Journal: Journal of Lipid Research

Article Title: Naturally occurring variant of mouse apolipoprotein A-I alters the lipid and HDL association properties of the protein

doi: 10.1194/jlr.M021154

Figure Lengend Snippet: T7-ApoA-I binding to J774 macrophages with and without CPT-cAMP induction of ABCA1 expression. J774 macrophages were incubated 15 h with CPT-cAMP to induce ABCA1 expression. C57 or FVB recombinant T7-ApoA-I was then incubated with cells for 2 h and removed by PBS wash. A: T7-ApoA-I binding was measured by western blot of the T7-ApoA-I remaining associated with the cell protein as described in Materials and Methods. B: ABCA1 levels postincubation with 2 μg/ml C57 or FVB recombinant T7-ApoA-I as measured by ABCA1 Western blot of cell protein as described in Materials and Methods. Data points show the mean ± SD of triplicate samples.

Article Snippet: ABCA1 levels from the same samples were run on a 4–12% gradient SDS-PAGE, transferred to Immobilon, and probed with rabbit-anti-mouse ABCA1 antibody (Novus Biologicals, Littleton, CO). rHDL cholesterol esterification assay rHDL was generated from C57 or FVB T7-ApoA-I using phosphatidylcholine, T7-ApoA-I, 3 H-cholesterol, and nonlabeled cholesterol (molar ratio 200:1.2:1:1).

Techniques: Binding Assay, Expressing, Incubation, Recombinant, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dysregulated cholesterol uptake and efflux of bone marrow-derived α-SMA + macrophages contribute to atherosclerotic plaque formation

doi: 10.1007/s00018-025-05655-3

Figure Lengend Snippet: α-SMA promotes the lipid uptake and inhibits lipid efflux in macrophages. A , B Flow cytometric analysis of binding and uptake of DiI-Ox-LDL in vector- or Acta2 hi RAW264.7 cells A , and BMDMs from Acta2 f/f or Acta2 MKO mice B . The quantification results are shown on the right. n = 3. * P < 0.05, *** P < 0.001, **** P < 0.0001 by unpaired Student’s t-test. C , D SR-A, ABCA1, ABCG1 and α-SMA protein levels in vector- and Acta2 hi RAW264.7 cells C , and BMDMs from Acta2 Flox or Acta2 MKO mice D . n = 3. * P < 0.05 by unpaired Student’s t-test. E–F Co-staining and quantification of SR-A + MOMA-2 + E or ABCA1 + MOMA-2 + F cells in aortic roots from Acta2 f/f or Acta2 MKO mice treated with AAV -PCSK9 DY followed by a 12-week HFD. n = 7. * P < 0.05 by unpaired Student’s t-test. G–H Flow cytometric analysis of binding G and uptake H of DiI-Ox-LDL in Acta2 hi RAW264.7 cells treated with an SR-A blocking antibody (20 μg/mL) or IgG control for 2 h were detected by flow cytometry. n = 3. * P < 0.05 by unpaired Student’s t-test. AAV, adeno-associated virus; HFD, high-fat diet

Article Snippet: Primary antibodies used included mouse anti-mouse α-SMA (BM0002, Boster, USA), goat anti-mouse SR-A (af1797, R&D, USA), rabbit anti-mouse ABCA1 (PB0490, Boster, USA), human anti-mouse ABCG1 (bs-23382R, Bioss, China), rabbit anti-human CD36 (ab133625, Abcam, USA), mouse GAPDH (AF0006, Beyotime, China), rabbit anti-mouse p-Smad3 (AF1759, Beyotime, China), and rabbit anti-mammalian Smad3 (sc-101154, SANTA CRUZ, USA).

Techniques: Binding Assay, Plasmid Preparation, Staining, Blocking Assay, Control, Flow Cytometry, Virus

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dysregulated cholesterol uptake and efflux of bone marrow-derived α-SMA + macrophages contribute to atherosclerotic plaque formation

doi: 10.1007/s00018-025-05655-3

Figure Lengend Snippet: AKT pathway is involved in α-SMA-induced lipid accumulation. A Abca1 mRNA levels in vector or Acta2 hi RAW264.7 cells treated with 50 μg/ml Ox-LDL for 0, 6, 12, or 24 h. n = 3. * P < 0.05 by unpaired Student’s t-test. B Top 15 KEGG pathways of genes downregulated in Acta2 hi RAW264.7 cells incubated with Ox-LDL for 6 h. C Protein levels of p-AKT, AKT, p-ERK, ERK, p-STAT3, STAT3, CD36, SR-A, ABCA1 and α-SMA treated with 50 μg/ml Ox-LDL for 6 h. n = 3. * P < 0.05 by one-way ANOVA. D – F Flow cytometric analysis of binding ( D and E ) and uptake ( D and F ) of DiI-Ox-LDL in vector or Acta2 hi RAW264.7 cells incubated with or without SC79 (2 μg/mL). n = 3. * P < 0.05, ** P < 0.01, **** P < 0.0001 by one-way ANOVA. G – I Protein levels of CD36, SR-A, ABCA1 and α-SMA in Acta2 hi or vector RAW264.7 cells incubated with or without SC79 (2 μg/mL) and treated with 50 μg/ml Ox-LDL for 2 h. n = 3. * P < 0.05, ** P < 0.01, **** P < 0.0001 by one-way ANOVA

Article Snippet: Primary antibodies used included mouse anti-mouse α-SMA (BM0002, Boster, USA), goat anti-mouse SR-A (af1797, R&D, USA), rabbit anti-mouse ABCA1 (PB0490, Boster, USA), human anti-mouse ABCG1 (bs-23382R, Bioss, China), rabbit anti-human CD36 (ab133625, Abcam, USA), mouse GAPDH (AF0006, Beyotime, China), rabbit anti-mouse p-Smad3 (AF1759, Beyotime, China), and rabbit anti-mammalian Smad3 (sc-101154, SANTA CRUZ, USA).

Techniques: Plasmid Preparation, Incubation, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dysregulated cholesterol uptake and efflux of bone marrow-derived α-SMA + macrophages contribute to atherosclerotic plaque formation

doi: 10.1007/s00018-025-05655-3

Figure Lengend Snippet: Schematic diagram illustrating the role of bone marrow-derived α-SMA + macrophages in atherosclerotic plaque formation. Macrophage-expressed α-SMA enhances SR-A expression via the AKT signaling pathway, leading to increased lipid binding and uptake. Concurrently, α-SMA suppresses ABCA1 expression, thereby reducing lipid efflux. This imbalance promotes lipid accumulation and contributes to atherosclerotic plaque formation

Article Snippet: Primary antibodies used included mouse anti-mouse α-SMA (BM0002, Boster, USA), goat anti-mouse SR-A (af1797, R&D, USA), rabbit anti-mouse ABCA1 (PB0490, Boster, USA), human anti-mouse ABCG1 (bs-23382R, Bioss, China), rabbit anti-human CD36 (ab133625, Abcam, USA), mouse GAPDH (AF0006, Beyotime, China), rabbit anti-mouse p-Smad3 (AF1759, Beyotime, China), and rabbit anti-mammalian Smad3 (sc-101154, SANTA CRUZ, USA).

Techniques: Derivative Assay, Expressing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH

doi: 10.3389/fimmu.2022.999871

Figure Lengend Snippet: PAR-2 signalling reduces sensitivity to IFNγ by enhancing SOCS3 expression. (A). MCSF-derived BMM cultured for 3 hours with 100 µM PAR-2 agonist or maintained in MCSF (control) prior to the addition of escalating concentrations of IFNγ. Cells were then analysed by intracellular flow cytometry for iNOS expression. Data shows results from 3 (PAR-2) and 5 (control) separate groups of experiments. (B). Representative flow cytometry profiles of experiment described in Fig1A. (C). MCSF-derived BMM incubated with increasing concentrations of IFNγ (control), or primed beforehand with thrombin for 24 hours (Thrombin) or with thrombin and 100 µM PAR-2 agonist prior to IFNγ stimulation. Cells were then analysed by intracellular flow cytometry for iNOS expression. Data shows results from 3 experiments. The significance levels shown are in comparison to MCSF control at the same concentration of IFN. (D). CD206 (white bars) or iNOS (black bars) expression on F480+CD11b+ cells analysed by intracellular flow cytometry after 24 hours culture with increasing concentrations of PAR2 agonist. (E). qPCR data of MCSF-derived BMM treated for 24 hours with 100μM PAR-2 agonist (n=5) or maintained in MCSF (control) (n=4). (F). ABCA1 expression in MCSF-derived BMM incubated for 24 hours with escalating concentrations of PAR-2 agonist before analysis by flow cytometry. Data represents percentage change from control (MCSF) expression. Data from at least 3 separate experiments. (G, H). Relative SOCS3 expression by qPCR analysis of MCSF-derived BMM. In F, cells maintained in MCSF (n=10) for 24 hours (circle), or after 24 hours incubation with 100μM PAR-1 agonist (TFLLR-NH2) (n=6) (Triangle) or 100μM PAR-2 agonist (n=8) (Square). In H, cells maintained in MCSF for 24 hours (n=8) (white bar), or after 24 hours incubation with 100μM PAR-2 agonist +/- pre incubation for 24 hours with either 30pmol control siRNA (n=4) (blue bar) or 30pmol siRNA specific for Gαs (n=3) (purple bar). (I). MCSF-derived BMM incubated for 24 hours with 30pmol siRNA to SOCS3 (white bar) (n=3) or negative control siRNA (blue bar) (n=3), before a 3-hour incubation with 100µM PAR-2 agonist followed by 24-hour incubation with IFNγ (1ng/ml). iNOS expression compared to baseline and shown as % change in iNOS expression from MCSF treated cells as analysed by flow cytometry. (J). Relative JAK2 expression by qPCR analysis of MCSF-derived BMM maintained in MCSF (n=5) (white bar), or after 24 hours incubation with 100μM PAR-2 agonist (n=3) (blue bar). The PAR-2 agonist used in all experiments is 2-Furoyl-LIGRLO-amide. All PCR data expression calculated relative to TBP. Samples were compared using a Mann Whitney U test with two tailed p-values (Fig 1I+J), and Kruskal–Wallis one-way anova for multiple comparisons (Fig1E-H). Bar data represents mean +SD. *P <0.05 **P < 0.01 ***P < 0.001. ns, non significant.

Article Snippet: Surface antibodies were FITC – F4/80, APC- CD11b, PE-ABCA1 (Santa Cruz Biotechnology, Heidelberg, Germany) FITC-TF (Biorbyt, Cambridge, United Kingdom) or FITC-TF (American Diagnostica).

Techniques: Expressing, Derivative Assay, Cell Culture, Control, Flow Cytometry, Incubation, Comparison, Concentration Assay, Negative Control, MANN-WHITNEY, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH

doi: 10.3389/fimmu.2022.999871

Figure Lengend Snippet: PAR-2 signalling delivers a protective phenotype in oxazolone induced delated type hypersensitivity. (A). Schematic illustrating the experimental protocol. C57BL/6 (WT) mice were sensitised on the abdomen with oxazolone on day 0 and re-challenged on day 5 on one of the ears with oxazolone, and on the other ear with vehicle alone. Immediately prior to re-challenge, mice received either IP saline (n= 8) or 10microM/g PAR-2 agonist (n= 7). 24 and 48 hours following re-challenge (days 6 and 7), ear swelling was quantified using a micrometer. Data are presented as Δ mean ear thickness (swelling of oxazolone-treated ear minus that of vehicle treated ear). At least 5 measurements were taken per ear and averaged. (B, C). Δ mean ear thickness at 24HRS (day 6) (B) and 48HRS (day 7) (C) post re-challenge with oxazolone. (D–M). Immunofluorescence of frozen sections through oxazolone-treated ears. Bars represent means + SD for saline treated mice (white) or PAR-2 agonist-treated mice (blue). (D). Infiltration of CD68 + cells: % area of the section occupied by CD68 + cells. (E). Representative two colour IF sections through oxazolone-painted ears. Images show staining with CD68 (red) and DAPI (blue). (F). Number of granuloma per section at 100x magnification: Granuloma was classified as an aggregation of cells co-localisation of DAPI and CD68 at an area of outpouching from the epidermis. (G). % CD206 expression within granulomata. (H–K). Proportion of CD68 + cells co-expressing iNOS (H+I) or ABCA1 (J+K). I+K Representative three colour IF sections through oxazolone-painted ears. Images show staining with CD68 (red) iNOS (green -I), ABCA1 (green -K) and DAPI (blue). (L, M). Proportion of whole section SCOS3 expression. Bars represent means + SD for saline treated mice (white) or PAR-2 agonist-treated mice (blue) (L, M) . Representative two colour IF sections through oxazolone-painted ears. Images show staining with SOCS3 (green) and DAPI (blue). For representative IF images dotted lines demarcate the auricular cartilage (AC). E= epidermis. All Samples were compared using a Mann Whitney U test with two tailed p-values. Bar data represents mean +SD. *P < 0.05 **P < 0.01 ****P < 0.0001). ns, non significant.

Article Snippet: Surface antibodies were FITC – F4/80, APC- CD11b, PE-ABCA1 (Santa Cruz Biotechnology, Heidelberg, Germany) FITC-TF (Biorbyt, Cambridge, United Kingdom) or FITC-TF (American Diagnostica).

Techniques: Saline, Immunofluorescence, Staining, Expressing, MANN-WHITNEY, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH

doi: 10.3389/fimmu.2022.999871

Figure Lengend Snippet: 3-MP inhibits IFNγ signalling through both PAR-1 antagonism and PAR-2 agonism. (A) The impact of combination PAR-1 antagonism and PAR-2 agonism on the outcome of Ox-DTH. Immediately prior to re-challenge with oxazolone, C57BL/6 (WT) mice received either IP saline (n= 8) or 10microM/g IP FLLRN (PAR-1 antagonist (α-agonist) (n=8), 2-Furoyl-LIGRLO-amide (PAR- 2 agonist) (n=7), FLLRN combined with 2-Furoyl-LIGRLO-amide (n=3), 3-MP (n=6) or 3-MP + FSLLRY-Amide (PAR-2 antagonist) (n=5). Data represented as Δ mean ear thickness. (B) qPCR data for day 5 MCSF-derived BMM incubated for 24 hours with 100μM PAR-2 agonist or 100μM 3- MP or maintained in MCSF alone. Expression calculated relative to TBP. Data from 3 separate experiments. (C) Day 5 MCSF-derived BMM were cultured for 2 hours with escalating concentrations of 3-MP prior to thrombin stimulation for 24 hours. Control cells were incubated in thrombin alone (white bar). Cells were then analysed by flow cytometry for surface ABCA1 expression. Data from 3 separate experiments. Significance compared to control conditions (white bar). (D). qPCR for SOCS3 expression by control MCSF-derived BMM maintained in MCSF for 24 hours (black circle) (n=10), or after 24 hours incubation with 100μM PAR-2 agonist (blue square) (n=8) or 3-MP (purple triangle) (n=3) with or without a 2-hour pre incubation with PAR-2 antagonist (αAg) (FSLLRY-NH2) (pink diamond) (n=4). Expression calculated relative to TBP. (E) MCSF-derived BMM incubated with increasing concentrations of IFNγ alone (=‘MCSF’), or primed with thrombin for 24 hours (Thrombin) or with thrombin and 100 µM 2-Furoyl-LIGRLO-amide (Thrombin +PAR-2 agonist), 3-MP (Thrombin + 3-MP) or FLLRN (Thrombin + PAR-1 antagonist), prior to IFNv stimulation (thrombin and PAR-2 agonist remained in culture). Cells were then analysed by intracellular flow cytometry for iNOS expression. Data shows results from 3 experiments. (F–I) Experimental groups and n numbers as described in Figure 5A. Immunofluorescence of frozen sections through oxazolone-treated ears. Bars represent means + SD for saline treated mice (white), IP FLLRN (PAR-1 antagonist) (blue), 2-Furoyl-LIGRLO-amide (PAR- 2 agonist) (purple), 3-MP (pink) or 3-MP and FSLLRY-NH2 (3-MP + PAR-2 antagonist- αAg) (red). (F) % area of the section occupied by CD68 + cells. (G) Representative two colour IF sections through oxazolone-painted ears. Images show staining with CD68 (red) and DAPI (blue). (H–I) : % of CD68 + cells co-expressing iNOS (H) or CD206 (I) . For ES data at least 5 measurements were taken per ear and the results averaged. Dotted lines demarcate the auricular cartilage (AC). E= epidermis. Samples were compared using Kruskal–Wallis one-way anova for multiple comparisons (Fig 5A,C,D,F,H+I) or two way anova (Fig5B) Bar data represents mean +SD. * P< 0.05 **P < 0.01 ***P < 0.001 **** P < 0.0001.

Article Snippet: Surface antibodies were FITC – F4/80, APC- CD11b, PE-ABCA1 (Santa Cruz Biotechnology, Heidelberg, Germany) FITC-TF (Biorbyt, Cambridge, United Kingdom) or FITC-TF (American Diagnostica).

Techniques: Saline, Derivative Assay, Incubation, Expressing, Cell Culture, Control, Flow Cytometry, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH

doi: 10.3389/fimmu.2022.999871

Figure Lengend Snippet: PTL0GC-1 provides a protective benefit in the outcome of type IV hypersensitivity greater than PTL060. (A) Schematic illustrating the experimental protocol. C57BL/6 (WT) mice were sensitised on the abdomen with oxazolone on day 0 and re-challenged on day 5 on one of the ears with oxazolone, and on the other ear with vehicle alone. On day 3 and 5 mice received either IV saline (n=5), or 10microM/g IV PTL060 (n=7) or equimolar PTL0GC-1 (n=5) or 3-MP (n=5). 3 hours after injection on day 5 mice were then re-challenged with oxazolone (on the right) or vehicle alone (on the left). (B) Results are expressed as change in mean ear thickness between treated (right ear) and untreated (left ear) at 24 hours. At least 5 measurements were taken per ear and the results averaged. (C–L) Immunofluorescence of frozen sections through oxazolone-treated ears (groups as described in Fig 6A). Bars represent means + SD for saline (black circle), PTL060 (blue square), PTL0GC-1 (purple triangle) or 3-MP (pink diamond) treated mice. (C) % area of the section occupied by CD68 + cells. D. Representative two colour IF sections through oxazolone-painted ears. Images show staining with CD68 (red) and DAPI (blue). (E–H) % of CD68 + cells co-expressing iNOS (E) , CD206 (G) . Representative three colour IF sections through oxazolone-painted ears. Images show staining with CD68 (red) iNOS (green - F)/CD206 (green - H) and DAPI (blue). (I–K) % area of the section occupied by IL10 (I) , ABCA1 (J) or SOCS3 (K) . At least 3 sections per mouse were analysed. (L) Evaluating other routes of delivery of PTL0GC-1 or 3-MP. On day 3 and 5 oxazolone primed mice were treated with saline (n=4) or 7.5 µg/g PTL0GC-1 IV (n=5), IP (n=5), SC (at double dose) (n=6) or 3-MP or IV (n=5), IP (n=5) or SC (n=5) 3-MP (at the equivalent molarity of PTL0GC-1). 3 hours after injection on day 5 mice were then re-challenged with oxazolone (on the right) or vehicle alone (on the left). Results are expressed as change in mean ear thickness between treated (right ear) and untreated (left ear) at 24 hours. For ES data at least 5 measurements were taken per ear and the results averaged. Dotted lines demarcate the auricular cartilage (AC). E= epidermis. All samples where compared using Kruskal–Wallis one-way anova for multiple comparisons. Bar data represents mean +SD. *P <0.05 **P < 0.01 *** P < 0.001 ****P < 0.0001.

Article Snippet: Surface antibodies were FITC – F4/80, APC- CD11b, PE-ABCA1 (Santa Cruz Biotechnology, Heidelberg, Germany) FITC-TF (Biorbyt, Cambridge, United Kingdom) or FITC-TF (American Diagnostica).

Techniques: Saline, Injection, Immunofluorescence, Staining, Expressing