Journal: bioRxiv

Article Title: Lysosome repair by ER-mediated cholesterol transfer

doi: 10.1101/2022.09.26.509457

Figure Lengend Snippet: A Representative movie stills from live-cell imaging experiments indicate recruitment of CHMP4B-eGFP to damaged lysosomes in control and U18666A treated cells. HeLa cells stably expressing CHMP4B-eGFP were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of CHMP4B-eGFP foci per cell. Error bars denote +/-SEM from n=4, >30 cells per experiment for each condition. B Representative immunofluorescence images illustrate Galectin-3 recruitment kinetics on the damaged lysosomes upon treatment with 2 µM U18666A for 17 h. HeLa cells were treated or not (Control) with U18666A and +/-LLOMe for the indicated time points, fixed and processed for immunofluorescence microscopy. Endogenous levels of Galectin-3 were visualized. C Number of Galectin-3 positive dots per cell was quantified from the dataset described in B. Error bars denote +/-SD from n=1, >50 cells per condition. Kruskal-Wallis, Dunn’s post hoc test, **** p<0.0001, ns=not statistically significant. D Representative movie stills from live-cell imaging experiments indicate recruitment of the PtdIns4P probe 2xSidM-eGFP to damaged lysosomes in control but not in the U18666A treated cells. HeLa cells were treated with 2 µM U18666A for 17 h before treatment with 250 µM LLOMe. The graph shows quantification of 2xSidM-eGFP foci per cell. Error bars denote +/-SD from n=2, >20 cells per experiment for each condition.

Article Snippet: Human Galectin-3 Alexa Fluor 488-conjugated antibody (cat. no. IC1154G) was purchased from R&D Systems.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Biomolecules

Article Title: Galectin-3 Is a Natural Binding Ligand of MCAM (CD146, MUC18) in Melanoma Cells and Their Interaction Promotes Melanoma Progression

doi: 10.3390/biom12101451

Figure Lengend Snippet: Galectin-3 is a natural ligand of MCAM in human melanoma cells. ( A , B ): MCAM and galectin-3 are naturally associated in human skin A375 ( A ) and Uveal 92.1 melanoma ( B ) cells. A375 and 92.1 cells were lysed and immunoprecipitated with antibodies against MCAM or galectin-3 and followed by immunoblotting. ( C ) MCAM and galectin-3 are co-localized on surface of melanoma cells. Localization of MCAM (green) and galectin-3 (red) in A375 and 92.1 cells was analysed by confocal microscopy following immunofluorescence staining (cell nucleus were stained by DAPI as blue). Representative images from at least two independent experiments are shown.

Article Snippet: Sub-confluent cells cultured on glass coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After two washes with PBS, the cells were incubated with a blocking buffer (2% BSA in PBS) before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech) (1:500 and 1:1000 dilution, respectively, in 1% BSA in PBS) for 2 h at room temperature.

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Immunofluorescence, Staining

Journal: Biomolecules

Article Title: Galectin-3 Is a Natural Binding Ligand of MCAM (CD146, MUC18) in Melanoma Cells and Their Interaction Promotes Melanoma Progression

doi: 10.3390/biom12101451

Figure Lengend Snippet: Galectin-3 induces MCAM dimerization and clustering on melanoma cell surface. ( A ) Galectin-3 induces MCAM dimerization in A375 cells. Cells were treated with 10 µg/mL galectin-3 or BSA (control) for 30 min before the cells were treated with BS3 cross-linker and analysed by MCAM immunoblotting. Representative blots are shown in ( A ) and percentage change of MCAM dimer bands in response to galectin-3 from two independent experiments is shown in ( B ). ( C ) Galectin-3 induces MCAM clustering on surface in A375 cells. Cells were treated with 10 µg/mL galectin-3 for 1 h, fixed and immunofluorescence-stained for MCAM (green) and galectin-3 (red), and imaged by confocal microscopy (cell nuclei were stained by DAPI as blue) (arrows point to clustering MCAM).

Article Snippet: Sub-confluent cells cultured on glass coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After two washes with PBS, the cells were incubated with a blocking buffer (2% BSA in PBS) before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech) (1:500 and 1:1000 dilution, respectively, in 1% BSA in PBS) for 2 h at room temperature.

Techniques: Western Blot, Immunofluorescence, Staining, Confocal Microscopy

Journal: Biomolecules

Article Title: Galectin-3 Is a Natural Binding Ligand of MCAM (CD146, MUC18) in Melanoma Cells and Their Interaction Promotes Melanoma Progression

doi: 10.3390/biom12101451

Figure Lengend Snippet: Galectin-3–MCAM interaction induces AKT activation in melanoma cells. ( A – C ) Galectin-3 induces time-dependent increase of AKT phosphorylation in A375 ( A ), Mel270 ( B ), and 92.1 ( C ) cells. Cells were treated with 10 µg/mL galectin-3 for different times before the cells were lysed, and p-AKT and AKT levels were analysed by immunoblotting. Band densities from two independent experiments were quantified and expressed as percentage p-AKT/AKT changes. ( D , E ) Full-length galectin-3, but not the truncated, C-terminal galectin-3 (Gal-3C), induces AKT activation in A375 cells in dose-dependent manner. Cells were treated with different concentrations of either full-length Gal-3 ( D ) or Gal-3C ( E ) for 1 h before cells were lysed and p-AKT and AKT levels were determined by immunoblotting.

Article Snippet: Sub-confluent cells cultured on glass coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After two washes with PBS, the cells were incubated with a blocking buffer (2% BSA in PBS) before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech) (1:500 and 1:1000 dilution, respectively, in 1% BSA in PBS) for 2 h at room temperature.

Techniques: Activation Assay, Western Blot

Journal: Biomolecules

Article Title: Galectin-3 Is a Natural Binding Ligand of MCAM (CD146, MUC18) in Melanoma Cells and Their Interaction Promotes Melanoma Progression

doi: 10.3390/biom12101451

Figure Lengend Snippet: Galectin-3 binds to MCAM via O -glycans. A375 cells were treated without or with N - or O -glycanase before cells were lysed and immunoprecipitated with MCAM antibody or control immunoglobulin. Immunoprecipitates were analysed by immunoblotting with antibodies against MCAM ( A ) and galectin-3 ( B ). Treatment of the cells with O -, but not N -, glycanase reduces presence of galectin-3 in MCAM immunoprecipitates.

Article Snippet: Sub-confluent cells cultured on glass coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After two washes with PBS, the cells were incubated with a blocking buffer (2% BSA in PBS) before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech) (1:500 and 1:1000 dilution, respectively, in 1% BSA in PBS) for 2 h at room temperature.

Techniques: Immunoprecipitation, Western Blot

Journal: Biomolecules

Article Title: Galectin-3 Is a Natural Binding Ligand of MCAM (CD146, MUC18) in Melanoma Cells and Their Interaction Promotes Melanoma Progression

doi: 10.3390/biom12101451

Figure Lengend Snippet: MCAM shRNA suppression inhibits AKT activation and proliferation of melanoma cells in response to galectin-3. ( A ) Generation of two subclones (55.6 and 55.3) of MCAM suppressed A375 cells with shRNA ( A ). ( B ) MCAM suppression reduces AKT activation in A375 cell in response to galectin-3. Moderately MCAM-suppressed (55.6) and highly MCAM-supressed (55.3) subclones of A375 cells were treated with 10 µg/mL galectin-3 for different time before the cells were lysed and levels of p-AKT and AKT were determined by immunoblotting. Representative blots from two independent analyses are shown. ( C – E ) Galectin-3–MCAM interaction increases melanoma cell proliferation. Proliferation of parental or MCAM suppressed A375 cells was analysed in the absence ( C ) or presence of Gal-3 ( D , E ) or lactose ( D ). Data are presented as mean ± SEM from three independent experiments, each in triplicate. * p < 0.05, ** p < 0.01.

Article Snippet: Sub-confluent cells cultured on glass coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After two washes with PBS, the cells were incubated with a blocking buffer (2% BSA in PBS) before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech) (1:500 and 1:1000 dilution, respectively, in 1% BSA in PBS) for 2 h at room temperature.

Techniques: shRNA, Activation Assay, Western Blot

Journal: Biomolecules

Article Title: Galectin-3 Is a Natural Binding Ligand of MCAM (CD146, MUC18) in Melanoma Cells and Their Interaction Promotes Melanoma Progression

doi: 10.3390/biom12101451

Figure Lengend Snippet: Galectin-3–MCAM interaction promotes melanoma cell adhesion, migration, invasion, and colony formation. ( A ) MCAM–galectin-3 interaction increases melanoma cell migration. Gaps of A375 and 55.3 cell monolayer were created by silicone inserts and imaged every 12 h in the presence of 10 µg/mL galectin-3 or BSA (control). Representative images are shown in ( A ), and percentage of the gaps in comparison with that at 0 h from two independent experiments are shown in ( B ). Gap of A375 cells treated with galectin-3 is 51% smaller than that of A375 cells without galectin after 24 h. ( C , D ) Galectin-3–MCAM interaction promotes melanoma cell adhesion to and invasion through Matrix proteins. A375 and 55.3 cell adhesion to or invasion through matrix proteins in the presence or absence of 10 µg/mL galectin-3 was assessed. ( E ) MCAM–galectin-3 interaction enhances melanoma colony formation. Number of colonies (>50 cells) after 10-day culture in the presence of 10 µg/mL galectin-3 or BSA were quantified. Data are presented as mean ± SEM of at least three independent experiments, each in triplicate. * p < 0.05, ** p < 0.01.

Article Snippet: Sub-confluent cells cultured on glass coverslips in 24-well plates were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After two washes with PBS, the cells were incubated with a blocking buffer (2% BSA in PBS) before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech) (1:500 and 1:1000 dilution, respectively, in 1% BSA in PBS) for 2 h at room temperature.

Techniques: Migration

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: Atherosclerosis regression is incomplete in Jak2 VF MPN mice with moderate cholesterol lowering. A: Study design created with BioRender.com. B: Plasma cholesterol (n = 34, 55, 55, 54, 16, 16 for Ctrl mice, n = 31, 55, 49, 39, 17, 16 for Jak2 VF mice, for weeks 0, 5, 11, 15, 17.5, 21 respectively). P = 0.028, <0.0001, <0.0001, <0.0001, 0.0005, <0.0001 (Ctrl vs. Jak2 VF at weeks 0, 5, 11, 15, 17.5, and 21, respectively). C: H&E images of aortic root lesions. Black lines , necrotic core. Scale bar, 200 μm. D: Lesion area, n = 15–23. P < 0.0001 (Ctrl Baseline vs. Jak2 VF Baseline; Ctrl LDL Lowering vs. Jak2 VF LDL Lowering), P = 0.052 ( Jak2 VF Baseline vs. LDL Lowering). E: Necrotic core area, n = 15–23. P = 0.0079 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.0002 (Ctrl LDL Lowering vs. Jak2 VF LDL Lowering), P = 0.0003 ( Jak2 VF Baseline vs. LDL Lowering). F: Picrosirius red-stained aortic root lesions. Scale bar, 200 μm. G: Collagen area as a percentage of lesion area, n = 15–24. P = 0.02 (Ctrl Baseline vs. LDL Lowering). H: Images of aortic root lesions stained for MAC2 ( Green ) and DAPI ( Blue ). Scale bar, 200 μm. I: Macrophage area, n = 15–24. P = 0.006 (Ctrl Baseline vs. LDL Lowering), P = 0.16 ( Jak2 VF Baseline vs. LDL Lowering). All quantifications shown as mean ± s.e.m. Two-way ANOVA with the Geisser-Greenhouse correction for sphericity and Tukey’s multiple comparisons test (B). Two-way ANOVA with Tukey’s multiple comparisons test (D, E, G, and I). DAPI, 4′,6-diamidino-2-phenylindole; LDL, low-density lipoprotein; MPN, myeloproliferative neoplasm.

Article Snippet: Following blocking, sections were incubated with the following primary antibodies at the indicated concentrations overnight at 4 °C in a humidified chamber: Absent in melanoma 2 (AIM2) (Abcam, ab119791, 1:250), Cleaved GasD (Cell Signaling, 10137, 10 μg/ml), c-Myc (Cell Signaling, 5605, 5.76 μg/ml), Ki67 (Abcam, ab15580, 9 μg/ml), MAC2 (Cedarlane, CL8942AP 1 μg/ml), MAC2 conjugated to Alexa Fluor 488 (Cedarlane, CL8942AF4, 1 μg/ml), mer proto-oncogene tyrosine kinase (MerTK) (R&D, BAF591, 2 μg/ml), pγH2AX (Cell Signaling, 9718, 0.74 μg/ml), ZsGreen (Thermo Fisher Scientific, TA180002, 10 μg/ml), triggering receptor expressed on myeloid cells 2 (TREM2) (Denali, 4D9 DC1847, 1:100).

Techniques: Clinical Proteomics, Staining

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: Aggressive cholesterol lowering normalizes regression in Jak2 VF MPN lesions. A: Study design, created with BioRender.com. B: Plasma cholesterol (n = 5, 38, 19, 18, and 18 for Ctrl mice, n = 5, 37, 15, 16, and 15 for Jak2 VF mice, for weeks 0, 4, 13, 15, and 18, respectively). P = 0.0041 for genotype effect by two-way ANOVA with Geisser-Greenhouse correction, P = 0.011, 0.048, 0.056, and 0.0008 (Ctrl vs. Jak2 VF at weeks 0, 4, 13, and 15, respectively). C: H&E images of aortic root lesions. Black lines , necrotic core. Scale bar, 200 μm. D: Lesion area, n = 13–20. P < 0.0001 (Ctrl Baseline vs. Jak2 VF Baseline; Ctrl LDL Lowering vs. Jak2 VF LDL Lowering). Figures 1 and 4 represent independent regression cohorts performed separately; therefore, absolute lesion area should be compared within each cohort rather than between figures. E: Necrotic core area, n = 13–20. P = 0.0003 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.0006 (Ctrl LDL Lowering vs. Jak2 VF LDL Lowering), P = 0.90 ( Jak2 VF Baseline vs. LDL Lowering). F: Picrosirius red-stained aortic root lesions. Scale bar, 200 μm. G: Collagen area as a percentage of lesion area, n = 13–20. P < 0.0001 (Ctrl Baseline vs. LDL Lowering), P = 0.0002 ( Jak2 VF Baseline vs. LDL Lowering), P = 0.02 (Ctrl LDL Lowering vs. Jak2 VF LDL Lowering). H: Images of aortic root lesions stained for MAC2 ( Green ) and DAPI ( Blue ). Scale bar, 200 μm. I: Macrophage area, n = 13–20. P < 0.0001 (Ctrl and Jak2 VF Baseline vs. LDL Lowering). All quantifications shown as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons test (D, G, and I). Two-way ANOVA with the Geisser-Greenhouse correction for sphericity and Tukey’s multiple comparisons test (B). Kruskal-Wallis test with Dunn’s multiple comparisons test (E). DAPI, 4′,6-diamidino-2-phenylindole; LDL, low-density lipoprotein; MPN, myeloproliferative neoplasm.

Article Snippet: Following blocking, sections were incubated with the following primary antibodies at the indicated concentrations overnight at 4 °C in a humidified chamber: Absent in melanoma 2 (AIM2) (Abcam, ab119791, 1:250), Cleaved GasD (Cell Signaling, 10137, 10 μg/ml), c-Myc (Cell Signaling, 5605, 5.76 μg/ml), Ki67 (Abcam, ab15580, 9 μg/ml), MAC2 (Cedarlane, CL8942AP 1 μg/ml), MAC2 conjugated to Alexa Fluor 488 (Cedarlane, CL8942AF4, 1 μg/ml), mer proto-oncogene tyrosine kinase (MerTK) (R&D, BAF591, 2 μg/ml), pγH2AX (Cell Signaling, 9718, 0.74 μg/ml), ZsGreen (Thermo Fisher Scientific, TA180002, 10 μg/ml), triggering receptor expressed on myeloid cells 2 (TREM2) (Denali, 4D9 DC1847, 1:100).

Techniques: Clinical Proteomics, Staining

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 lowering suppresses Jak2 VF macrophage proliferation and DNA damage. A: Study design created with BioRender.com. B: Plasma cholesterol (n = 79, 78, and 78 for Baseline mice for weeks 3, 11, and 15, respectively; n = 13, 14 for Ctrl Progression mice, n = 16, 16 for Ctrl LDL Lowering mice, n = 15, 15 for Jak2 VF Progression mice, n = 16, 16 for Jak2 VF LDL Lowering mice, for weeks 18 and 23, respectively). C: Images of aortic root lesions for MAC2 ( Green ), Cleaved GasD ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. D: Log 10 transformed cleaved GasD mean fluorescence intensity (MFI) in lesions with the addition of constant 1, n = 14–16. P = 0.01 (Baseline vs. Jak2 VF Progression), P = 0.051 ( Jak2 VF Progression vs. Jak2 VF LDL Lowering). E: Log 10 transformed cleaved GasD MFI in the necrotic core with the addition of constant 1, n = 14–16. P = 0.04 (Baseline vs. Jak2 VF Progression), P = 0.013 ( Jak2 VF Progression vs. Jak2 VF LDL Lowering). F: Images of aortic root lesions stained for MAC2 ( Green ), Cleaved GasD ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , cleaved GasD + macrophages. G: Log 10 transformed cleaved GasD positive macrophages per section with the addition of constant 1, n = 14–16. H: Images of aortic root lesions for MAC2 ( Red ), Ki67 ( White ), and ZsGreen ( Green ). Scale bar, 60 μm. White arrows , macrophages double positive for Ki67 and ZsGreen. I: Macrophages positive for both Ki67 and ZsGreen per section, n = 13–15. P = 0.0019 (Baseline vs. Jak2 VF Progression), P = 0.0006 (Ctrl Progression vs. Jak2 VF Progression), P = 0.0003 ( Jak2 VF Progression vs. Jak2 VF LDL Lowering). J: Macrophages positive for Ki67 but negative for ZsGreen per section, n = 13–16. K: Images of aortic root lesions stained for pγH2AX ( Red ), ZsGreen ( Green ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows, pγH2AX positive cells. L: Log 10 transformed cells double positive for pγH2AX and ZsGreen in lesions with the addition of constant 1, n = 13–16. P = 0.0024 (Baseline vs. Jak2 VF Progression), P = 0.0019 ( Jak2 VF Progression vs. Jak2 VF LDL Lowering). M: Log 10 transformed cells positive for pγH2AX but negative for ZsGreen in lesions with the addition of constant 1, n = 13–16. All quantifications shown as mean ± s.e.m. One-way ANOVA with Holm–Sidak’s multiple comparisons test (D and E). Kruskal–Wallis test with Dunn’s multiple comparison’s test (G, I, J, L, and M). DAPI, 4′,6-diamidino-2-phenylindole; GasD, gasdermin D; LDL, low-density lipoprotein; pγH2AX, phosphorylated histone H2A.X.

Article Snippet: Following blocking, sections were incubated with the following primary antibodies at the indicated concentrations overnight at 4 °C in a humidified chamber: Absent in melanoma 2 (AIM2) (Abcam, ab119791, 1:250), Cleaved GasD (Cell Signaling, 10137, 10 μg/ml), c-Myc (Cell Signaling, 5605, 5.76 μg/ml), Ki67 (Abcam, ab15580, 9 μg/ml), MAC2 (Cedarlane, CL8942AP 1 μg/ml), MAC2 conjugated to Alexa Fluor 488 (Cedarlane, CL8942AF4, 1 μg/ml), mer proto-oncogene tyrosine kinase (MerTK) (R&D, BAF591, 2 μg/ml), pγH2AX (Cell Signaling, 9718, 0.74 μg/ml), ZsGreen (Thermo Fisher Scientific, TA180002, 10 μg/ml), triggering receptor expressed on myeloid cells 2 (TREM2) (Denali, 4D9 DC1847, 1:100).

Techniques: Clinical Proteomics, Transformation Assay, Fluorescence, Staining

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: Moderate cholesterol lowering reverses macrophage AIM2 inflammasome activation, DNA damage, and proliferation in Jak2 VF MPN lesions. A: Images of aortic root lesions stained for MAC2 ( Green ), Cleaved GasD ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. B: Log 10 transformed cleaved GasD mean fluorescence intensity (MFI) in lesions with the addition of constant 1, n = 15–24. P = 0.021 (Ctrl Baseline vs. Jak2 VF Baseline). C: Log 10 transformed cleaved GasD mean fluorescence intensity (MFI) in necrotic cores with the addition of constant 1, n = 15–24. P = 0.0008 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.032 (Ctrl LDL Lowering vs. Jak2 VF LDL Lowering). D: Images of aortic root lesions stained for MAC2 ( Green ), Cleaved GasD ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , cleaved GasD + macrophages. E: Log 10 transformed cleaved GasD positive macrophages per section with the addition of constant 1, n = 15–24. P = 0.024 ( Jak2 VF Baseline vs. LDL Lowering). F: Images of aortic root lesions stained for MAC2 ( Green ), AIM2 ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , AIM2 positive macrophages. G: Log 10 transformed AIM2 positive macrophages per section with the addition of constant 1, n = 15–23. P = 0.0001 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.03 ( Jak2 VF Baseline vs. LDL Lowering). H: Images of aortic root lesions stained for MAC2 ( Green ), pγH2AX ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , pγH2AX positive cells. I: Log 10 transformed pγH2AX positive cells per section with the addition of constant 1, n = 15–23. P = 0.0019 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.045 ( Jak2 VF Baseline vs. LDL Lowering). J: Images of aortic root lesions stained for MAC2 ( Green ), Ki67 ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , Ki67 positive macrophages. K: Log 10 transformed Ki67 positive macrophages per section with the addition of constant 1, n = 15–23. P = 0.0019 ( Jak2 VF Baseline vs. LDL Lowering). MAC2 intensity differences reflect independent staining and imaging sessions across panels. All quantifications shown as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons test (B and C). Kruskal–Wallis test with Dunn’s multiple comparisons test (E, G, I, and K). AIM2, absent in melanoma 2; DAPI, 4′,6-diamidino-2-phenylindole; GasD, gasdermin D; LDL, low-density lipoprotein; MPN, myeloproliferative neoplasm; pγH2AX, phosphorylated histone H2A.X.

Article Snippet: Following blocking, sections were incubated with the following primary antibodies at the indicated concentrations overnight at 4 °C in a humidified chamber: Absent in melanoma 2 (AIM2) (Abcam, ab119791, 1:250), Cleaved GasD (Cell Signaling, 10137, 10 μg/ml), c-Myc (Cell Signaling, 5605, 5.76 μg/ml), Ki67 (Abcam, ab15580, 9 μg/ml), MAC2 (Cedarlane, CL8942AP 1 μg/ml), MAC2 conjugated to Alexa Fluor 488 (Cedarlane, CL8942AF4, 1 μg/ml), mer proto-oncogene tyrosine kinase (MerTK) (R&D, BAF591, 2 μg/ml), pγH2AX (Cell Signaling, 9718, 0.74 μg/ml), ZsGreen (Thermo Fisher Scientific, TA180002, 10 μg/ml), triggering receptor expressed on myeloid cells 2 (TREM2) (Denali, 4D9 DC1847, 1:100).

Techniques: Activation Assay, Staining, Transformation Assay, Fluorescence, Imaging

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: Moderate cholesterol lowering reverses impaired efferocytosis in Jak2 VF MPN lesions while increasing TREM2 Hi macrophages in control and Jak2 VF MPN lesions. A: Images of aortic root lesions stained for MAC2 ( Green ), MerTK ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. B: Percentage of MAC2 positive area double positive for MerTK and MAC2, n = 15–23. P = 0.02 (Ctrl Baseline vs. Jak2 VF Baseline), P < 0.0001 ( Jak2 VF Baseline vs. LDL Lowering). C: Images of aortic root lesions stained for MAC2 ( Green ), TREM2 ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. D: Percentage of MAC2 positive area double positive for TREM2 and MAC2, n = 15–24. P < 0.0001 (Ctrl and Jak2 VF Baseline vs. LDL Lowering). E: Images of in situ efferocytosis in aortic root lesions: MAC2 ( Green ), TUNEL ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , nuclei double positive for TUNEL and MAC2. White wedges , nuclei positive for TUNEL but negative for MAC2. F: Percentage of TUNEL positive nuclei also positive for MAC2. P = 0.011 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.021 ( Jak2 VF Baseline vs. LDL Lowering). MAC2 intensity differences reflect independent staining and imaging sessions across panels, n = 13–24. All quantifications shown as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons test (D). Kruskal-Wallis test with Dunn’s multiple comparisons test (B and F). DAPI, 4′,6-diamidino-2-phenylindole; LDL, low-density lipoprotein; MerTK, mer proto-oncogene tyrosine kinase; MPN, myeloproliferative neoplasm; TREM2, triggering receptor expressed on myeloid cells 2.

Article Snippet: Following blocking, sections were incubated with the following primary antibodies at the indicated concentrations overnight at 4 °C in a humidified chamber: Absent in melanoma 2 (AIM2) (Abcam, ab119791, 1:250), Cleaved GasD (Cell Signaling, 10137, 10 μg/ml), c-Myc (Cell Signaling, 5605, 5.76 μg/ml), Ki67 (Abcam, ab15580, 9 μg/ml), MAC2 (Cedarlane, CL8942AP 1 μg/ml), MAC2 conjugated to Alexa Fluor 488 (Cedarlane, CL8942AF4, 1 μg/ml), mer proto-oncogene tyrosine kinase (MerTK) (R&D, BAF591, 2 μg/ml), pγH2AX (Cell Signaling, 9718, 0.74 μg/ml), ZsGreen (Thermo Fisher Scientific, TA180002, 10 μg/ml), triggering receptor expressed on myeloid cells 2 (TREM2) (Denali, 4D9 DC1847, 1:100).

Techniques: Control, Staining, In Situ, TUNEL Assay, Imaging

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: Aggressive cholesterol lowering more strongly decreases macrophage pyroptosis and DNA damage in Jak2 VF MPN lesions. A: Images of aortic root lesions stained for MAC2 ( Green ), cleaved GasD ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. B: Log 10 transformed cleaved GasD MFI in the necrotic core with the addition of constant 1, n = 14–20. P = 0.0064 for genotype effect and P = 0.021 for treatment effect by two-way ANOVA. C: Images of aortic root lesions stained for MAC2 ( Green ), Cleaved GasD ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , cleaved GasD + macrophages. D: Log 10 transformed cleaved GasD positive macrophages per section with the addition of constant 1, n = 13–20. P = 0.023 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.005 ( Jak2 VF Baseline vs. LDL Lowering). E: Representative immunoblot analysis of full-length and cleaved GasD in CD11b + splenocytes. F: Log 10 transformed densitometric quantification of the ratio of cleaved GasD to full-length GasD from E with the addition of constant 1. n = 7–8 biological replicates. P = 0.0003 (Ctrl Baseline vs. Jak2 VF Baseline), P = 0.0046 ( Jak2 VF Baseline vs. LDL Lowering). G: Images of aortic root lesions stained for MAC2 ( Green ), pγH2AX ( Red ), and DAPI ( Blue ). Scale bar, 60 μm. White arrows , pγH2AX positive cells. H: Log 10 transformed pγH2AX positive cells per section with the addition of constant 1, n = 14–20. P = 0.016 (Ctrl Baseline vs. LDL Lowering), P = 0.0016 (Ctrl Baseline vs. Jak2 VF Baseline), P < 0.0001 ( Jak2 VF Baseline vs. LDL Lowering). All quantifications shown as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons test (B, D, F, and H). DAPI, 4′,6-diamidino-2-phenylindole; GasD, gasdermin D; LDL, low-density lipoprotein; MFI, mean fluorescence intensity; MPN, myeloproliferative neoplasm; pγH2AX, phosphorylated histone H2A.X.

Article Snippet: Following blocking, sections were incubated with the following primary antibodies at the indicated concentrations overnight at 4 °C in a humidified chamber: Absent in melanoma 2 (AIM2) (Abcam, ab119791, 1:250), Cleaved GasD (Cell Signaling, 10137, 10 μg/ml), c-Myc (Cell Signaling, 5605, 5.76 μg/ml), Ki67 (Abcam, ab15580, 9 μg/ml), MAC2 (Cedarlane, CL8942AP 1 μg/ml), MAC2 conjugated to Alexa Fluor 488 (Cedarlane, CL8942AF4, 1 μg/ml), mer proto-oncogene tyrosine kinase (MerTK) (R&D, BAF591, 2 μg/ml), pγH2AX (Cell Signaling, 9718, 0.74 μg/ml), ZsGreen (Thermo Fisher Scientific, TA180002, 10 μg/ml), triggering receptor expressed on myeloid cells 2 (TREM2) (Denali, 4D9 DC1847, 1:100).

Techniques: Staining, Transformation Assay, Western Blot, Fluorescence