Journal: bioRxiv

Article Title: Efferocytosis Remodels Osteoclast Metabolism and Enhances Antibacterial Defense

doi: 10.64898/2026.10.13.759003

Figure Lengend Snippet: a , Overview of glucose metabolism and associated metabolic pathways, with a heatmap summarizing transcriptional signatures of glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation across BMMs, osteoclasts, and efferocytic osteoclasts. Bulk RNA-seq analyses used n = 3 biological replicates per group. b , Immunoblot analysis of HIF-1α, GLUT1, and proteins detected using an OXPHOS antibody cocktail in BMMs and osteoclasts before and after apoptotic neutrophil efferocytosis, with densitometric quantification. c , Immunoblot analysis of MERTK expression in BMMs and osteoclasts derived from OC(ΔMertk) mice and control littermates, with densitometric quantification normalized to β-actin. d , Immunofluorescence analysis of apoptotic neutrophil uptake by BMMs and osteoclasts derived from OC(ΔMertk) mice and control littermates. Engulfed apoptotic neutrophils were quantified per 100 µm². Scale bars, 20 µm (BMMs) and 50 µm (osteoclasts). e,f , Extracellular flux analysis of glycolytic activity and mitochondrial respiration following apoptotic neutrophil efferocytosis. Glycolytic activity was assessed by extracellular acidification rate (ECAR, mpH/min) ( e ), and mitochondrial respiration was assessed by oxygen consumption rate (OCR, pmol/min) ( f ). The oligomycin-sensitive component of OCR represents ATP-linked respiration. Data are presented as mean ± s.d. Statistical significance was assessed using two-tailed unpaired t -tests ( d ) and one-way ANOVA followed by Tukey’s multiple comparisons test ( b,c,e,f ). n = 3 independent biological samples per group ( b–d ); each point represents one biological sample. n = 7–9 measured wells per group ( e,f ); each point in the summary plots represents one well.

Article Snippet: Membranes were blocked with 5% nonfat milk in TBST (TBS containing 0.1% Tween-20) for 1 h at room temperature and then incubated overnight at 4°C with the indicated primary antibodies against GAS6 (MedChemExpress; HY-P85881), MERTK (Proteintech; 27900-1-AP), AXL (MedChemExpress; HY-P81198), HIF-1α (Proteintech; 82989-4-RR), GLUT1 (Proteintech; 21829-1-AP), and OXPHOS Cocktail (Proteintech; PK30006).

Techniques: Phospho-proteomics, RNA Sequencing, Western Blot, Analysis, Expressing, Derivative Assay, Control, Immunofluorescence, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Efferocytosis Remodels Osteoclast Metabolism and Enhances Antibacterial Defense

doi: 10.64898/2026.10.13.759003

Figure Lengend Snippet: a , Schematic illustrating osteoclast metabolic rewiring toward increased glycolytic activity after efferocytosis. b , Lactate concentration in the culture media of BMMs and osteoclasts derived from OC(ΔMertk) mice and control littermates before and after efferocytosis. c,d , Functional assessment of sodium lactate supplementation. Osteoclast differentiation was evaluated by TRAP staining ( c ). Scale bars, 100 µm. Antibacterial activity was assessed by CFU quantification after Staphylococcus aureus challenge ( d ). e–g , LDH isoenzyme profiling of BMMs and osteoclasts before and after efferocytosis. Representative agarose gel electrophoresis images showing LDH isoenzyme patterns detected by in-gel NAD⁺/NBT-PMS activity staining ( e ), densitometric quantification of individual isoenzymes ( f ), and activity-derived estimates of LDHA/LDHB ratios ( g ). h , RT–qPCR analysis of Slc2a1 , Slc16a1 , and Slc16a3 expression before and after apoptotic neutrophil efferocytosis. Expression levels were normalized to Actb . i,j , Effects of pharmacological inhibition of MCT1, MCT4, and LDHA on viable bacterial counts in the presence or absence of apoptotic neutrophils in osteoclasts ( i ) and macrophages ( j ), assessed using CFU quantification after Staphylococcus aureus challenge. k , Proposed model of distinct lactate-handling programs in macrophages and osteoclasts after efferocytosis. The depicted transport directions and metabolic fates represent mechanistic hypotheses informed by the expression and isoenzyme profiles. Data are presented as mean ± s.d. Statistical significance was assessed using two-tailed unpaired t -tests ( c,i,j ) and one-way ANOVA followed by Tukey’s multiple comparisons test ( b,d,g,h ). n = 8 samples per group (b); n = 3 ( c,g,h ); n = 6–7 per group ( d ); n = 5–6 per group ( i ); n = 6 per group ( j ). Each point represents one independent biological sample.

Article Snippet: Membranes were blocked with 5% nonfat milk in TBST (TBS containing 0.1% Tween-20) for 1 h at room temperature and then incubated overnight at 4°C with the indicated primary antibodies against GAS6 (MedChemExpress; HY-P85881), MERTK (Proteintech; 27900-1-AP), AXL (MedChemExpress; HY-P81198), HIF-1α (Proteintech; 82989-4-RR), GLUT1 (Proteintech; 21829-1-AP), and OXPHOS Cocktail (Proteintech; PK30006).

Techniques: Activity Assay, Concentration Assay, Derivative Assay, Control, Functional Assay, Staining, Profiling, Agarose Gel Electrophoresis, Quantitative RT-PCR, Analysis, Expressing, Inhibition, Two Tailed Test