Journal: medRxiv

Article Title: Impairment of endothelial MerTK accelerates atherosclerosis development

doi: 10.1101/2025.04.14.25325845

Figure Lengend Snippet: ( A ) Body weight measurement for two months. ( B - C ) The visible formation of atherosclerotic plaque and H&E staining in aortic arch. ( D - G ) Immunostaining for the expression of IL-1β, MCP-1, ApoE, p22 phox and NF-κB in aortic arch from MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice of atherosclerosis model.

Article Snippet: The information of antibodies is shown as follows: IL-1β, p22 phox , p38 and α-SMA were from Cell Signaling; MCP-1 and TNF-α were from Invitrogen; ApoE was from Proteintech; p47 phox and gp91 phox were from SantaCruz Biotechnology; IFN-γ was from BiossUSA; and JNK was from Proteintech.

Techniques: Staining, Immunostaining, Expressing

Journal: medRxiv

Article Title: Impairment of endothelial MerTK accelerates atherosclerosis development

doi: 10.1101/2025.04.14.25325845

Figure Lengend Snippet: ( A - D ) Immunostaining for the expression of inflammation markers (TNF-α and IFN-γ), NADPH oxidase subunits (p47 phox and gp91 phox ), and MAPK family (ERK, p38 and JNK) in aortic arch from MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice of atherosclerosis model.

Article Snippet: The information of antibodies is shown as follows: IL-1β, p22 phox , p38 and α-SMA were from Cell Signaling; MCP-1 and TNF-α were from Invitrogen; ApoE was from Proteintech; p47 phox and gp91 phox were from SantaCruz Biotechnology; IFN-γ was from BiossUSA; and JNK was from Proteintech.

Techniques: Immunostaining, Expressing

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 2. Clinically relevant concentrations of ATO stimulate phosphorylation of phospho p47phox in NB4 but not in U937 or THP-1 cells.NB4 (A, D), U937 (B, E) or THP-1 (C, F) cells were incubated for 6 h with 0.5 or 1 µM ATO. In other experiments, NB4 (G) or U937 (H) cells were treated for 6 h with 2.5 NaAsO2 or 5 µM ATO, respectively. PMA (0.162 µM, 15 min) was used as a positive control. After treatments, the cells were analyzed for phospho p47phox expression (A-H). The blot, representative of three separate experiments, was re-probed for p47phox. Representative micrographs providing immunocytochemical evidence of p47phox phos phorylation are also reported in panel D (NB4 cells), E (U937 cells) and F (THP-1 cells). After treatments, the cells were fixed and processed as detailed in Material and Methods. Scale bar represents 20 µm. Results represent the means ± SD calculated from three separate experiments. *P < 0.05, * *P < 0.01, compared with untreated cells (one-way ANOVA followed by Dunnett’s test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Incubation, Positive Control, Expressing

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 3. Pharmacological inhibition of NOX 2 activity suppresses mitochondrial ROS formation induced by ATO in NB4 cells.NB4 cells were pretreated for 5 min with the vehicle, 10 µM apocynin, or 1 µM DPI, and exposed for 15 min to 0.162 µM PMA. After treatments, the cells were analyzed for phospho p47phox expression (A) or DHR-fluorescence (B). NB4 cells pretreated as indicated above or with rotenone, were exposed for 6 h to 1 µM ATO. After treatments, the cells were analyzed for phospho p47phox expression (C), MitoSOX red- (D) or DHR- (E) fluorescence. In other experiments, the cells were pretreated with the vehicle, rotenone, apocynin or DPI, incubated for 6 h with 2.5 (F, G) or 5 µM (H-L) NaAsO2 and finally analyzed for MitoSOX red- (F, I) and DHR- (G, L) fluorescence, or for phospho p47phox

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Inhibition, Activity Assay, Expressing, Fluorescence, Incubation

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 4. ATO fails to promote mitochondrial ROS formation in NB4 cells with downregulated p47phox expression.NB4 control cells, transfected with scrambled or p47phox siRNA, were lysed and analyzed by Western blotting 48 h post-transfection using anti-p47phox antibody. The relative band intensity of p47phox is depicted in the top bar chart (A). GADPH was accounted for loading control. Results represent the means ± SD calculated from three separate experiments. *P < 0.05, compared with control cells (one-way ANOVA followed by Dunnett’s test).Cells transfected with scrambled or p47phox specific siRNA were pretreated for 5 min with the vehicle, apocynin, or DPI and exposed for either 15 min to PMA (B) or 6 h to 1 µM ATO (C, D). In some experiments, rotenone was added to the cultures 5 min before ATO. After treatments, the cells were analyzed for DHR- (B, C) or MitoSOX red- (D) fluorescence. Results represent the means ± SD calculated from three separate ex periments. *P < 0.05, * *P < 0.01, compared with untreated cells. #P < 0.05, ##P < 0.01 compared with ATO treated cells. $P < 0.05, $$P < 0.01 compared with treated scrambled siRNA cells (ANOVA followed by Tukey test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Transfection, Western Blot, Fluorescence

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 5. Low mitochondrial ROS formation induced by ATO in resistant cells is due to poor activation of NOX 2.U937 (A), THP-1 (B) or p47phox siRNA trans fected NB4 (C) cells were pretreated for 5 min with the vehicle, rotenone, apocynin or DPI and incubated for 6 h with 0.5 or 1 µM ATO alone or associated PMA. After treatments, the cells were analyzed for MitoSOX red fluorescence. Results represent the means ± SD calculated from three separate experiments. * *P < 0.01, compared with untreated cells. ##P < 0.01 compared with ATO/ PMA treated cells (ANOVA followed by Tukey test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Activation Assay, Incubation, Fluorescence

Journal: Pharmacological research

Article Title: Unveiling the link between NADPH oxidase 2 activation and mitochondrial superoxide formation in leukemic cell killing induced by arsenic trioxide.

doi: 10.1016/j.phrs.2024.107554

Figure Lengend Snippet: Fig. 6. Clinically relevant concentrations of ATO promote mitochondrial permeability transition-dependent apoptosis in susceptible NB4 cells. NB4 control cells, and NB4 cells previously transfected with scrambled or specific p47phox siRNA, were pretreated for 5 min with the vehicle, rotenone, apocynin or 0.5 µM CsA and incubated for 6 (A), 9 (B, C) or 14 (D, E, F) h with 1 µM ATO. After treatments, the cells were analyzed for NAO-fluorescence (A) MitoTracker red CMXRos- fluorescence (B), cytochrome c (Cyt c) localization (C), caspase 3 activity (D) and apoptotic DNA fragmentation/condensation by both the Hoechst (E) and comet (F) assays. * *P < 0.01, compared with untreated cells. ##P < 0.01 compared with ATO treated cells. $$P < 0.01 compared with ATO treated scrambled siRNA cells (one-way ANOVA followed by Tukey test).

Article Snippet: NB4 cells were transfected with a pool of 4 target-specific p47phox siRNAs (sc-29422, Santa Cruz Biotechnology).

Techniques: Permeability, Control, Transfection, Incubation, Fluorescence, Activity Assay

Journal: Redox biology

Article Title: Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H 2 O 2 , and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

doi: 10.1016/j.redox.2023.102915

Figure Lengend Snippet: Fig. 2. Clozapine blunts NOX-2-dependent ROS in SW872 cells. Confluent SW872 cells were induced to differentiate for 3 days with or without CLZ. (A) ROS formation detected with a flow cytometric assay using DCF. (B) Representative micrographs showing phosphorylated p47phox localization (white arrows). In these experiments, the cells were fixed and analysed for immunocy tochemical detection of phosphorylated p47phox by confocal microscopy, with all three panels having identical excitation and exposure conditions. Magnification 60X; Scale bar represents 25 μm. (C) Representative flow cytometry histograms of phosphorylated p47phox fluorescence. (D) Statistical analysis of the results obtained in (C). (E–F) Statistical analysis of flow cytometry histograms of p47phox and p67phox fluorescence. T3 CTR received DMSO alone. Data are expressed in % respect to T0 condition. Results represent the means ± SEM calculated from at least three independent determinations. *p < 0.05, **p < 0.01, as compared to T0; (**)p < 0.01, as compared to T3. ns: not significant (one-way ANOVA followed by Dunnett’s test).

Article Snippet: Anti-phosphorylated p47phox (STJ91180; St. John’s Laboratory, UK), anti-p67phox (CQA1036; Cohesion Biosciences) and anti-p47phox (SAB4502810; Merck Life Science s.r.l) were incubated for 30 min at 4 ◦C; after a washing step, anti-rabbit IgG-FITC antibody was added (sc-2012; Santa Cruz Biotech, Santa Cruz CA), as indicated in the manufacturer’s instructions.

Techniques: Flow Cytometry, Confocal Microscopy, Fluorescence

Journal: Science Advances

Article Title: Sphingolipid biosynthesis is essential for metabolic rewiring during T H 17 cell differentiation

doi: 10.1126/sciadv.adk1045

Figure Lengend Snippet: ( A ) WT and KO naïve CD4 + T cells were differentiated under T H 17 conditions for 12 hours and mitochondrial ROS measured using Mitosox red. The left panel is a representative histogram, and the right panel shows the cumulative data (MFI) from n = 3 biologically independent samples. ( B ) WT and KO naïve CD4 + T cells were differentiated under T H 17 conditions for 4 days with or without DPI (10 nM) and scored for intracellular cytokines. The left panel is a representative flow figure, and the right panel shows the cumulative data from n = 4 biologically independent samples. ( C ) WT and KO naïve CD4 + T cells cross-linked with anti-CD3 for 15 min with DCFDA and read in flow cytometry. The left panel is a representative histogram, and the right panel shows the cumulative data (MFI) from n = 3 biologically independent samples. ( D ) WT and KO naïve CD4 + T cells cross-linked as in (C) and membrane fraction immunoblotted for p47 phox . Each lane is a pooled sample from four to eight mice. The left panel shows representative blot, and the right panel is the quantitative data from three independent experiments. ( E ) CD4 + T cells cross-linked as in (C) and stained for gp91 phox (green) and p47 phox (red) and analyzed using confocal microscopy. The left panel shows the representative confocal picture, and the right panel shows the number of colocalization spots per cell. n = 3 biologically independent samples. ( F ) WT and KO Naïve CD4 + T cells were nucleofected with control or p47 phox siRNA, differentiated into T H 17 cells, and scored for intracellular cytokines. The left panel is a representative flow figure, and the right panel shows the cumulative data. Each dot represents cells pooled from three mice. n = 3 independent experiment. All data presented as means ± SEM: * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant.

Article Snippet: Pellets were washed twice with ice-cold PBS and resuspended in RIPA lysis buffer containing protease and phosphatase inhibitors and subjected to Western blot analysis using monoclonal antibody to p47 phox (Santa Cruz Biotechnology, sc-17845, clone D-10).

Techniques: Flow Cytometry, Membrane, Staining, Confocal Microscopy, Control

Journal: Journal of medical microbiology

Article Title: Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin.

doi: 10.1099/jmm.0.018192-0

Figure Lengend Snippet: Fig. 4. Fumagillin inhibits the translocation of p47phox from the cytosol to the membrane. Neutrophils (1108) were incubated in the presence or absence of 2 mg fumagillin ml”1

Article Snippet: The cells were incubated with primary antibody against p47phox or against MPO (Genway) overnight at 4 uC.

Techniques: Translocation Assay, Membrane, Incubation

Journal: Journal of medical microbiology

Article Title: Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin.

doi: 10.1099/jmm.0.018192-0

Figure Lengend Snippet: Fig. 5. Visualization of p47phox in neutrophils. Neutrophils were incubated in the presence of 2 mg fumagillin ml”1 for 25 min, washed, PMA- stimulated, fixed and permeabilized as described in Methods. Untreated cells showed a wide dispersal of p47phox compared to PMA- stimulated cells, which showed a high level of translocation to the membrane. Neutrophils which had been incubated with fumagillin showed some translocation to the membrane but retained most p47phox in the cytosol.

Article Snippet: The cells were incubated with primary antibody against p47phox or against MPO (Genway) overnight at 4 uC.

Techniques: Incubation, Translocation Assay, Membrane