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: Scientific Reports

Article Title: Plasmid-mediated gene transfer of Cas9 induces vector-related but not Sp Cas9-related immune responses in human retinal pigment epithelial cells

doi: 10.1038/s41598-022-17269-x

Figure Lengend Snippet: Plasmid-mediated gene transfer of Cas9 induces HLA-ABC and CD54 upregulation in ARPE-19 cells. ARPE 19 cells were transfected with the Sp Cas9-encoding plasmids or the corresponding stuffer plasmids, or treated with the SpCas9 plasmid or LTX alone or left unstimulated. IFN-γ (1000 IU/ml) served as a positive stimulation control. At 24 h after transfection cells were analyzed by flow cytometry. Living ARPE-19 cells were gated as shown in Fig. C and analyzed for the expression of the surface markers HLA-ABC, HLA-DR and CD54. Surface marker expression following transfection with the Sp Cas9 plasmid or the stuffer plasmid ( A , B ) and the EGFP- Sp Cas9 plasmid or the EGFP-stuffer plasmid ( C , D ). ( A , C ) Surface marker expression presented as overlays of single-color histograms of log10 mean fluorescence intensity (MFI) obtained with fluorescence minus one (FMO) control (filled histograms) and specific antibodies against respective surface marker (open histograms). ( B , D ) Graphical analysis of the surface marker expression showing increases of HLA-ABC and CD54 after transfection with the Sp Cas9-encoding plasmids or the corresponding stuffer plasmids. Bars represent means + SD of pooled mean values from three independent experiments measured in duplicates. Data was analyzed by one-way ANOVA followed by Bonferroni’s comparison tests. Asterisks indicate differences in comparison to unstimulated ARPE-19 cells. Kruskal–Wallis-test followed by pairwise comparisons was used to analyze non-normally distributed data. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For flow cytometric characterization of cells, the following antibodies, peptides or reagents were used: 7-aminoactinomycin D (7-AAD), Anti-HLA-DR, APC-Cy™7 (1:1; AB_2868692, clone L243), APC Mouse Anti-Human HLA-ABC (1:100; AB_398603, clone G46-2.6), PE Mouse Anti-Human CD54 (1:100; AB_395901, clone HA58), Human BD Fc Block™ (1:40; AB_2869554) and Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (all BD Biosciences, Heidelberg, Germany).

Techniques: Plasmid Preparation, Transfection, Flow Cytometry, Expressing, Marker, Fluorescence