Journal: International Journal of Immunopathology and Pharmacology

Article Title: Targeted chelation therapy decreases NLRP3 expression by vascular cells and acts as senomorphic in chronic kidney disorder induced vascular calcification

doi: 10.1177/03946320251391142

Figure Lengend Snippet: (a) EDTA is Senomorphic and not Senolytic—Immortalized human vascular smooth muscle cells cultured under high phosphate conditions for 3 days and were treated with either EDTA or ABT 263 (senolytic agent) for 24 h. IHC-based Expression level analysis of Caspase 3 was used as a marker for senolytic (inducing selective apoptosis of senescent cells) activity. We observed that in comparison to ABT 263, EDTA treatment significantly brought down Caspase 3 activity, indicating that EDTA does not induce apoptosis. Experiment performed in triplicate, repeated four times, results represented as Mean ± S.D and (b) EDTA but not ABT263 Decreases NLRP3 expression—Immortalized human vascular smooth muscle cells cultured under high phosphate conditions for 3 days and were treated with either EDTA or ABT 263 (senolytic agent) for 24 h. IHC-based Expression level analysis of NLRP3 reveals that EDTA treatment and not ABT 263 decreased NLRP3 activity. Justifying the anti-apoptotic and senomorphic nature of EDTA treatment. Experiment performed in triplicate, repeated four times, results represented as Mean ± S.D.

Article Snippet: 130127, Madison, WI), EDTA (Sigma, E6511), vitamin D3 (VitD3, Cholecalciferol, Sigma, #C9756-5G), NaCl (sigma, S3014), KCl (Sigma P9541), NaHCO3 (Sigma, S5761), glucose (Sigma, G7021), NaH2PO4 (Sigma, S0876), dPBS (Gibco, 10010-031), DMEM (Gibco-1885-076), FBS, Pen-Sterp, Human Aortic Smooth Muscle Cells (SMCs) (Promocell, C12533 ), SMC Medium II (Promocell 22062), ABT263, HSA (Evonik Birmingham Laboratory, 777HSA097), HEPES (Sigma, H3375), absolute ethanol (Fisher BP 2818500), mPEGNHS, MW 2000 (BOC Sciences), Traut’s reagent (G-Biosciences, Saint Louis, MO, BC95), 5-Dodecanoylaminofluorescein di-β-D-Galactopyranoside (C12FDG, MedChem Express HY-126839), Cell Staining buffer (BioLegend 00-4222-26), anti-CD16/32 mouse monoclonal antibody (BioLegend Cat#156604), anti-CD45-APC (eBiosciences Cat#17-0461-82) DAPI (BioLegend Cat#422801), Rabbit anti-Rat Caspase-3 (Cell Signaling Technology, C8487), NLRP3 (Novus Biologicals, NBP2-12446), and PiT-1 (Santa Cruz Biotechnology, SC98824), anti-rabbit IgG secondary antibody Cy5 (Invitrogen, A10523) cDNA synthesis Kit (Bio-Rad, Cat# 1725034), iTaq—Universal SYBR Green Super mix 12 reagent (Bio-Rad, Cat# 172512), SA-βGal Staining Kit (Cell Signaling, 9860), Rat IL-6 ELISA Kit (RnD SYSTEMS DY-506), Rat IL-1β ELISA Kit (RnD SYSTEMS SRLB00), T-Per protein extraction buffer (ThermoFischer, 78510), BCA assay kit (Thermo Fisher Scientific, A55865).

Techniques: Cell Culture, Expressing, Marker, Activity Assay, Comparison

Journal: Scientific Reports

Article Title: Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation

doi: 10.1038/srep15720

Figure Lengend Snippet: Levels of striatal dopamine and 5-HT and their metabolites 9 days after treatment with MPTP.

Article Snippet: Muscimol hydrobromide (1 mg/kg, G019, Sigma-Aldrich), baclofen (20 mg/kg, B5399, Sigma-Aldrich), and tiagabine hydrochloride (5 mg/kg, T436400, Toronto Research Chemicals) were intraperitoneally injected 1 h before the first MPTP injection.

Techniques:

Journal: Scientific Reports

Article Title: Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation

doi: 10.1038/srep15720

Figure Lengend Snippet: ( A ) Immunohistochemical staining showing TH-ir cells in the substantia nigra (scale bar: 0.2 mm). Stereological counting of TH-ir cells (n = 3–4) and Nissl-positive neurons (n = 3–4) in both sides in the control, LPS and LPS+ tiagabine groups is shown in the right panel. All data are presented as the mean ± SEM. * p < 0.05 and * * p < 0.01. ( B ) Immunofluorescence staining for TH (red) and Iba-1 (green) in the SN (scale bar: 0.1 mm). Stereological counting of Iba-1-positive cells in both sides in the control, LPS and LPS+ tiagabine groups is shown in the right panel. All data are presented as the mean ± SEM. * * p < 0.01 (n = 3–4).

Article Snippet: Muscimol hydrobromide (1 mg/kg, G019, Sigma-Aldrich), baclofen (20 mg/kg, B5399, Sigma-Aldrich), and tiagabine hydrochloride (5 mg/kg, T436400, Toronto Research Chemicals) were intraperitoneally injected 1 h before the first MPTP injection.

Techniques: Immunohistochemical staining, Staining, Control, Immunofluorescence

Journal: Scientific Reports

Article Title: Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation

doi: 10.1038/srep15720

Figure Lengend Snippet: In the left panel, MPP + (the toxic metabolite of MPTP) causes immediate injury to striatal TH activity and TH + fibers and the activation of striatal microglial cells. The damage to TH + neurons and microglial activation in the SN are observed later. Tiagabine inhibits microglial activation and consequently protects dopaminergic neurons. In the right panel, LPS directly stimulates microglia and induces an inflammatory reaction. Tiagabine inhibits this processes and thereby protects against dopaminergic neuron loss.

Article Snippet: Muscimol hydrobromide (1 mg/kg, G019, Sigma-Aldrich), baclofen (20 mg/kg, B5399, Sigma-Aldrich), and tiagabine hydrochloride (5 mg/kg, T436400, Toronto Research Chemicals) were intraperitoneally injected 1 h before the first MPTP injection.

Techniques: Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a-c , NINJ1 oligomerization (Native-PAGE) and LDH-release from WT and NINJ1 KO iPSDMs pretreated with glycine or inhibitors as indicated and infected with Mtb mc²6206, MOI 20 for 4h ( a, b ), Mtb H37Rv, 7.5 MOI for 24h ( c ), or treated with cell death stimuli for 4h: d , pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950; e , apoptosis – venetoclax; f , necroptosis – Z-VAD-FMK+BV-6+TNFa (zBT) w/wo RIPK3 inhibitor GSK’872; g , ferroptosis – RSL-3 w/wo Ferrostatin-1. Data are means (bars) ± s.e.m. of at least three independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test of treatments within each cell line and stimuli between cell lines. For gel source data, see Supplementary Figure 1.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Clear Native PAGE, Infection

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, Lysates of WT, NINJ1 KO and GSDMD KO iPSC-derived monocytes (m) and macrophages (M) were subjected to SDS-PAGE and immunoblotted for NINJ1, GSDMD and β-actin. A3 and C6 are NINJ1 KO clones; A7, B8 are GSDMD KO clones. b, Human monocyte-derived macrophages were transfected with siRNA targeting NINJ1 or a non-targeted control and infected with Mtb mc²6206, MOI 20, for 4h. Shown is LDH-release and NINJ1 expression level by qPCR. Data are presented as mean ± s.e.m of four donors (shown with individual symbols). *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with uncorrected Fisher’s LSD. c-j, LDH-release from iPSDM WTs and NINJ1 KO clones A3, C6 ( c-f ) or GSDMD KO clones A7, B8 ( g-j ) pretreated with glycine or inhibitors as indicated and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( c, g ); apoptosis – venetoclax ( d, h ); necroptosis – Z-VAD-FMK+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( e, i ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( f, j ). ( c-f ) Data are means (bars) ± s.e.m. of three (A3) or two (C6) independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test. ( g-j ) Data are means (bars) ± s.d. of two or three technical replicates (circles) from 1 independent experiment.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Derivative Assay, SDS Page, Clone Assay, Transfection, Control, Infection, Expressing

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a-d , Cytokine release from WT iPSDMs pretreated with glycine or inhibitors as indicated for 30 min and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( a ); apoptosis – venetoclax ( b ); necroptosis – zVAD+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( c ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( d ). Cytokine levels were assessed by multiplex ELISA. Data are means (bars) of two individual experiments (circles), each with two replicates.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a , Representative images of NINJ1 KO THP-1 cells expressing hNINJ1-mNG (THP1-NINJ1-mNeonGreen) pretreated with glycine ( a , right column) before cell death stimuli venetoclax (apoptosis), LPS + nigericin (LPS+N, pyroptosis), RSL-3 (ferroptosis) for 4h, or infection with Mtb mc26206-BFP for 20h. Time-lapse TIRF (mNG), widefield (DRAQ7 and BFP) and brightfield microscopy images of NINJ1-mNG (green), DRAQ7 (red) and Mtb (blue), and morphology, respectively. Scale bars 10 mm. b , Quantification of NINJ1-mNG puncta densities at 4h (LPS+N, RSL-3) or 20h (Mtb) as represented in ( a ). Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c , Representative time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 uptake (red) upon Mtb mc26206-BFP infection (blue) temporally aligned by start of NINJ1 clustering (t=0). Time in min. Scale bars 10 mm. d, e , Fitted curves ( d ) and t50 values (x value where y=50% max.) of sigmoidal fits ( e ) of NINJ1 oligomerization kinetics during Mtb-infection, apoptosis (venetoclax), pyroptosis (LPS+N) or ferroptosis (RSL-3) monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) or 5 min (Mtb-infection). 3-5 cells analyzed per condition. Individual kinetic curves and representative images for RCD induction are shown in Extended Data Figure 3.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Expressing, Infection, Microscopy

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, Quantification of NINJ1-mNG puncta densities monitored by TIRF microscopy ( a ) and phagocytosed bacteria monitored by intracellular BFP intensities ( b ) before NINJ1 inhibition by glycine and after 4h of RCD induction (Venetoclax, LPS+N, RSL-3) or after 3h and 20h of Mtb mc²6206-BFP infection. Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c,d, Kinetics of NINJ1 puncta densities and DRAQ7 intensities monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) with (c) representative Time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 (WF, red) aligned by start of NINJ1 clustering. Time in sec. Scale bars 10 μm. Intensity values were normalized to the highest value for each cell and aligned by the start of NINJ1 oligomerization. Data are means ± s.d. for 3-5 cells per condition with sigmoidal fits for NINJ1-mNG puncta density increase (green) and mono-exponential increase for DRAQ7 intensities (red).

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Microscopy, Bacteria, Inhibition, Infection

Journal: World Journal of Oncology

Article Title: Mitochondria of T Lymphocytes Promote Anti-Pulmonary Tumor Immune Response

doi: 10.14740/wjon1841

Figure Lengend Snippet: Treatment of BH3-mimetic drugs in HCC1195 and HCC1438 cells. (a-c) Cytosol and mitochondrial proteins were extracted from HCC1195, HCC1438, CD3 + T, pulmonary fibroblast cells, and expression of Bcl-2 or Bcl-xL was determined using western blotting. Beta-tubulin was used as a positive marker for the cytosol, while COX IV was used as a positive marker for the mitochondrial fraction. (d) Cell death was measured following treatment with 3 nM ABT199, 19 nM A115463, or 15 µM ABT737 for 1 h, or incubation with 200 pg/µL granzyme B and 100 pg/µL perforin for 2 h. (e, f) The cells were fixed up to 5 h, and apoptosis were determined using ELISA kit. (g) Structures of ABT199 and ABT199-BODIPY. (h) Following treatment of synthetic ABT199-BODIPY, refractive index images of HCC1195 were enlarged at 28, 30, or 32 min, and ABT199-BODIPY were colored in green. (i) Quantified mean fluorescent intensity of the BODIPY signal in cells was measured at 512 nm. Results are the means ± standard error (SE) of six experiments in each group. *Significantly different from cytosol fraction, P < 0.05. # Significantly different from treatment of ABT199-BODIPY at 0 min, P < 0.05. COX IV: cytochrome c oxidase subunit IV; BH3: Bcl-2 homology 3; Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma extra-large; ELISA: enzyme-linked immunoassay; BODIPY: boron dipyrromethene.

Article Snippet: The cells were treated with ABT199 (3 nM, 6960, Tocris, UK), A115463 (19 nM, S7800, Selleck Chemicals, USA), or ABT737 (15 µM, 6835, Tocris, UK) for 1 h. Apoptosis was induced following combinations of recombinant perforin (100 pg/µL, ENZ-PRT313-0010, Enzo Life Science, USA) and granzyme B (200 pg/µL, 10345-H08H, Sinobiological, China).

Techniques: Expressing, Western Blot, Marker, Incubation, Enzyme-linked Immunosorbent Assay, Refractive Index

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: Mass detector parameters used for the analysis of vitamin C, lopinavir, ketoprofen, and valsartan (IS).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques:

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: In vitro hairless mouse skin permeation profiles of lopinavir after the application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) on the mouse skin fixed in the diffusion cells (A) and the arterial plasma concentration versus time profiles of lopinavir after the transdermal application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) in rats (B). Bullet symbols and their error bars represent the means and standard deviations ( n = 3–4). The asterisk (*) represents a value of the RED group significantly different from that of the control group ( p < .05).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: In Vitro, Control, Diffusion-based Assay, Clinical Proteomics, Concentration Assay

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: In vitro skin permeation parameters of lopinavir after the application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) on the hairless mouse skin fixed in the diffusion cells ( n = 3–4).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: In Vitro, Control, Diffusion-based Assay

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: Pharmacokinetic parameters of lopinavir after the transdermal application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) in rats ( n = 3–4).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: Control

Journal: Cell Death Discovery

Article Title: 5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

doi: 10.1038/s41420-023-01576-x

Figure Lengend Snippet: A MK2-deficient MEFs transduced with MK2 expression or control vector were treated with different doses of 5-ITu in the presence or absence of TNF (10 ng/ml) for 6 h. B Cells of the indicated genotype were treated with 10 µM 5-ITu for 6 and 24 h and cell viability was quantified. C MK2-deficient MEFs transduced with MK2 expression or control vector were treated with indicated small molecules (5 µM ABT-702, 100 nM gemcitabine, 5 µM etoposid, 5 µM doxorubicin and 5 µM staurosporine) in the presence or absence of TNF for 6 h and cell viability was assessed. D MK2-deficient MEFs transduced with MK2 expression or control vector were treated as indicated and viability was quantified after 6 h treatment. E , F RAW264.7 cells were treated as indicated to monitor the effect of 5-ITu in LPS-induced necroptosis ( E ) and SM-mediated autocrine TNF-dependent death ( F ). Average values of n = 3 independent wells are plotted ± SD (** p ≤ 0.001, *** p ≤ 0.0001, **** p ≤ 0.0001).

Article Snippet: The following reagents were used for cell treatments at given concentrations: LPS ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml −1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 μM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 μM), Staurosporine (#81590, Cayman, 5 μM), Etoposide (#E1383, Sigma, 5 μM), Doxorubicin (#15007, Cayman, 5 μM), Gemcitabine (#S1714, Selleckchem, 100 nM).

Techniques: Transduction, Expressing, Control, Plasmid Preparation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Ablation of Myeloid ADK (Adenosine Kinase) Epigenetically Suppresses Atherosclerosis in ApoE −/− (Apolipoprotein E Deficient) Mice

doi: 10.1161/atvbaha.118.311806

Figure Lengend Snippet: Figure 3. ADK (adenosine kinase) knockdown decreases ox-LDL (oxidized low-density lipoprotein)–induced foam cell formation in vitro. A and B, Oil Red O staining of ox-LDL–induced foam cells in bone marrow-derived macrophage (BMDMs) from ADKWT and ADKMAC-KO mice incubated with ox-LDL (100 μg/mL) for 24 h and quantitative data. **P<0.01, n=6. C, Total cholesterol content of BMDMs from ADKWT and ADKMAC-KO mice incubated with ox-LDL (100 μg/mL) for 24 h. ***P<0.001, n=6–8. D and E, Oil Red O staining of ox-LDL–induced foam cells in BMDMs pretreated with dimethyl sulfoxide (DMSO) or ABT702 (20 μM) after incubation with ox-LDL (100 μg/mL) for 24 h and quantitative data. ***P<0.001, n=6. F, Total cholesterol content of BMDMs pretreated with DMSO or ABT702 (20 μM) after incubation with ox-LDL (100 μg/mL) for 24 h. ***P<0.001, n=6. Data in bar graphs are expressed as mean±SEM. Statistical significance was determined by unpaired Student t test (for B and E) and 1-way ANOVA with Bonferroni post hoc tests (for C and F).

Article Snippet: In some experiments, 50 or 100 μg/mL ox-LDL (oxidized low-density lipoprotein; YB-002; Yiyuan Biotech, Guangzhou, Guangdong, China), 20 μM ABT702 (2372; Tocris Bioscience, Bristol, United Kingdom), 5 μM ZM241385 (1036; Tocris Bioscience, Bristol, United Kingdom), 2 μM MRS1754 (2752; Tocris Bioscience, Bristol, United Kingdom), 2 μM 5-Aza-2′deoxycytidine (5′-Aza-dc; A3656; Sigma-Aldrich, Saint Louis, MO), or 20 μM adenosine periodate oxidized (A7154; Sigma-Aldrich, Saint Louis, MO) was added to the culture medium to treat macrophages.

Techniques: Knockdown, In Vitro, Staining, Derivative Assay, Incubation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Ablation of Myeloid ADK (Adenosine Kinase) Epigenetically Suppresses Atherosclerosis in ApoE −/− (Apolipoprotein E Deficient) Mice

doi: 10.1161/atvbaha.118.311806

Figure Lengend Snippet: Figure 4. The repressed foam cell formation in ADK (adenosine kinase)-deficient macrophages is adenosine receptor independent. A, Western blot anal- ysis and densitometric quantification of ADK protein level in bone marrow-derived macrophage (BMDMs) from ADKWT and ADKMAC-KO mice. ***P<0.001, n=6. B, HPLC (high-performance liquid chromatography) analysis of intracellular adenosine in BMDMs from ADKWT and ADKMAC-KO mice. **P<0.01, n=6. C, HPLC analysis of intracellular S-adenosyl homocysteine (SAH) in BMDMs from ADKWT and ADKMAC-KO mice. *P<0.05, n=6. D, Oil Red O staining of BMDMs pretreated with dimethyl sulfoxide (DMSO), ABT702 (20 μM), ABT702 (20 μM) and ZM241385 (5 μM), ABT702 (20 μM) and MRS1754 (2 μM), ABT702 (20 μM) and ZM241385 (5 μM), and MRS1754 (2 μM) after incubation with ox-LDL (oxidized low-density lipoprotein; 100 μg/mL) for 24 h and quantitative data. ***P<0.001, n=6. Data in bar graphs are expressed as mean±SEM. Statistical significance was determined by unpaired Student t test (for A–C) and 1-way ANOVA with Bonferroni post hoc tests (for D).

Article Snippet: In some experiments, 50 or 100 μg/mL ox-LDL (oxidized low-density lipoprotein; YB-002; Yiyuan Biotech, Guangzhou, Guangdong, China), 20 μM ABT702 (2372; Tocris Bioscience, Bristol, United Kingdom), 5 μM ZM241385 (1036; Tocris Bioscience, Bristol, United Kingdom), 2 μM MRS1754 (2752; Tocris Bioscience, Bristol, United Kingdom), 2 μM 5-Aza-2′deoxycytidine (5′-Aza-dc; A3656; Sigma-Aldrich, Saint Louis, MO), or 20 μM adenosine periodate oxidized (A7154; Sigma-Aldrich, Saint Louis, MO) was added to the culture medium to treat macrophages.

Techniques: Western Blot, Derivative Assay, High Performance Liquid Chromatography, Staining, Incubation