Fig 2: Mechanistic insight into HTTQ94-induced ALOX5 activation. (A) Western blot analysis of FLAP and HTTQ94 or HTTQ19 in HEK293 cells transfected with an HA-FLAP-expressing plasmid and either an empty vector, HTTQ94-GFP-expressing vector, or HTTQ19-GFP-expressing vector. (B) Western blot analysis of endogenous FLAP and ALOX5 in HTTQ94 tet-on SK-N-BE(2)C cells incubated with 0.5 μg/mL doxycycline for 4, 8, and 16 h. (C) Densitometry quantification of FLAP protein levels calculated using ImageJ software and plotted for half-life determination corresponding to Supplemental Figure S5B. (D) Co-IP of GFP-tagged HTTQ94/HTTQ19 and FLAG-tagged FLAP in HEK293 cells. Cell lysates were immunoprecipitated with anti-FLAG-coupled beads (M2), followed by Western analysis of HTTQ94, HTTQ19, and FLAP. (E) Ubiquitination analysis of FLAP in the presence of HTTQ94 or HTTQ19. HEK293 cells were cotransfected with FLAG-FLAP and HA-ubiquitin (Ub) in the presence of HTTQ94 or HTTQ19. Cell lysates were immunoprecipitated with anti-FLAG-coupled beads (M2), followed by Western blot analysis of HA, FLAP, and HTT proteins. (F) Cell death assay for HTTQ94 tet-on SK-N-BE(2)C cells with FLAP knockdown. Cells were transfected with control siRNA (ctrl) or FLAP-specific siRNA and then preincubated with 0.5 µg/mL doxycycline for 16 h, followed by 350 μM TBH treatment for 24 h. (G) Cell death assay. HTTQ94 tet-on HT-22 cells preincubated with 0.5 μg/mL doxycycline for 4 h were treated with 10 μM TBH for 8 h in the presence or absence of 2 μM Ferr-1, 10 μM zileuton, or 10 μM MK886. Cell deaths were calculated from three replicates. Data shown in F and G are the means ± SD. P-values were derived from two-tailed unpaired t-test. (***) P ≤ 0.001.

Fig 3: Quantitation of Enzyme Positive Cells during Fracture Healing.The total number of each cell type was counted from before till 14 days after fracture as described in the Materials and Methods and are shown as cells per mm or cells per mm2 (circles; right axis). The number of periosteal (triangles, right axis) and endosteal (inverted triangles, right axis) osteoclasts were also counted (Panels A and B, respectively). The percentage of cells positive for COX-1 (green), COX-2 (red), 5-LO (blue), or LTA4H (yellow) was calculated and mean values (+S.E.M) are shown (left axis). Cell types are indicated for each panel. For Panel D: Intramedullary Fracture Site Cells and Panel F: Callus Periosteal Leukocytes, no data were available before fracture. For Panel G: Chondrocytes and Panel H: Callus Leukocytes, no chondrocytes or external callus marrow containing leukocytes were present prior to day 4 after fracture and so no values were entered for earlier time points. Note that the X-axis scales (Days after Fracture) are not linear with respect to time.

Fig 4: Epigenetic modification is regulated by arachidonic acid (AA) pathway and metabolic rewiring, but metabolic rewiring is not affected by the AA pathway.(A-B) Monocytes were pre-treated with zileuton (ZLT), an inhibitor of ALOX5, or 2DG, a glycolysis inhibitor, followed by stimulation with IS for 24 hours. After a resting period of 5 days, the enrichment of H3K4me3 on the promoters of TNFA and IL6 loci was assessed. Normalization was performed using 2% input. (C) Monocytes were pre-treated with zileuton (ZLT) and stimulated with IS for 24 hr. Cell lysates were immunoblotted for phosphorylated S6 Kinase, with β-actin serving as a normalization control. Band intensities in the immunoblots were quantified using densitometry. (D) A schematic representation of the mechanistic framework underlying IS-trained immunity. Bar graphs show the mean ± SEM. * = p < 0.05, ** = p < 0.01, and *** = p < 0.001 by two-tailed paired t-test.

Fig 5: Liver lipid metabolism. A: protein levels of lipogenic enzymes. ACC, acetyl coA carboxylase; PDH, pyruvate dehydrogenase; PPARγ, peroxisome proliferator activator receptor γ; FAS, fatty acid synthase; SREBP1, sterol regulatory element-binding protein 1. B: protein levels of lipolytic enzymes. PPARα, peroxisome proliferator activator receptor α; LPL, lipoprotein lipase; ATGL, adipose triglyceride lipase; 5-LO, 5-lipoxygenase; CYP4A14, cytochrome P-450 4A14. C: total liver lipid content determined from 100 mg liver tissue. D: representative Oil-red-O (ORO) and hematoxylin-and-eosin (H&E) histology images. Scale bar is 50 μm. E: quantification of lipid droplet staining. Data are presented as means ± SE for control (white bar) and Mat-Ob (gray bar) offspring. n = 6–8 per group; *P < 0.05, ***P < 0.001.