR primerACTCTTTGCTTCGCCGAGAT
Sequence-based reagentHmox1_FThis paperqPCR primerCAGAGCCGTCTCGAGCATAG
Sequence-based reagentHmox1_RThis paperqPCR primerCAAATCCTGGGGCATGCTGT
Sequence-based reagentIl1b_FThis paperqPCR primerAGCTTCAGGCAGGCAGTATC
Sequence-based reagentIl1b_RThis paperqPCR primerAAGGTCCACGGGAAAGACAC
Sequence-based reagentLdha_FThis paperqPCR primerGCGTCTCCCTGAAGTCTCTT
Sequence-based reagentLdha_RThis paperqPCR primerGCCCAGGATGTGTAACCTTT
Sequence-based reagentMgl2_FThis paperqPCR primerccttgcgtttgtcaaaacatgac
Sequence-based reagentMgl2_RThis paperqPCR primerctgaggcttatggaactgaggc
Sequence-based reagentMmsdh_FThis paperqPCR primerGAGGCCTTCAGGTGGTTGAG
Sequence-based reagentMmsdh_RThis paperqPCR primerGATAGATGGCATGGTCTCTCCC
Sequence-based reagentNfe2l2_FThis paperqPCR primerGGTTGCCCACATTCCCAAAC
Sequence-based reagentNfe2l2_RThis paperqPCR primerGCAAGCGACTCATGGTCATC
Sequence-based reagentNfkb1_FThis paperqPCR primerCCTGCTTCTGGAGGGTGATG
Sequence-based reagentNfkb1_RThis paperqPCR primerGCCGCTATATGCAGAGGTGT
Sequence-based reagentNqo1_FThis paperqPCR primerTCTCTGGCCGATTCAGAGTG
Sequence-based reagentNqo1_RThis paperqPCR primerTGCTGTAAACCAGTTGAGGTTC
Sequence-based reagentNr1d2_FThis paperqPCR primerTCATGAGGATGAACAGGAACCG
Sequence-based reagentNr1d2_RThis paperqPCR primerCGGCCAAATCGAACAGCATC
Sequence-based reagentPpard_FThis paperqPCR primerCAGCCTCAACATGGAATGTC
Sequence-based reagentPpard_RThis paperqPCR primerTCCGATCGCACTTCTCATAC
Sequence-based reagentPparg_FThis paperqPCR primerCAGGAGCCTGTGAGACCAAC
Sequence-based reagentPparg_RThis paperqPCR primerACCGCTTCTTTCAAATCTTGTCTG
Sequence-based reagentSlc7a2_FThis paperqPCR primerCCCGGGATGGCTTACTGTTT
Sequence-based reagentSlc7a2_RThis paperqPCR primerAGGCCATCACAGCAGAAATGA
Sequence-based reagentSlc7a8_FThis paperqPCR primerGAACCACCCGGGTTCTGAC
Sequence-based reagentSlc7a8_RThis paperqPCR primerTGATGTTCCCTACAATGATACCACA
Sequence-based reagentSlc7a11_FThis paperqPCR primerATCTCCCCCAAGGGCATACT
Sequence-based reagentSlc7a11_RThis paperqPCR primerGCATAGGACAGGGCTCCAAA
Sequence-based reagentStat3_FThis paperqPCR primerTGGCAGTTCTCGTCCAC
Sequence-based reagentStat3_RThis paperqPCR primerCCAGCCATGTTTTCTTTGC
Sequence-based reagentBmal1_FThis paperCloning primersGGCGAATTCGCGGACCAGAGAATGGAC
Sequence-based reagentBmal1_RThis paperCloning primersGGGCTCGAGCTACAGCGGCCATGGCAA
Cell line (Mus musculus)RAW264.7 macrophagesATCCqPCR primerTIB-71
Cell line (Mus musculus)B16-F10 melanoma cellsATCCqPCR primerCRL-6475
Recombinant DNA reagentpBABE-puro retroviral expression vector (plasmid)AddgenePlasmid #1764
Recombinant DNA reagentpBABE-Bmal1 (plasmid)This studyFor stable overexpression of mouse Bmal1
Chemical compound, drugLipopolysaccharides from Escherichia coli K-235Sigma-AldrichCat# L2143Final concentration used varied as indicated
Chemical compound, drugDimethyl malonateSigma-AldrichCat# 136441;
CAS: 108-59-8
Used 10 mM final concentration
Chemical compound, drugSodium succinate dibasicSigma-AldrichCat# 14160;
CAS: 150-90-3
Used 10 mM final concentration
Chemical compound, drugN-acetyl-L-cysteineSigma-AldrichCat# A9165;
CAS: 616-91-1
Final concentration used varied as indicated.
Chemical compound, drug2-deoxy-D-glucoseSigma-AldrichCat# D8375;
CAS: 154-17-6
Used 50 mM final concentration
Chemical compound, drugQuant-iT ribogreen RNA reagentThermoFisher ScientificCat# R11491
Chemical compound, drugMitotracker green FMThermoFisher ScientificCat# M7514
CAS: 201860-17-5
Used 100 nM final concentration
Chemical compound, drugMitosox red superoxide indicatorThermoFisher ScientificCat# M36008Used 5 μM final concentration
Chemical compound, drug2-NBDGThermoFisher ScientificCat# N13195Used 10 μM final concentration
Chemical compound, drugFixable viability dye eFluor 455uvThermoFisher ScientificCat# 65-868-14Flow cytometry (0.5 μL per test)
Chemical compound, drugFixable viability dye eFluor 506ThermoFisher ScientificCat# 65-0866-14Flow cytometry (0.5 μL per test)
Chemical compound, drugOligomycinAbcamCat# ab141829; CAS: 1404-19-9Used 2 μM final concentration
Chemical compound, drugRotenoneAbcamCat# ab143145
CAS: 83-79-4
Used 1 μM final concentration
Chemical compound, drugAntimycin ASigma-AldrichCat# A8674;
CAS: 1397-94-0
Used 1 μM final concentration
Chemical compound, drugCarbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)Santa Cruz BiotechnologyCat# sc203578
CAS: 370-86-5
Used 0.5 μM final concentration
Chemical compound, drugPhorbol 12-myristate 13-acetate (PMA)Sigma-AldrichCat# P8139;
CAS: 16561-29-8
Used 20 ng/mL final concentration
Chemical compound, drugIonomycin calcium salt from Streptomyces conglobatusSigma-AldrichCat# I0634;
CAS: 56092-82-1
Used 1 μg/mL final concentration
Chemical compound, drugBrefeldin ACell Signaling TechnologyCat# 9972S;
CAS: 20350-15-6
Used 10 μg/mL final concentration
Chemical compound, drugBCA protein assay kitThermoFisher ScientificCat# 23227
Chemical compound, drugSeahorse XF24 FluxPakAgilentCat# 102070–00
Chemical compound, drugFoxp3/Transcription factor staining buffer setThermoFisher ScientificCat# 00-5523-00
Chemical compound, drugTruSeq stranded mRNA library prep kitIlluminaCat# RS-122–2101
Chemical compound, drugDynabeads sheep anti-rat IgGThermoFisher ScientificCat # 11035
Software, algorithmSTRINGvon Mering, 2004https://string-db.org/
Software, algorithmDAVIDHuang et al., 2009https://david-d.ncifcrf.gov/
OtherThioglycollate mediumSigma-AldrichCat# T9032
OtherBovine serum albumin, fatty acid freeGemini Bio-ProductsCat# 700–107P
OtherCollagen I protein, rat tailThermoFisher ScientificCat# A1048301
OtherCollagenase type IVThermoFisher ScientificCat# 17104019
OtherRecombinant DNase I, RNase-freeAffymetrixCat# 78411

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  1. Ryan K Alexander
  2. Yae-Huei Liou
  3. Nelson H Knudsen
  4. Kyle A Starost
  5. Chuanrui Xu
  6. Alexander L Hyde
  7. Sihao Liu
  8. David Jacobi
  9. Nan-Shih Liao
  10. Chih-Hao Lee
(2020)
Bmal1 integrates mitochondrial metabolism and macrophage activation
eLife 9:e54090.
https://doi.org/10.7554/eLife.54090