33444s  (Cell Signaling Technology Inc)

Journal: Journal of Lipid Research

Article Title: Aggressive cholesterol lowering normalizes atherosclerosis regression in Jak2 V617F mice

doi: 10.1016/j.jlr.2026.101003

Figure Lengend Snippet: Cholesterol increases AIM2 inflammasome activation and nuclear dsDNA breaks via increased mitochondrial ROS in Jak2 VF macrophages. A: EdU + BMDMs as a percentage of total BMDMs after overnight incubation with acLDL (25 μg/ml) in base media without LCM or recombinant M-CSF during the proliferation assay. n = 4 biological replicates. P = 0.032 for acLDL effect. B: Representative immunoblot analysis of pγH2AX in BMDMs after overnight incubation with acLDL (25 μg/ml) in base media without LCM or recombinant M-CSF during the proliferation assay. C: Densitometric quantification of pγH2AX normalized to β-Actin from B. Each data point represents a cell-well technical replicate from one mouse (1 biological replicate, 3 technical replicates). P = 0.022 (Ctrl Vehicle vs. Jak2 VF Vehicle), P = 0.038 (Ctrl acLDL vs. Jak2 VF acLDL). Statistical comparisons in this panel were performed across cell-well technical replicates from a single mouse (1 biological replicate; n = 3 wells per condition) to assess within-experiment reproducibility. D: EdU + BMDMs as a percentage of total BMDMs after overnight incubation with oxPAPC (50 μg/ml) in base media without LCM or recombinant M-CSF during the proliferation assay. Each data point represents a cell-well technical replicate from one mouse (1 biological replicate, 3 technical replicates). P = 0.022 (Ctrl Vehicle vs. Ctrl oxPAPC), P < 0.0001 ( Jak2 VF Vehicle vs. Jak2 VF oxPAPC), P = 0.0004 (Ctrl oxPAPC vs. Jak2 VF oxPAPC). Statistical comparisons in this panel were performed across cell-well technical replicates from a single mouse (1 biological replicate; n = 3 wells per condition) to assess within-experiment reproducibility. E: Representative immunoblot analysis of pγH2AX in BMDMs after overnight incubation with oxPAPC (50 μg/ml) in base media without LCM or recombinant M-CSF during the proliferation assay. F: Densitometric quantification of pγH2AX normalized to β-Actin from E. Each data point represents a cell-well technical replicate from one mouse (1 biological replicate, 3 technical replicates). P = 0.026 for oxPAPC effect. Statistical comparisons in this panel were performed across cell-well technical replicates from a single mouse (1 biological replicate; n = 3 wells per condition) to assess within-experiment reproducibility. G: ELISA quantification of total IL-1β in cell culture media of BMDMs incubated with MβCD-cholesterol (30 μg/ml) alone, mitoTEMPO (10 μM) alone, or MβCD-cholesterol (30 μg/ml) in combination with mitoTEMPO (10 μM) for 4 h after priming with LPS (100 ng/ml) for 3 h in complete media containing 20 ng/ml recombinant M-CSF. Data are shown as technical replicates from n = 2–3 independent experiments (biological replicates). Wells failing prespecified quality-control criteria in which the coefficient of variation (CV) exceeded 20% were excluded. P = 0.0007 ( Jak2 VF Vehicle vs. Jak2 VF MβCD-cholesterol), P = 0.019 ( Jak2 VF MβCD-cholesterol vs. Jak2 VF MβCD-cholesterol + mitoT), H: ELISA quantification of total IL-1β in cell culture media of BMDMs incubated with MβCD-cholesterol (30 μg/ml) for 4 h after priming with LPS (100 ng/ml) for 3 h in complete media containing 20 ng/ml recombinant M-CSF. n = 6 cell-well technical replicates from a single mouse (1 biological replicate). No biological replicates were available. P = 0.0017 ( Jak2 VF Vehicle vs. Jak2 VF MβCD-cholesterol), P = 0.0026 ( Jak2 VF MβCD-cholesterol vs. Jak2 VF Aim2 −/− MβCD-cholesterol). Statistical comparisons in this panel were performed across cell-well technical replicates from a single mouse (1 biological replicate; n = 6 wells per condition) to assess within-experiment reproducibility. I: Representative immunoblot analysis of pγH2AX in BMDMs after a 4-h incubation with MβCD-cholesterol (30 μg/ml) in complete media containing 20 ng/ml recombinant M-CSF or LCM. Irrelevant lanes were removed from the blot. Vertical lines indicate where lanes were spliced from the same membrane and exposure. J: Densitometric quantification of pγH2AX normalized to β-Actin from H. n = 8 biological replicates for the vehicle groups and n = 4 biological replicates for the MβCD-cholesterol groups. P = 0.0019 (Ctrl Vehicle vs. Ctrl MβCD-cholesterol), P = 0.0028 ( Jak2 VF Vehicle vs. Jak2 VF MβCD-cholesterol). P = 0.019 for genotype effect. P < 0.0001 for MβCD-cholesterol effect. Ctrl Vehicle data include earlier assessments of this end point in untreated cells, prior to the initiation of the treatment arm. All samples were processed and analyzed using the same protocol. K: EdU + BMDMs as a percentage of total BMDMs after a 4-h incubation with MβCD-cholesterol (30 μg/ml) in complete media containing 20 ng/ml recombinant M-CSF. n = 3 biological replicates. P = 0.043 for genotype effect. All quantifications shown as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons test (A, C, D, F, H, J, and K). Kruskal–Wallis test with Dunn’s multiple comparisons test (G). acLDL, acetylated LDL; AIM2, absent in melanoma 2; BMDM, bone marrow-derived macrophage; IL-1β, interleukin-1β; LPS, lipopolysaccharide; MitoTEMPO, mitochondria-targeted TEMPO (a triphenylphosphonium-conjugated nitroxide); MβCD, methyl-β-cyclodextrin; oxPAPC, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; pγH2AX, phosphorylated histone H2A.X; ROS, reactive oxygen species.

Article Snippet: BMDMs were primed in complete media containing 20 ng/ml recombinant M-CSF (Cell Signaling 33444S) and 100 ng/ml lipopolysaccharide (Cell Signaling 14011) for 3 h at 37°C and 5% CO 2 before treating with complete media containing vehicle, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO) (MedChemExpress, HY-112879) (10 μM), methyl-β-cyclodextrin-cholesterol (MβCD-Cholesterol) (Sigma-Aldrich, C4951) (30 μg/ml) or a combination of mitoTEMPO (10 μM) and MβCD -Cholesterol (30 μg/ml) for 4 h at 37°C and 5% CO 2 .

Techniques: Activation Assay, Incubation, Recombinant, Proliferation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Membrane, Derivative Assay