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

Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of testicular sections from mice under four different treatments in the short term (W2, 2 weeks) and long term (W8, 8 weeks) experiments. Scale bar, 200 μm. b ELISA of CSF1 in mice serum of the four different groups in W2 and W8 experiments. c RT-qPCR detected Csf1 expression in mice testicular tissue under the four different treatments in W2 and W8 experiments. d CSF1 expression level detected by western blot analysis in the four groups in W2 and W8. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. (The results are shown as the mean ± S.E.M of at least three mice ( n = 3) and the statistical significance was expressed as follows: * p < 0.05; ** p < 0.01). C control group, M normal group treated with MLT, D diabetic group, DM MLT-treated diabetic group

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of MLTC-1 under three different ERS states (C control, Tm Tm treatment induced excessive ERS, Tm + 4PBA Tm treatment in combination with 4PBA for alleviated ERS). b CSF1 concentration in culture medium of MLTC-1 under the three different treatments was detected using ELISA. c RT-qPCR detected the mRNA expression level of Csf1 in MLTC-1 under the three treatments. d Expression of CSF1 in MLTC-1 under different treatments was measured by Western blot. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. e A graphical conclusion of this study is displayed: DM-induced high glucose activated ERS response factors (Grp78 and CHOP) in Leydig cells, resulting in apoptosis of Leydig cells and growth arrest of SSCs in testes. In response to these changes, melatonin treatment alleviated the apoptosis of Leydig cells via inhibition of ERS and recovered the CSF1 secretion in testes. CSF1 then resumed the self-renewal capacity of SSCs. (The results are expressed as the mean ± S.E.M of three separated wells of cells ( n = 3) in at least three different experiments and the statistical significance is expressed as follows: * p < 0.05; ** p < 0.01)

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Inhibition