Journal: bioRxiv

Article Title: Macrophage signaling and function are regulated by distinct sterol biochemistries

doi: 10.1101/2025.08.07.669025

Figure Lengend Snippet: Inhibition of sterol synthesis induces biochemical changes in macrophages. (A) Cholesterol biosynthesis is composed of multiple reactions catalyzed by enzymes (gray text) which can be targeted with small molecule inhibitors (red text). (B) Quantified GC-MS analyses of BMDMs treated for 48 h with small molecule inhibitors of cholesterol biosynthesis show reduced cholesterol and accumulation of sterol precursor molecules (mean ± SEM; n = 3 biological replicates from 3 independent experiments).

Article Snippet: Mature BMDMs were found adhered to the dish after another 48 h. Culture medium for BMDMs includes DMEM (ATCC, Cat. 30-2002), 20% (v/v) fetal bovine serum (FBS; Corning), 0.1% (v/v) penicillin/streptomycin (Life Technologies, 15140122), 50 ng/mL colony-stimulating factor 1 (CSF1; BioLegend, 574804), and 57.6 nM β-mercaptoethanol (Life Technologies, 21985023).

Techniques: Inhibition, Gas Chromatography-Mass Spectrometry

Journal: bioRxiv

Article Title: Macrophage signaling and function are regulated by distinct sterol biochemistries

doi: 10.1101/2025.08.07.669025

Figure Lengend Snippet: Macrophage surface proteins are altered by sterol change. (A) Representative distribution of forward (FSC) and side scatter (SSC) events showing gating position for detecting live BMDMs with flow cytometry. (B) Representative distribution of live and dead populations of BMDMS used for flow cytometry analyses. (C) Cell surface expression of CD11b in cholesterol biosynthesis inhibited BMDMs (mean ± SEM; n = 3-4 biological replicates from 4 independent experiments). One-way ANOVA (F 5,17 = 16.66, p < 0.0001) with Dunnett’s multiple comparisons test (*p ≤ 0.05; ***p ≤ 0.001). (D) Surface expression of CD11c n cholesterol biosynthesis inhibited BMDMs (mean ± SEM; n = 3 biological replicates from 3 independent experiments). One-way ANOVA (F 5,12 = 32.35, p < 0.0001) with Dunnett’s multiple comparisons test (*p ≤ 0.05; ****p < 0.001). (E) CD16/32 surface expression in BMDMs upon cholesterol biosynthesis inhibition (mean ± SEM; n = 2-4 biological replicates from 4 independent experiments). One-way ANOVA (F 4,15 = 19.65, p < 0.0001) with Dunnett’s multiple comparisons test (*p ≤ 0.05; ***p ≤ 0.001). (F) CD80 surface expression in BMDMs targeted for cholesterol biosynthesis inhibition (mean ± SEM; n = 4 biological replicates from 4 independent experiments). One-way ANOVA (F 5,18 = 15.82, p < 0.0001) with Dunnett’s multiple comparisons test (****p < 0.001). (G) Surface expression of CD115 in BMDMs in cholesterol replete compared to cholesterol inhibited conditions (mean ± SEM; n = 4 biological replicates from 4 independent experiments). One-way ANOVA (F 5,18 = 4.706, p ≤ 0.0064) with Dunnett’s multiple comparisons test (*p ≤ 0.05).

Article Snippet: Mature BMDMs were found adhered to the dish after another 48 h. Culture medium for BMDMs includes DMEM (ATCC, Cat. 30-2002), 20% (v/v) fetal bovine serum (FBS; Corning), 0.1% (v/v) penicillin/streptomycin (Life Technologies, 15140122), 50 ng/mL colony-stimulating factor 1 (CSF1; BioLegend, 574804), and 57.6 nM β-mercaptoethanol (Life Technologies, 21985023).

Techniques: Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Macrophage signaling and function are regulated by distinct sterol biochemistries

doi: 10.1101/2025.08.07.669025

Figure Lengend Snippet: Macropinocytosis is regulated by both total sterol levels and sterol-mediated membrane phase separation. (A) Illustration of experimental design for analysis of cholesterol-biosynthesis inhibition impacts on macropinocytosis in BMDMs. (B) Representative histograms showing LY expression in BMDMs after cholesterol-targeted treatment paradigms. (C) Quantified LY uptake in BMDMs following impaired cholesterol biosynthesis (mean ± SEM; n = 3 biological replicates from 3 independent experiments). One-way ANOVA (F 4,10 = 10.9, p < 0.0011) with Dunnett’s multiple comparisons test (*p ≤ 0.05 compared to LPDS culture). Unpaired t-test (LPDS vs +Simvastatin: t 4 = 3.595, *p ≤ 0.05; LPDS vs +AY9944: t 4 = 18.82, p < 0.0001; LPDS vs +U18666A: t 4 = 4.525, *p ≤ 0.05). (D) Illustrated experimental design for acute modification of plasma membrane sterol content through cholesterol removal with MβCD followed by replacement with sterols of interest. Inset shows sterol structural differences (in red) relative to cholesterol. (E) Representative histograms of LY uptake in BMDMs after cholesterol removal and reloading of distinct sterols. (F) Quantified LY uptake following cholesterol removal and reloading of distinct sterols which support phase separation (cholesterol, 7DHC, or desmosterol) versus non-supportive sterols (cholestenone or cholesterol sulfate) (mean ± SEM; n = 3 biological replicates from 3 independent experiments). One-way ANOVA (F 6,14 = 106.8, p < 0.0001) with Sidak’s multiple comparisons test (*p ≤ 0.05, ****p < 0.0001 compared to LPDS treatment).

Article Snippet: Mature BMDMs were found adhered to the dish after another 48 h. Culture medium for BMDMs includes DMEM (ATCC, Cat. 30-2002), 20% (v/v) fetal bovine serum (FBS; Corning), 0.1% (v/v) penicillin/streptomycin (Life Technologies, 15140122), 50 ng/mL colony-stimulating factor 1 (CSF1; BioLegend, 574804), and 57.6 nM β-mercaptoethanol (Life Technologies, 21985023).

Techniques: Membrane, Inhibition, Expressing, Modification, Clinical Proteomics

Journal: bioRxiv

Article Title: Macrophage signaling and function are regulated by distinct sterol biochemistries

doi: 10.1101/2025.08.07.669025

Figure Lengend Snippet: Genetic disruption of Dhcr7 impairs macropinocytosis in macrophages. (A) Representative histograms of LY uptake in Dhcr7 +/+ , Dhcr7 T93M/+ , and Dhcr7 T93M/β BMDMs. (B) Quantified LY uptake in wild-type and Dhcr7 mutant BMDMs (mean ± SEM; n = 4-6 biological replicates from 4 independent experiments). One-way ANOVA (F 2,11 = 47.67, p < 0.0001) with Dunnett’s multiple comparisons test (***p ≤ 0.001; ****p < 0.0001 compared to Dhcr7 +/+ ). (C) Representative histograms of BMDM LY uptake including replacement of LPDS with FBS or cholesterol-loaded MβCD (+MβCD-chol). (D) Quantified LY internalization in response to cholesterol-repletion in wild-type and Dhcr7 mutant BMDMs (mean ± SEM; n = 3 biological replicates from 3 independent experiments). One-way ANOVA ( Dhcr7 +/+ : F 2,6 = 46.78, p ≤ 0.0002; Dhcr7 T93M/Δ3-5 : F 2,6 = 9.777, p ≤ 0.0129) with Dunnett’s multiple comparisons test (**p ≤ 0.01, ***p ≤ 0.001 compared to Dhcr7 +/+ LPDS).

Article Snippet: Mature BMDMs were found adhered to the dish after another 48 h. Culture medium for BMDMs includes DMEM (ATCC, Cat. 30-2002), 20% (v/v) fetal bovine serum (FBS; Corning), 0.1% (v/v) penicillin/streptomycin (Life Technologies, 15140122), 50 ng/mL colony-stimulating factor 1 (CSF1; BioLegend, 574804), and 57.6 nM β-mercaptoethanol (Life Technologies, 21985023).

Techniques: Disruption, Mutagenesis

Journal: bioRxiv

Article Title: Macrophage signaling and function are regulated by distinct sterol biochemistries

doi: 10.1101/2025.08.07.669025

Figure Lengend Snippet: Sterol homeostatic change induces both shared and biochemistry-distinct cellular signaling deficits in macrophages. (A) Venn diagram illustrating differentially expressed transcripts that are shared and distinct between cholesterol-depleted conditions (n = 4 biological replicates per condition; displays genes for p-values < 0.05, log 2 fold change > ±0.5). (B) Volcano plot indicating differentially expressed transcripts LPDS to FBS conditions (n = 4 biological replicates per condition). Red data points indicate differentially expressed transcripts ( p < 0.05, log 2 fold change ≥ ±0.5). (C) Volcano plot indicating differentially expressed transcripts when comparing LPDS to AY9944 treated conditions (n = 4 biological replicates per condition). Fold changes correspond to AY9944 expression relative to LPDS. Red data points indicate differentially expressed transcripts ( p < 0.05, log 2 fold change ≥±0.5. (D) Pathway analysis comparing LPDS to FBS conditions reveals alterations in immune response behavior in BMDMs (n = 4 biological replicates per condition). (E) Pathway analysis comparing AY9944 to LPDS conditions shows enhanced BMDM immunoreactive profile after AY9944 treatment (n = 4 biological replicates per conditions). (F) Transcriptional changes associated with chemokine signalling are significantly altered across treatments in BMDMs (mean ± SEM; n = 4 biological replicates per condition).

Article Snippet: Mature BMDMs were found adhered to the dish after another 48 h. Culture medium for BMDMs includes DMEM (ATCC, Cat. 30-2002), 20% (v/v) fetal bovine serum (FBS; Corning), 0.1% (v/v) penicillin/streptomycin (Life Technologies, 15140122), 50 ng/mL colony-stimulating factor 1 (CSF1; BioLegend, 574804), and 57.6 nM β-mercaptoethanol (Life Technologies, 21985023).

Techniques: Expressing

Journal: bioRxiv

Article Title: Macrophage signaling and function are regulated by distinct sterol biochemistries

doi: 10.1101/2025.08.07.669025

Figure Lengend Snippet: Sterol disruption induces immunoreactivity and morphological changes in macrophages. (A) Representative images for late endosomes/lysosomes (Rab7), immunoreactivity (CD68), and F-actin filaments (phalloidin-647) in BMDMs after 48 h exposure to LPDS or AY9944 treatment. Scale bar, 25 µm. (B) Simvastatin, 17α-OHP, TASIN-1, AY9944, and avasimibe promote accumulation of Rab7-positive late endosomes/endosomes (mean ± SEM; n = 16-23 images taken from 2 independent experiments). One-way ANOVA (F 7,146 = 34.55, p < 0.0001) with Dunnett’s multiple comparisons test (***p ≤ 0.001; ****p < 0.0001). (C) Quantified CD68 expression in cholesterol metabolism inhibited BMDMs (mean ± SEM; n = 20-28 images taken from 3 independent experiments). One-way ANOVA (F 7,183 = 19.38, p < 0.0001) with Dunnett’s multiple comparisons test (***p ≤ 0.001; ****p < 0.0001). (D) Quantified phalloidin-647 expression observed after disruption of cholesterol metabolism. One-way ANOVA (F 7,183 = 16.71, p < 0.0001) with Dunnett’s multiple comparisons test (*p ≤ 0.05; **p ≤ 0.01; ****p < 0.0001).

Article Snippet: Mature BMDMs were found adhered to the dish after another 48 h. Culture medium for BMDMs includes DMEM (ATCC, Cat. 30-2002), 20% (v/v) fetal bovine serum (FBS; Corning), 0.1% (v/v) penicillin/streptomycin (Life Technologies, 15140122), 50 ng/mL colony-stimulating factor 1 (CSF1; BioLegend, 574804), and 57.6 nM β-mercaptoethanol (Life Technologies, 21985023).

Techniques: Disruption, Expressing

Journal: bioRxiv

Article Title: Macrophage signaling and function are regulated by distinct sterol biochemistries

doi: 10.1101/2025.08.07.669025

Figure Lengend Snippet: Biochemical profiles in macrophages from Dhcr7 deficient mice are driven by increased immunoreactivity. (A) Quantified GC-MS analyses of BMDMs from Dhcr7 T93M/+ and Dhcr7 T93M/β3-5 show reduced cholesterol but no 7DHC accumulation (mean ± SEM; n = 2 biological replicates from 2 independent experiments). (B) Quantified GC-MS analyses of BMDMs treated with AY9944 alongside IFNγ/LPS (mean ± SEM; n = 2 biological replicates from 2 independent experiments). (C) Representative images of Rab7 and CD68 from Dhcr7 +/+ , Dhcr7 T93M/+ , and Dhcr7 T93M/β BMDMs in LPDS conditions. Scale bars, 25 µm. (D) Quantified Rab7 expression in wild-type and Dhcr7 mutant BMDMs (mean ± SEM; n = 24 images taken from 3 independent experiments). Two-way ANOVA (media effect: F 1,138 = 20.65, p < 0.0001; genotype effect: F 2,138 = 10.02, p < 0.0001) with Sidak’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01 relative to Dhcr7 +/+ LPDS). (E) Quantified CD68 expression in Dhcr7 +/+ versus Dhcr7 mutant ( Dhcr7 T93M/+ , Dhcr7 T93M/β ) BMDMs (mean ± SEM; n = 24 images taken from 3 independent experiments). Two-way ANOVA (media effect: F 1,138 = 12.34, p ≤ 0.0006; genotype effect: F 2,138 = 38.94, p < 0.0001) with Sidak’s multiple comparisons test (*p ≤ 0.05, ****p < 0.0001 relative to Dhcr7 +/+ LPDS).

Article Snippet: Mature BMDMs were found adhered to the dish after another 48 h. Culture medium for BMDMs includes DMEM (ATCC, Cat. 30-2002), 20% (v/v) fetal bovine serum (FBS; Corning), 0.1% (v/v) penicillin/streptomycin (Life Technologies, 15140122), 50 ng/mL colony-stimulating factor 1 (CSF1; BioLegend, 574804), and 57.6 nM β-mercaptoethanol (Life Technologies, 21985023).

Techniques: Gas Chromatography-Mass Spectrometry, Expressing, Mutagenesis

Journal: JCI Insight

Article Title: Single-cell transcriptomics–based MacSpectrum reveals macrophage activation signatures in diseases

doi: 10.1172/jci.insight.126453

Figure Lengend Snippet: (A) Pathways significantly enriched in the 435 AMDSGs. (B) Macrophage distributions along the AMDI scale. (C and D) Single-cell trajectory of M0, M1, and M2 BMDMs (4736 cells/sample) with Monocle (30) using PSGs and AMDSGs colored by BMDM population (C) or pseudotime (D). (E) Heatmap showing smoothened relative expression of PSGs and AMDSGs along pseudotime progression, from M0 to M1 and M2 branches of the single-cell trajectory. Known signature genes are indicated. Hierarchical clustering generated 6 expression groups: groups I and III enriched in M1, group II enriched in M2 or M1 and M2, groups V and VI enriched in M0, group IV enriched in M0 or M2.

Article Snippet: Cells were induced for differentiation to monocytes in BMDM growth medium (Iscove’s Modified Dulbecco’s Medium [IMDM] + 10% FBS + 15% L-929 cell [ATCC, CCL-1]) for 7 days; fresh BMDM growth medium was replaced on day 3.

Techniques: Expressing, Generated