Journal: Science signaling

Article Title: Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) Side view of A1M-S1P complex with and without the phospholipids, highlighting the S1P binding pocket in both the ApoM-S1P and ApoA1-fused systems. Monomer 1 - ApoA1 in sienna, linker in brown, and ApoM in beige. Monomer 2 - ApoA1 in sage, linker in turquoise, and ApoM in teal. Detail of the binding pocket in A1M-S1P and ApoM-S1P are shown in the right panel. (B) Projection of protein atoms along the top two dominant eigenvectors (eigenvector 1 - PC1 and eigenvector 2 – PC2) is responsible for over 40% of the dominant collective variances in the structure set, generating 5 clusters for which the three-dimensional structure of the most representative member of each cluster is shown. Permanence times: cluster 1 – 9.6%, cluster 2 – 52.9%, cluster 3 – 18.7%, cluster 4 – 12.2%, cluster 5 – 6.5%. (C) Distribution of the proportion of variance between the PCs.

Article Snippet: The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Binding Assay

Journal: Science signaling

Article Title: Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) HMEC-1 cells expressing an NF-κB-luciferase reporter were assayed for TNFα-induced NF-κB reporter activity in the presence of ApoA1, A1M, A1M-S1P, and ApoM-Fc-S1P (N = 3 biological replicates per group). Data are presented as means + S.D. **P < 0.01, ***P < 0.001, ****P < 0.0001 by ordinary two-way ANOVA with Tukey’s multiple comparisons test. TNFα is the reference group. (B) HUVECs were starved for 1 h, pre-treated for 10 minutes with ApoM-Fc-S1P (100 nM), iloprost (200 nM), both ApoM-Fc-S1P and iloprost, A1M (200 μg/mL), A1M-S1P (200 μg/mL), or A1M-iloprost (200 μg/mL) and induced with TNFα (10 ng/mL) for 5 hours. Lysates were subjected to immunoblot analysis for ICAM-1. Quantification of immunoblots was analyzed from 3 biological replicates per group. Data are presented as means ± S.D. *P < 0.05, **P < 0.01, ****P < 0.0001 by ANOVA with post-hoc Holm-Sidak’s multiple comparisons test. (C) Cholesterol efflux in response to human HDL (hHDL), human ApoA1 protein (hApoA1), and bacterial A1M (bA1M) in PMA-induced THP-1 cells (N ≥ 5 independent experiments). Data are presented as means + S.D. *P < 0.05, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Expressing, Luciferase, Activity Assay, Western Blot

Journal: Signal transduction and targeted therapy

Article Title: Porphyromonas gingivalis aggravates atherosclerotic plaque instability by promoting lipid-laden macrophage necroptosis.

doi: 10.1038/s41392-025-02251-6

Figure Lengend Snippet: Fig. 3 Oxidative stress serves as a principal driver of Pg–induced necroptosis. a Dihydroethidium (DHE) staining and CD45-ACTA2 co-staining of serial sections of the aortic roots isolated from Apoe–/– mice infected with or without Pg for 8 weeks. Nuclei were labeled using DAPI. Scale bar = 100 μm. b The co-localization of NOX2 and CD45-labled macrophages in the aortic root plaques of Apoe–/– mice infected with or without Pg for 8 weeks. Nuclei were labeled using DAPI. Scale bar = 20 μm. c Flow cytometry analyses of the reactive oxygen species (ROS) level in macrophages infected by Pg in different MOIs, and treated with or without ox-LDL (60 μg/mL). n = 4 per group. d Relative mRNA expression levels of Nox2, Cox2, Nos2, Gpx1, Sod1, and Sod2 in ox-LDL (60 μg/mL)-loaded macrophages treated with or without Pg (MOI = 100) for 24 h. n = 4 per group. Flow cytometry analyses of ROS production (e) and ratio of PI+ cells (f) in ox-LDL (60 μg/mL)-loaded macrophages pre- administrated with EUK134 (10 μM) and infected with Pg (MOI = 100). n = 4 per group. g Western blot analyses of RIPK3, p-MLKL, MLKL, and GAPDH expression in ox-LDL (60 μg/mL)-loaded macrophages pre-administrated with EUK134 (10 μM) and infected with Pg (MOI = 100) for 24 h. GAPDH was used as the loading control. h H&E, Masson, Oil Red O staining, and CD45 and F4/80 co-immunohistochemical staining of atherosclerotic plaques in aortic roots of Apoe−/−mice untreated or challenged by Pg under the administration of EUK134 for 8 weeks. Scale bar = 200 μm in H&E, scale bar = 100 μm in the rest images. i Quantitative analyses of plaque size, necrotic area, Oil Red O+ area, and CD45 and F4/80-positive areas of the plaques in h. n = 6 per group. Results were represented as mean ± SD (c, d, e, f) or mean ± SEM (i). Data were analyzed by two-way ANOVA (c, d), or one-way ANOVA (e, f, i). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05

Article Snippet: Cholesterol efflux assay Macrophages incubated with 1 μg/mL BODIPY-cholesterol (HY125746, MedChemExpress) for 6 h. Then cellular monolayers were rinsed with PBS and underwent 24-h incubation with 5 μg/mL apo-AI (HY-P72833, MedChemExpress) and 20 μg/mL HDL (YB-003, Yiyuan biotechnology) as cholesterol receptors in serum-free medium, along with Pg (MOI= 100) or PBS co-stimulation.

Techniques: Staining, Isolation, Infection, Labeling, Flow Cytometry, Expressing, Western Blot, Control, Immunohistochemical staining

Journal: Signal transduction and targeted therapy

Article Title: Porphyromonas gingivalis aggravates atherosclerotic plaque instability by promoting lipid-laden macrophage necroptosis.

doi: 10.1038/s41392-025-02251-6

Figure Lengend Snippet: Fig. 4 Gingipains play important roles in Pg-aggravated oxidative stress. Flow cytometry analyses of the ROS production (a) and the ratio of PI+ cells (b) in ox-LDL (60 μg/mL) loaded macrophages infected with Pg or KDP136 (MOI = 100). n = 4 per group. c Western blot analyses of RIPK3, p-MLKL, MLKL, and GAPDH expression in ox-LDL (60 μg/mL)-loaded macrophages infected with Pg or KDP136 (MOI = 100) for 24 h. GAPDH was used as the loading control. Flow cytometry analyses of the ox-LDL uptake (d), the ROS production (e), and the ratio of PI+ cells (f) in ox-LDL-loaded macrophages exposed to RgpA, RgpB, or Kgp (1 μg/mL). n = 5 per group in (d), n = 4 per group in (e, f). g Western blot analyses of RIPK3, p-MLKL, MLKL, and GAPDH expression in ox-LDL (60 μg/mL)-loaded macrophages treated with RgpA, RgpB, or Kgp (1 μg/ mL) for 24 h. GAPDH was used as the loading control. h The co-staining of RgpA and CD68 (macrophage marker) in the coronary plaques of human. Scale bar = 20 μm. i H&E, Oil Red O staining, and CD45 and F4/80 co-staining of the aortic root plaques from Apoe−/−mice infected with or without Pg or KDP136 for 8 weeks. Scale bar = 200 μm in H&E, scale bar = 100 μm in the rest images. j Quantitative analyses of plaque size, necrotic area, Oil Red O+ area, CD45, and F4/80-positive areas of the plaques in i. n = 6 per group. Results were presented as mean ± SD (a, b, d, e, f) or mean ± SEM (j). All data were analyzed by one-way ANOVA. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05

Article Snippet: Cholesterol efflux assay Macrophages incubated with 1 μg/mL BODIPY-cholesterol (HY125746, MedChemExpress) for 6 h. Then cellular monolayers were rinsed with PBS and underwent 24-h incubation with 5 μg/mL apo-AI (HY-P72833, MedChemExpress) and 20 μg/mL HDL (YB-003, Yiyuan biotechnology) as cholesterol receptors in serum-free medium, along with Pg (MOI= 100) or PBS co-stimulation.

Techniques: Flow Cytometry, Infection, Western Blot, Expressing, Control, Staining, Marker

Journal: Signal transduction and targeted therapy

Article Title: Porphyromonas gingivalis aggravates atherosclerotic plaque instability by promoting lipid-laden macrophage necroptosis.

doi: 10.1038/s41392-025-02251-6

Figure Lengend Snippet: Fig. 6 Proteolysis of FOXO3 by gingipains upregulates the transcription of MSR1. a GO enrichment analysis of differential proteins between ox-LDL (60 μg/mL)-loaded macrophages treated with Pg (MOI = 100) versus PBS for 24 h (n = 4 per genotype). P < 0.05 represented statistically significant difference, and upregulated or downregulated proteins were identified with a fold-change (Pg/PBS) > 1.15 or <0.87. qRT-PCR (b) and Western blot (c) analyses of TPT1, BNIP3, FOXO3, CD74, HDAC4, PDCD4, and TAX1BP1 expression in ox-LDL (60 μg/mL)-loaded macrophages infected with Pg or KDP136 (MOI = 100) for 24 h. β-actin was used as control, and n = 4 per group (b). GAPDH was employed as the loading control in (c). Western blot analyses (d) and immunohistochemical staining (e) of FOXO3 in ox-LDL (60 μg/mL)-loaded macrophages infected with Pg, KDP136 (MOI = 100), RgpA, RgpB, Kgp, or Pg-LPS (1 μg/mL) for 24 h. GAPDH was employed as the loading control in (d). Scale bar = 10 μm in (e). The co-staining of FOXO3 and F4/80 (macrophage marker) in the plaques of rabbit aortic arches (f), and mouse aortic roots (g). Scale bar = 20 μm. h Silver staining of recombinant human FOXO3 (rFOXO3, 207.9 μM) incubated with RgpA, RgpB or Kgp (2.1 μM) for 60 min at 37 °C. Red arrowhead points to original rFOXO3. Blue arrowhead points to RgpA. Orange arrowhead points to RgpB. Green arrowhead points to Kgp. Dashed lines encircle rFOXO3 fragments. i Western blot analyses of MSR1 expression in Foxo3 siRNA (si-Foxo3) transfected macrophages loaded with ox-LDL (60 μg/mL) and stimulated with RgpA, RgpB, or Kgp (1 μg/mL) for 24 h. GAPDH was used as control. Flow cytometry analyses of the ox-LDL uptake (j), ROS production (k), and the ratio of PI+ cells (l) in si-Foxo3 transfected macrophages treated with RgpA, RgpB, or Kgp (1 μg/mL) in the presence of ox-LDL (60 μg/mL). n = 4 per group. m Flow cytometry analyses of the ratio of PI+ cells in si-Foxo3 transfected macrophages pre-administrated with or without fucoidan (40 μg/mL) and exposed to ox-LDL (60 μg/mL) for 24 h. n = 4 per group. n Flow cytometry analyses of the proportion of PI+ cells in Msr1 and/or Foxo3 knockdown macrophages treated with ox- LDL (60 μg/mL) for 24 h. n = 4 per group. Results were expressed as mean ± SD. Data were analyzed by two-way ANOVA (b) or one-way ANOVA (j–n). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05

Article Snippet: Cholesterol efflux assay Macrophages incubated with 1 μg/mL BODIPY-cholesterol (HY125746, MedChemExpress) for 6 h. Then cellular monolayers were rinsed with PBS and underwent 24-h incubation with 5 μg/mL apo-AI (HY-P72833, MedChemExpress) and 20 μg/mL HDL (YB-003, Yiyuan biotechnology) as cholesterol receptors in serum-free medium, along with Pg (MOI= 100) or PBS co-stimulation.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Infection, Control, Immunohistochemical staining, Staining, Marker, Silver Staining, Recombinant, Incubation, Transfection, Flow Cytometry, Knockdown

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Cholesterol efflux from ABAE and SMCs . Cells were first labeled with [ 3 H]-cholesterol (0.5 μCi/mL) for 36 h at 37°C and then equilibrated for 24 h in DMEM/0.05% BSA at 37°C. Cellular [ 3 H]-cholesterol loads in DPM per μg of proteins were compared between ABAE and SMCs (A) . Data represent the mean ± SEM (in DPM/μg of proteins), with n = 6 from two sets of experiments. Paired- t -test was performed, *** p < 0.001 when compared with ABAE condition. [ 3 H]-cholesterol released from both cell types was then measured 6 h after medium supplementation with either BSA (control condition) or cholesterol acceptors [ApoA-I (20 μg/mL) or HDL (50 μg/mL)] at 37°C (B) . Data represent the mean ± SEM (in DPM/μg of proteins), with n = 6 from two sets of experiments. One-way ANOVA test followed by Bonferroni correction was performed in which *** p < 0.001 refers to the control condition and ### p < 0.001 refers to the matched HDL condition in ABAE.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Control

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Cholesterol release from ABAE (A,C) and from SMCs (B,D) upon CD-treatment . Cells were first labeled with [ 3 H]-cholesterol (0.5 μCi/mL) for 36 h at 37°C and then equilibrated in DMEM/0.05% BSA for 24 h at 37°C. [ 3 H]-cholesterol released in the medium from both cell types was then measured 6 h after medium supplementation with cholesterol acceptors [ApoA-I (20 μg/mL) or HDL (50 μg/mL)] at 37°C. The radioactivity contained in the culture medium, that corresponds to the cholesterol released from cells was measured. Data are expressed as the mean ± SEM (in DPM/μg of proteins, n = 6 from two sets of experiments). Statistical analysis: a one-way ANOVA followed by Dunnett's test for multiple comparisons in which * p < 0.05; *** p < 0.001 compared with the control condition (open bars) without any CD treatment.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Radioactivity, Control

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Effect of 10 μM T0901317 on cholesterol efflux to ApoA-I and HDL in ABAE (A) and SMCs (B) treated or nor with Rameβ . Cells were first labeled with [ 3 H]-cholesterol as previously described and incubated in the presence of 1 mM Rameβ, 10 μM T0901317 or both during 24 h. Then, [ 3 H]-cholesterol released in the medium from both cell types was measured 6 h after medium supplementation with either ApoA-I (20 μg/mL) or HDL (50 μg/mL). Data are expressed as the mean ± SEM (in DPM/μg of proteins, n = 6 from two sets of experiments). Control condition (without any treatment) is used as the reference level and set to 100%. Statistical analysis: a one-way ANOVA followed by Dunnett's test for multiple comparisons in which * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the untreated condition (Control condition, open bars) and in which ## p < 0.01 when Rameβ + T0901317 condition was compared with Rameβ condition.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Incubation, Control

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Antibodies, lectins and proteins, as well as their molecular weights, transfer time and incubate time.

Article Snippet: Rabbit anti-ceruloplasmin (CerP) antibody, rabbit anti-transferrin (TF) antibody and rabbit anti-Apolipoprotein A1 (ApoA1) antibody were purchased from Boster Corporation (Wuhan, China).

Techniques: Molecular Weight, High Molecular Weight

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Comparison of the re-probed ability of PVDF membrane and NC membrane. The pooled sera proteins (3.0, 1.5, 0.7, 0.3 and 0.1 μg) were separated by 8% SDS-PAGE, and transferred to PVDF membranes (up) and NC membrane (down), respectively. ( a ) Staining with AAL and then re-probed with PHA-E; ( b ) staining with ApoA1 and then re-probing with IgG; ( c ) Staining with PHA-E and then re-probing with A2M; ( d ) staining with A2M and then re-probing with PHA-E. Band intensities were statistically analyzed (n = 3 individual experiments) and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. Pink bar, PVDF membrane; Blue bar, NC membrane. Band intensities were analyzed *Significantly different p < 0.05, ** p < 0.01. N.S., not significant. All values are means ± S.E. (error bars).

Article Snippet: Rabbit anti-ceruloplasmin (CerP) antibody, rabbit anti-transferrin (TF) antibody and rabbit anti-Apolipoprotein A1 (ApoA1) antibody were purchased from Boster Corporation (Wuhan, China).

Techniques: Comparison, Membrane, SDS Page, Staining, Software

Journal: Cells

Article Title: Geniposide Ameliorated Dexamethasone-Induced Cholesterol Accumulation in Osteoblasts by Mediating the GLP-1R/ABCA1 Axis

doi: 10.3390/cells10123424

Figure Lengend Snippet: GEN ameliorated DEX-induced cholesterol accumulation by increasing ABCA1 expression. ( A , B ) The in vivo immunohistochemical examination of ABCA1 expression was conducted. ( C – E ) The protein expression of ABCA1 and ApoA-I was detected by western blot in DEX-treated MC3T3-E1 cells. ( F ) The level of the total intracellular cholesterol was measured using the ELISA kit in MC3T3-E1 cells co-treated with DEX and DIDS (200 μM). ( G – I ) The protein expression of ABCA1 and ApoA-I was detected by western blot in DEX/DIDS-treated MC3T3-E1 cells. ( J ) The ALP staining and the Alizarin Red S staining assays were conducted (×100 magnification). ( K – M ) The protein expression of RUNX2 and OPN was detected by western blot in DEX/DIDS-treated MC3T3-E1 cells. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. OIM, osteogenic induction medium. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN.

Article Snippet: After being blocked in TBS containing 5% skimmed milk for 1 h, the membranes were co-incubated with the primary antibodies at 4 °C overnight against RUNX2 (1:1000 dilution; MyBioSource, cat.no.127554, San Diego, CA, USA), OPN (1:1000 dilution; Proteintech, cat.no.22952-1-AP, Rosemont, IL, USA), GLP-1R (1:1000 dilution; ABGENT, cat.no.AP52040), ABCA1 (1:1000 dilution; Affinity, cat.no.DF8233), apoA-I (1:1000 dilution; Affinity, cat.no.DF6264), and GAPDH (1:1000 dilution; Affinity, cat.no.AF7021), and then with HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution; Boster Biological Technology, Wuhan, China).

Techniques: Expressing, In Vivo, Immunohistochemical staining, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Negative Control

Journal: Cells

Article Title: Geniposide Ameliorated Dexamethasone-Induced Cholesterol Accumulation in Osteoblasts by Mediating the GLP-1R/ABCA1 Axis

doi: 10.3390/cells10123424

Figure Lengend Snippet: GEN promoted ABCA1-mediated cholesterol metabolism in a GLP-1R-dependent manner. ( A , B ) The in vivo immunohistochemical examination of GLP-1R expression was conducted. ( C , D ) The protein expression of GLP-1R was determined by western blot in DEX-treated MC3T3-E1 cells. ( E ) The ALP staining and the Alizarin Red S staining assay were performed in EX-treated MC3T3-E1 cells (×100 magnification). ( F ) The level of the total intracellular cholesterol was measured using the ELISA kit. ( G – K ) The protein expression of GLP-1R, ABCA1, RUNX2, and OPN was detected by western blot. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN; EX, Exendin9-39.

Article Snippet: After being blocked in TBS containing 5% skimmed milk for 1 h, the membranes were co-incubated with the primary antibodies at 4 °C overnight against RUNX2 (1:1000 dilution; MyBioSource, cat.no.127554, San Diego, CA, USA), OPN (1:1000 dilution; Proteintech, cat.no.22952-1-AP, Rosemont, IL, USA), GLP-1R (1:1000 dilution; ABGENT, cat.no.AP52040), ABCA1 (1:1000 dilution; Affinity, cat.no.DF8233), apoA-I (1:1000 dilution; Affinity, cat.no.DF6264), and GAPDH (1:1000 dilution; Affinity, cat.no.AF7021), and then with HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution; Boster Biological Technology, Wuhan, China).

Techniques: In Vivo, Immunohistochemical staining, Expressing, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Negative Control