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: Cardiovascular Drugs and Therapy

Article Title: Insulin Rescued MCP-1-Suppressed Cholesterol Efflux to Large HDL2 Particles via ABCA1, ABCG1, SR-BI and PI3K/Akt Activation in Adipocytes

doi: 10.1007/s10557-021-07166-2

Figure Lengend Snippet: MCP-1 inhibited the mRNA, total, and cell-surface protein expression of ABCA1, ABCG1, and SR-B1 in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were treated with either increasing concentrations of MCP-1 (0–80 ng/ml) for 48 h or with MCP-1 at 40 ng/ml for increasing times (0, 24, 48, 72 h). (a) Dose response of MCP-1 on ABCA1, ABCG1, and SR-B1 mRNA expression. (b) Time course of MCP-1 on ABCA1, ABCG1, and SR-B1 mRNA expression. Total proteins and cell surface proteins were extracted from the cultured cells, and the protein expressions of ABCA1, ABCG1, and SR-B1 were detected as described in “Materials and Methods.” The quantitative analysis of proteins was performed using Image-J software. (c) Dose response of MCP-1 on the total protein expression of ABCA1, ABCG1, and SR-B1. (d) Time course of MCP-1 on the total protein expression of ABCA1, ABCG1, and SR-B1. (e) Dose response and time course of MCP-1 on the cell surface protein expression of ABCA1, ABCG1, and SR-B1. (f) The effect of MCP-1 on the expression of ABCA1, ABCG1, and SR-B1 by confocal microscopy. Differentiated 3T3-L1 adipocytes grown on glass cover slips were serum-starved for 6 h, followed by incubation in serum-free medium in the absence or presence of MCP-1 (40 ng/ml) for 48 h. ABCA1, ABCG1, and SR-BI were labeled with Alexa 546 (red), Alexa 488 (green), and Alexa 633 (pink), respectively. The protein expression of ABCA1, ABCG1, and SR-BI was analyzed using confocal microscopy (LSM780) (×63), as described in “Materials and Methods.” ( (a–f) , n = 3). *P < 0.05 compared with untreated cells

Article Snippet: The adipocytes were incubated at 4 °C overnight with a 1:25 dilution of mouse anti-mouse ABCA1 antibody, a 1:200 dilution of rabbit anti-mouse ABCG1 antibody and a 1:50 dilution of goat anti-mouse SR-BI antibody (Novus Biologicals, CO, USA) in blocking buffer.

Techniques: Expressing, Cell Culture, Software, Confocal Microscopy, Incubation, Labeling

Journal: Cardiovascular Drugs and Therapy

Article Title: Insulin Rescued MCP-1-Suppressed Cholesterol Efflux to Large HDL2 Particles via ABCA1, ABCG1, SR-BI and PI3K/Akt Activation in Adipocytes

doi: 10.1007/s10557-021-07166-2

Figure Lengend Snippet: Insulin rescues MCP-1-mediated mRNA, total and surface protein suppression of ABCA1, ABCG1, and SR-B1 via PI3K/Akt activation in differentiated 3T3-L1 adipocytes. Cells were serum-starved for 6 h, followed by pretreatment with (+) or without (−) the PI3K inhibitor wortmannin for 45 min. The cells were then incubated with (+) or without (−) insulin (100 nM) and MCP-1 (40 ng/ml). (a–c) Insulin rescues MCP-1 mediated mRNA suppression of ABCA1, ABCG1and SR-B1. (d, e) Insulin rescues the total protein expression of the three receptors. (f) Insulin rescues the surface protein expression of the three receptors. Cell surface receptor levels and Na + /K + ATPases were directly extracted using cell surface biotinylation and then measured using western blotting, as described in “Materials and Methods.” (g) Insulin rescues the expression of the three receptors by confocal microscopy (×63). ABCA1, SR-BI, and ABCG1 labeled with Alexa 546 (red), Alexa 633 (pink), and Alexa 488 (green), respectively, were detected by confocal microscopy as describe in “Materials and Methods.” Note that insulin reversed the MCP-1-mediated decreases in ABCA1, ABCG1, and SR-BI mRNA and protein levels, and this correction was inhibited by wortmannin. ( (a–g) : n = 3). * P < 0.05 compared with untreated cells; # P < 0.05 compared with MCP-1- or insulin-treated cells; + P < 0.05 compared with MCP-1 or insulin-treated cells

Article Snippet: The adipocytes were incubated at 4 °C overnight with a 1:25 dilution of mouse anti-mouse ABCA1 antibody, a 1:200 dilution of rabbit anti-mouse ABCG1 antibody and a 1:50 dilution of goat anti-mouse SR-BI antibody (Novus Biologicals, CO, USA) in blocking buffer.

Techniques: Activation Assay, Incubation, Expressing, Cell Surface Receptor Assay, Western Blot, Confocal Microscopy, Labeling

Journal: Cardiovascular Drugs and Therapy

Article Title: Insulin Rescued MCP-1-Suppressed Cholesterol Efflux to Large HDL2 Particles via ABCA1, ABCG1, SR-BI and PI3K/Akt Activation in Adipocytes

doi: 10.1007/s10557-021-07166-2

Figure Lengend Snippet: Proposed model of the effect of Insulin on MCP-1-mediated suppression of cholesterol efflux to large HDL2 particles and regulatory mechanism in 3T3-L1 adipocytes. Increased MCP-1 level has a significant, negative correlation with HDL2-C in CAD patients with obesity and overweight. MCP-1 decreases the expression of ABCA1, ABCG1, and SR-B1, leading to the reduction of cholesterol efflux. Insulin rescues the expression of the three receptors via PI3K/Akt activation and restores the cholesterol efflux to HDL2. (+), Activation; (−), Inhibition

Article Snippet: The adipocytes were incubated at 4 °C overnight with a 1:25 dilution of mouse anti-mouse ABCA1 antibody, a 1:200 dilution of rabbit anti-mouse ABCG1 antibody and a 1:50 dilution of goat anti-mouse SR-BI antibody (Novus Biologicals, CO, USA) in blocking buffer.

Techniques: Expressing, Activation Assay, Inhibition

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Primers used for real-time qPCR and antibodies used for Western blot analysis

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques: Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Primary and secondary antibodies

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Trimethylamine (TMA) lyase inhibition alters the hepatic expression of key genes involved in sterol and bile acid metabolism. At 8 wk of age, wild-type C57BL/6J male mice were switched from standard rodent chow to 1 of 4 experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol with or without the microbe-targeted TMA lyase inhibitor iodomethylcholine (IMC; 0.06%, wt/wt). After 4 wk on these diets, liver RNA was extracted and gene expression was measured using quantitative real-time PCR. A: relative mRNA expression of sterol regulatory-binding protein 2 (Srebp2), HMG-CoA reductase (HMG CoA-red), HMG-CoA synthase (HMG CoA-syn), squalene synthase (Squalene-syn), ATP-binding cassette transporters (Abca1, Abcg5, and Abcg8), bile acid modifying cytochrome-P450 family members (Cyp7a1, Cyp8b1, and Cyp27a1), bile salt export pump (Bsep), and small heterodimeric partner (Shp) were quantified using the ΔΔCT method. B and C: Western blot analysis HMG-CoA reductase and Cyp7a1 with densitometric quantification. Data shown in A represent the means ± SE for n = 5 mice per group, whereas data in B and C represent the means ± SE for n = 3 mice per group; *significantly different than the nondrug-treated mice within each diet group. AU, arbitrary units.

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Trimethylamine (TMA) lyase inhibition alters the host intestinal expression of key genes involved in sterol and bile acid transport and metabolism. At 8 wk of age, wild-type C57BL/6J male mice were switched from standard rodent chow to 1 of 4 experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol with or without the microbe-targeted TMA lyase inhibitor iodomethylcholine (IMC; 0.06%, wt/wt). After 4 wk on these diets, RNA was extracted from either the jejunum (A) or ileum (B), and gene expression was measured using quantitative real-time PCR. The mRNA abundance of HMG-CoA reductase (HMG Reduc.), HMG-CoA synthase (HMG Synth.), ATP-binding cassette transporters (Abca1, Abcg5, and Abcg8), Niemann-pick C1-like 1 (Npc1l1), farnesoid X receptor (Fxr), apical sodium bile acid transporter (Asbt), organic solute transporter α and β (Ostα and Ostβ), fibroblast growth factor 15 (Fgf15), and ileal bile acid-binding protein (I-babp) were quantified using the ΔΔCT method. C: Western blot analysis of Abca1, Npc1l1, and Gapdh with densitometric quantification. Data shown in A represent the means ± SE for n = 5 mice per group, whereas data in B and C represent the means ± SE for n = 3 mice per group; *significantly different than the nondrug-treated mice within each diet group.

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: Nature Communications

Article Title: ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

doi: 10.1038/ncomms12313

Figure Lengend Snippet: ( a ) Heat map representation of gene expression from microarray data comparing mouse peritoneal macrophages incubated with or without Ac-LDL (120 μg ml −1 ) for 24 h. Left panel shows upregulated genes ( P <0.05, unpaired t -test; fold change≥1.4) and right panel shows downregulated genes ( P <0.05, unpaired t -test; fold change≥2.08) upon Ac-LDL treatment compared with non-treated cells. Samples were analysed in triplicate. ( b ) qRT-PCR validation of selected genes upregulated in the microarray in mouse peritoneal macrophages treated with or without Ac-LDL for 24 h. qRT-PCR analysis of Angptl4 expression levels in macrophages ( c , left) and whole aorta ( c , right) from WT and Ldlr −/− mice fed a WD (left) ( n =4 per group), and in human peripheral blood mononuclear cells ( d ) treated with or without Ac-LDL (120 μg ml −1 ) for 24 h. Abca1 and Hmgcr genes were used as control genes for cholesterol loading and Cd68 was used as a marker for macrophages. All data represent the mean±s.e.m. and are representative of three experiments in duplicate; * P ≤0.05 compared with untreated macrophages ( b , d ) and WT mice ( c ) by unpaired t -test. ( e ) Immunostaining of ANGPTL4 (red) and macrophage marker CD68 (green) and their co-localization in atherosclerotic plaques of Ldlr −/− mice fed a WD. Scale bar, 400 μm. ( f ) Immunohistochemistry staining of ANGPTL4 and CD68 in human atherosclerotic plaques. Scale bar, 200 μm. ( g ) Comparison of ANGPTL4 mRNA expression in NCP and corresponding culprit (CP) human atherosclerotic plaques ( n =9). ( h ) Representative western blot showing comparison of ANGPTL4 expression in NCP and corresponding CP. Lower panel shows densitometry analysis for the 50 kDa bands of the western blots ( n =7). * P ≤0.05 compared with NCP by unpaired t -test; a.u. arbitrary units. Full scans of westerns blots are provided in .

Article Snippet: Following overnight transfer of the proteins onto nitrocellulose membranes, membranes were probed using the following antibodies (catalogue numbers and dilutions used are indicated within parentheses): rabbit polyclonal antibodies against ANGPTL4 (#40-9800; 1:100), ABCG1 (#NB400-132; 1:1000), COX-2 (#160106; 1:1000) and iNOS (#2982; 1:1000) were obtained from Invitrogen, Novus, Cayman and Cell Signaling Technology respectively; a mouse monoclonal antibody against ABCA1 (clone AB.H10; # ab18180, 1:1000) and a rabbit polyclonal antibody against CD36 (#ab124515; 1:1000) were obtained from Abcam; a goat polyclonal antibody against SR-A (#sc-20444; 1:1000) was from Santa Cruz and mouse monoclonal antibodies against HSP-90 (clone 68/HSP90; #610419, 1:1000) and GAPDH (clone GT239; #GTX627408, 1:5000) were purchased from BD Biosciences and GeneTex, respectively.

Techniques: Expressing, Microarray, Incubation, Quantitative RT-PCR, Marker, Immunostaining, Immunohistochemistry, Staining, Western Blot

Journal: Nature Communications

Article Title: ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

doi: 10.1038/ncomms12313

Figure Lengend Snippet: ( a ) Representative pictures from WT and Angptl4 −/− mouse peritoneal macrophages incubated with or without Ac- LDL (120 μg ml −1 ) for 24 h and stained with BODIPY 493/503 (1 μg ml −1 ) and DAPI (Green and blue, respectively). Scale bar, 5 μm. Quantification of the mean average intensity is in the right panel. ( b ) Total cholesterol content in peritoneal macrophages isolated from WT and Angptl4 −/− mice incubated with or without Ac-LDL (120 μg ml −1 ) for 24 h. ( c ) Flow cytometry analysis of DiI-Ox-LDL binding in peritoneal macrophages incubated with DiI-Ox-LDL (30 μg cholesterol per ml) for 30 min at 4 °C. At the end of the incubation period, cells were washed and incubated in RPMI 10% FBS media for 15 min at 37 °C to allow the internalization. ( d ) Flow cytometry analysis of DiI-Ox-LDL uptake in peritoneal macrophages incubated with DiI-Ox-LDL (30 μg cholesterol per ml) for 2 h at 37 °C. The results are expressed in terms of specific MFI after subtracting auto-fluorescence of cells incubated in the absence of DiI-Ox-LDL. ( e ) Cholesterol efflux to apolipoprotein A1 (ApoA1) in peritoneal macrophages isolated from WT and Angptl4 −/− mice stimulated with or without T0901317 (T090). ( f ) Western blot analysis of indicated proteins in peritoneal macrophages from WT and Angptl4 −/− mice incubated with or without Ac-LDL (120 μg ml −1 ) for 24 h. ( g ) Western blot analysis (representative of three blots) of ABCA1 expression in WT and Angptl4 −/− peritoneal macrophages incubated with Ac-LDL for 24 h. Surface ABCA1 was isolated using biotinylation followed by incubation with neutravidin. HSP90 is used as loading control (f and g). Full scans of westerns blots are provided in . ( h ) Representative confocal images of mouse peritoneal macrophages from WT and Angptl4 −/− mice incubated with Ac-LDL for 24 h and stained with cholera toxin B (CTxB), ABCA1 and DAPI. Quantification of co-localization of CTxB and ABCA1 is on the right panel. Scale bar, 10 μm. ( i ) Representative images of WT and Angptl4 −/− macrophages cultured on coverslips and treated with or without Ac-LDL (120 μg ml −1 ) in combination with ACAT inhibitor (58035) for 24 h to induce lipid-loading-induced apoptosis (scale bars, 200 μm). Apoptosis was detected using Annexin-V staining. Right panel shows the quantification of percentage of apoptotic cells from four random fields from each cover slip. All data represent the mean±s.e.m. from at least three experiments in duplicate; * P <0.05 compared with WT macrophages by unpaired t -test. MFI, median intensity of fluorescence.

Article Snippet: Following overnight transfer of the proteins onto nitrocellulose membranes, membranes were probed using the following antibodies (catalogue numbers and dilutions used are indicated within parentheses): rabbit polyclonal antibodies against ANGPTL4 (#40-9800; 1:100), ABCG1 (#NB400-132; 1:1000), COX-2 (#160106; 1:1000) and iNOS (#2982; 1:1000) were obtained from Invitrogen, Novus, Cayman and Cell Signaling Technology respectively; a mouse monoclonal antibody against ABCA1 (clone AB.H10; # ab18180, 1:1000) and a rabbit polyclonal antibody against CD36 (#ab124515; 1:1000) were obtained from Abcam; a goat polyclonal antibody against SR-A (#sc-20444; 1:1000) was from Santa Cruz and mouse monoclonal antibodies against HSP-90 (clone 68/HSP90; #610419, 1:1000) and GAPDH (clone GT239; #GTX627408, 1:5000) were purchased from BD Biosciences and GeneTex, respectively.

Techniques: Incubation, Staining, Isolation, Flow Cytometry, Binding Assay, Fluorescence, Western Blot, Expressing, Cell Culture

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