Journal: Frontiers in Cardiovascular Medicine

Article Title: Hematopoietic Cell-Specific SLC37A2 Deficiency Accelerates Atherosclerosis in LDL Receptor-Deficient Mice

doi: 10.3389/fcvm.2021.777098

Figure Lengend Snippet: SLC37A2 deficiency promotes oxidized LDL (OxLDL)-induced macrophage inflammation in vitro . (A) SLC37A2 protein expression analyzed by Western blotting in WT and Slc37a2 −/− bone marrow-derived macrophages (BMDMs) treated with 50 μg/ml oxLDL for 0-24 h. (B) Quantification of cellar total cholesterol (TC), free cholesterol (FC), and cholesterol ester (CE) in WT and Slc37a2 −/− BMDMs after treated with or without 25 or 50 μg/ml oxLDL for 24 h. (C) Relative transcript level of cytokines in WT and Slc37a2 −/− BMDMs stimulated with 50 μg/ml oxLDL for 0-24 h, measured by qPCR. Data are representative of two independent experiments with three samples per group (mean ± SEM). * p < 0.05; unpaired, two-tailed Student's t -test.

Article Snippet: To induce foam cell formation, BMDMs were treated with 25 or 50 μg/ml oxLDL (Athens Research & Technology) for 0-24 h. In some experiments, BMDMs were pretreated for 30 min with hexokinase inhibitor 2-deoxy-D-glucose (2-DG; 10 mM, Sigma-Aldrich), actin polymerization inhibitor cytochalasin D (10 μM, Cayman), or fatty acid β-oxidation inhibitor etomoxir (50 μM, Cayman) and subsequently treated with apoptotic cells for an additional 4 h in the presence of each inhibitor.

Techniques: In Vitro, Expressing, Western Blot, Derivative Assay, Two Tailed Test

Journal: Cardiovascular diabetology

Article Title: Klotho inhibits renal ox-LDL deposition via IGF-1R/RAC1/OLR1 signaling to ameliorate podocyte injury in diabetic kidney disease.

doi: 10.1186/s12933-023-02025-w

Figure Lengend Snippet: Fig. 6 Klotho regulated podocytic ox-LDL deposition via IGF-1R/RAC1/OLR1 signal axis. A IF analysis of the expression of Nephrin and RAC1 following administration of the inhibitor (PPP) and agonist (IGF2) of IGF-1R on KL+/− DKD and TgKL DKD, respectively. B Western blot analysis was conducted to determine the expression of RAC1, Podocin, and Cleaved Caspase3 following administration of the inhibitor (PPP) and agonist (IGF2) of IGF-1R on KL+/− DKD and TgKL DKD, respectively. C Representative western blot and summarized data showing the effects of administration of the inhibitor (PPP) and agonist (IGF2) of IGF-1R in the absence or presence of Klotho on the relative protein levels of OLR1, RAC1, Podocin, WT1 and Cleaved Caspase3. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: The antibodies used were listed below: anti-ox-LDL antibody (1:100, orb10973, Biorybt), anti-Podocin antibody (1:200, PA5-79757, Invitrogen), anti-Klotho antibody (1:100, PA5-88303, Invitrogen), anti-WT1 antibody (1:50, ab89901, Abcam), anti-PDGFRβ antibody (1:50, ab89901, Abcam), anti-GATA3 antibody (1:2400, ab199428, Abcam), anti-CXCL16 antibody (1:200, 60123- 1-Ig, Proteintech), anti-Parkin antibody (1:100, YT3593, ImmunoWay), anti-RAC1 antibody (1:20, AF385-SP, R&D), anti-Nephrin antibody (1:10, sc-376522, Santa Cruz), anti-OLR1 antibody (1:100, 11837-1-AP, Proteintech), anti-SYNPO antibody (1:200, 21064-AP, Proteintech), anti-IGF-1R antibody (1:10, sc-81464, Santa Cruz), goat polyclonal secondary antibody to mouse Alexa fluor 488 (1:400, ab150113, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 555 (1:400, ab150078, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 647 (1:400, ab150079, Abcam), goat antimouse Alexa fluor 568 (1:400, ab175473, Abcam), DAPI (1:1000, C1002, Beyotime).

Techniques: Expressing, Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The Scavenger Receptor CD68 Regulates Platelet Mediated Oxidized Low-Density Lipoprotein (oxLDL) Deposition in Atherosclerotic Vessels at an Early Stage of Atherosclerosis in LDLR -/- /ApoBec -/- Mice.

doi: 10.33594/000000048

Figure Lengend Snippet: Fig. 1. (A) Schematic representation of the domain structure of Fc-CD68 fusion proteins. IgK: Ig Kappa leader sequence, hinge region, Fragments crystalizable (Fc) from human IgG1 or IgG2 were fused with the extracellular domain of CD68 using different linkers. L: short linker (GGR), Hel. L: linker with supposed rigid, helical conformation, Flex. L: linker with supposed flexible structure. (B) Coomassie-stained SDS-PAGE and Western-Blot analysis of the purified Fc-CD68 fusion proteins. A 4-20% PAA gradient gel was used for all analysis. A: Coomassie staining. B: Western Blot with an anti human Fc antibody. C: Western-Blot with an anti CD68 antibody. (C) Binding ELISA with lipoproteins oxLDL, HDL and LDL (1 µg/well) immobilized on a 96-well Maxisorp plate. Dilution series of the different Fc-fusion proteins in the soluble phase were tested for binding. The Means ± SEM of 4 independent experiments. (D) Binding ELISA with immobilized oxLDL. A dilution series of oxLDL (0.004-3 µg/well) was immobilized on a 96-well Maxisorp plate and binding of the different Fc-CD68 fusion proteins was tested using a fixed concentration in the soluble phase (10 µg/ ml). The Means ± SEM of 4 independent experiments. (E) Binding ELISA with immobilized fusion proteins. A dilution series of the Fc-CD68 fusion proteins (0,004 - 3 µg/well) was immobilized. oxLDL (10 µg/ml) in the soluble phase was tested for binding. The Means ± SEM of 4 independent experiments are shown.

Article Snippet: OxLDL content of the serum samples was measured using the ox-LDL/MDA Addukt ELISA kit (Immundiagnostik, #k 7810) or ELISA Kit for oxLDL for mice (cloud-clone Corp, Caty, Tx, USA) according to manufacturer’s protocol.

Techniques: Sequencing, Staining, SDS Page, Western Blot, Purification, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Experimental and Therapeutic Medicine

Article Title: P2X7R antagonist protects against renal injury in mice with adriamycin nephropathy

doi: 10.3892/etm.2021.11084

Figure Lengend Snippet: Effect of A438079 on the expression of P2X7R, ox-LDL, CXCL16, Bax, caspase-3 and NLRP3 in the glomeruli. Expression of (A and G) P2X7R, (B and H) ox-LDL, (C and I) CXCL16, (D and J) Bax, (E and K) caspase-3 and (F and L) NLRP3 in glomeruli with different groups. ▲ P<0.01 vs. NC group; # P<0.05 and ## P<0.01 vs. ADR group. ADR, adriamycin; A100/200/300, A438079 100/200/300 µmol/kg; CXCL16, C-X-C motif chemokine ligand 16; NC, negative control; ox-LDL, oxidized low density lipoprotein.

Article Snippet: After blocking the sections with 10% goat serum (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at 37˚C, the sections were incubated with rabbit anti-mouse P2X7R antibody (pAb; 1:300; cat. no. ab109054; Abcam), C-X-C motif chemokine ligand 16 (CXCL16; 1:100; cat. no. bs-1441R; BIOSS), ox-LDL (1:100; cat. no. bsm-1698M; BIOSS), Bax (1:200; cat. no. bsm-52316R; BIOSS), caspase-3 (1:200; cat. no. bsm-33277M; BIOSS) and NLRP3 (1:200; cat. no. ab270449; Abcam) at 4˚C overnight, followed by incubation with HRP-conjugated secondary goat anti-rabbit (1:500; cat. no. ab6721; Abcam)/mouse (1:500; cat. no. ab6789; Abcam) antibody at 37˚C for 30 min.

Techniques: Expressing, Negative Control