Journal: International Journal of Molecular Sciences

Article Title: Voghera Sweet Pepper: A Potential Ally against Oxidative Stress and Aging

doi: 10.3390/ijms24043782

Figure Lengend Snippet: Double immunolabeling for SOD2 protein (green fluorescence) and mitochondria (red fluorescence); DNA was stained with Hoechst 33258 (blue fluorescence). Magnification: 60×; scale bar: 10 μm. Histograms representing the analysis of the fluorescence intensity of SOD2; Anova one-way test: p = 0.02; ** p < 0.01, statistical significance between untreated young NHDF (Y-NC) and old untreated NHDF (O-NC); $$ p < 0.01, statistical significance between old NHDF treated with VP and old NHDF untreated.

Article Snippet: SOD2 , Rabbit monoclonal anti-SOD2 (Cell Signaling Technology, Danvers, MA, USA) , 1:200.

Techniques: Immunolabeling, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Voghera Sweet Pepper: A Potential Ally against Oxidative Stress and Aging

doi: 10.3390/ijms24043782

Figure Lengend Snippet: Primary antibodies used for immunofluorescence reactions.

Article Snippet: SOD2 , Rabbit monoclonal anti-SOD2 (Cell Signaling Technology, Danvers, MA, USA) , 1:200.

Techniques: Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Hydrogen peroxide attenuates rhinovirus-induced anti-viral interferon secretion in sinonasal epithelial cells

doi: 10.3389/fimmu.2023.1086381

Figure Lengend Snippet: The expression levels of SOD1 (A, C) and SOD2 ( B, D) in cultured sinonasal epithelial cells pretreated with H 2 O 2 at 50, 100, and 150 uM and then followed by RV 16 infection (A, B) and poly (I:C) treatment (C, D) , which were evaluated by western blot. Data are mean ± SEM from 7 different epithelial donors. Control indicates non-treated normal epithelial cells. Rhino indicates epithelial cells infected with RV 16. H 2 O 2 +rhino indicates the epithelial cells pretreated with H 2 O 2 at 50, 100, and 150 uM followed by RV 16 infection. NAC+ H 2 O 2 +rhino indicates the cells which were pretreated with NAC at 5 mM for 1 h and then followed by H 2 O 2 treatment at 100 uM and subsequently infected with RV 16. Poly (I:C) indicates epithelial cells treated with poly (I:C). H 2 O 2 +poly (I:C) indicates the epithelial cells pretreated with H 2 O 2 at 50, 100, and 150 uM followed by poly (I:C) treatment. NAC+ H 2 O 2 +poly (I:C) indicates the cells which were pretreated with NAC at 5 mM for 1 h and then followed by H 2 O 2 treatment at 100 uM and subsequently treated with poly (I:C). Rhino indicates RV 16. NAC indicates N-acetyl-L-cysteine.

Article Snippet: Thereafter the membranes were rinsed and probed with the primary antibodies in the refrigerator overnight at 4 °C; the primary antibodies employed for Western blots were anti-β actin (1:5000) which was obtained from Santa Cruz, USA, anti-viperin, anti-OAS, anti-Mx, anti-TLR3, anti-RIG1, anti-MDA5, anti-SOD1, anti-SOD2, anti-IRF3, and anti-phospho-IRF3 which were obtained from Cell Signaling Technology and used at a dilution of 1:1000.

Techniques: Expressing, Cell Culture, Infection, Western Blot

Journal: Biomedicines

Article Title: Multidrug-Loaded Lipid Nanoemulsions for the Combinatorial Treatment of Cerebral Cavernous Malformation Disease

doi: 10.3390/biomedicines11020480

Figure Lengend Snippet: ( A ) Immunoblot analysis and representative histogram of the oxidative stress marker forkhead box class O1 (FoxO1) in KRIT1-knockout (KRIT1−/−) and KRIT1-overexpressing (KRIT1+/+) mouse embryonic fibroblasts (MEFs) untreated (Ctrl) or treated for 24 h with injectable nanoemulsions (ILs) loaded with rapamycin (Rapa), bevacizumab (Bvz), yeast avenanthramide I (Avn), or with a combination of all three compounds (herein referred to as Mix) to a final concentration of: Avn = 15 μg/mL (50 μM), Rapa = 0.46 μg/mL (500 nM), Bvz = 10 μg/mL (70 nM). After treatment, cells were lysed, as described in Materials and Methods, and analyzed for the indicated proteins by Western blot (WB) analysis. ( B ) Immunoblot analysis and histogram representing the quantitative evaluation by densitometric analysis of superoxide dismutase 2 (SOD2) protein expression levels. Quantifications are relative protein level units referring to the average value obtained for KRIT1+/+ samples. α-tubulin was used as internal loading control for WB normalization. Statistical analysis: treatments vs. NT in KRIT−/− MEFs: # p < 0.1; * p < 0.05; ** p > 0.01; *** p < 0.005; **** p < 0.0001.

Article Snippet: Primary antibodies used in the present study include the following: rabbit anti-KRIT1 mAb (ab196025), 1:1000 dilution (Abcam, San Francisco, CA, USA); mouse anti-α-tubulin mAb (B-5-1-2), 1:5000 dilution (Sigma-Aldrich, Milan, Italy); rabbit anti-SQSTM1/p62 mAb (D1Q5S), 1:1000 dilution (Cell Signaling, Danvers, MA, USA); mouse anti-SOD2 (2A1) mAb (ab16956), 1:2000 dilution (Abcam); rabbit anti-VEGF pAb (ab46154), 1:1000 dilution (Abcam).

Techniques: Western Blot, Marker, Knock-Out, Concentration Assay, Expressing

Journal: Biomedicines

Article Title: Multidrug-Loaded Lipid Nanoemulsions for the Combinatorial Treatment of Cerebral Cavernous Malformation Disease

doi: 10.3390/biomedicines11020480

Figure Lengend Snippet: Schematic representation of the molecular targets considered in order to develop the proposed nanomedicine-based combinatorial therapeutic approach for CCM disease. The most significant results have been obtained by targeting the oxidative stress with lipid nanoemulsions containing avenanthramide, both alone and in combination with rapamycin. Conversely, the efficacy of rapamycin as an autophagy promoter needs to be improved in the nanoemulsion, and the effect of the bevacizumab-loaded nanoemulsion in rescuing the abnormal angiogenesis looks contradictory, probably due to the in vitro model employed. Abbreviations: Avn: yeast avenanthramide I; Akt: protein kinase B; Bvz: bevacizumab; CCM: cerebral cavernous malformation; CD40: cluster of differentiation 40; ERK: extracellular-signal-regulated kinase; FoxO1: forkhead box class O1; HIF: hypoxia-induced factor; KRIT1: Krev interaction trapped protein 1; mTOR: mammalian target of rapamycin; PTEN: phosphatase and tensin homolog; Rapa: rapamycin; ROS: reactive oxygen species; SOD2: superoxide dismutase 2; VEGF: vascular endothelial growth factor.

Article Snippet: Primary antibodies used in the present study include the following: rabbit anti-KRIT1 mAb (ab196025), 1:1000 dilution (Abcam, San Francisco, CA, USA); mouse anti-α-tubulin mAb (B-5-1-2), 1:5000 dilution (Sigma-Aldrich, Milan, Italy); rabbit anti-SQSTM1/p62 mAb (D1Q5S), 1:1000 dilution (Cell Signaling, Danvers, MA, USA); mouse anti-SOD2 (2A1) mAb (ab16956), 1:2000 dilution (Abcam); rabbit anti-VEGF pAb (ab46154), 1:1000 dilution (Abcam).

Techniques: In Vitro

Journal: Biomedicines

Article Title: Multidrug-Loaded Lipid Nanoemulsions for the Combinatorial Treatment of Cerebral Cavernous Malformation Disease

doi: 10.3390/biomedicines11020480

Figure Lengend Snippet: Effects of Mix and IL-Mix on the main CCM molecular phenotypes. Abbreviations: Avn: yest avenanthramide I; Bvz: bevacizumab; CCM: cerebral cavernous malformation; NT: not-treated control; FoxO1: forkhead box class O-1; IL: injectable nanoemulsion; LC3-II: light chain 3-II; Mix: yeast avenanthramide I, bevacizumab, rapamycin combination; N.D.: not determined; NT: untreated; p62: Sequestosome-1; Rapa: rapamycin; SOD2: superoxide dismutase 2; VEGF: vascular endothelial growth factor; VEGFR2: vascular endothelial growth factor receptor 2.

Article Snippet: Primary antibodies used in the present study include the following: rabbit anti-KRIT1 mAb (ab196025), 1:1000 dilution (Abcam, San Francisco, CA, USA); mouse anti-α-tubulin mAb (B-5-1-2), 1:5000 dilution (Sigma-Aldrich, Milan, Italy); rabbit anti-SQSTM1/p62 mAb (D1Q5S), 1:1000 dilution (Cell Signaling, Danvers, MA, USA); mouse anti-SOD2 (2A1) mAb (ab16956), 1:2000 dilution (Abcam); rabbit anti-VEGF pAb (ab46154), 1:1000 dilution (Abcam).

Techniques:

Journal: Scientific Reports

Article Title: Caloric restriction increases the resistance of aged heart to myocardial ischemia/reperfusion injury via modulating AMPK–SIRT 1 –PGC 1a energy metabolism pathway

doi: 10.1038/s41598-023-27611-6

Figure Lengend Snippet: Expression of cardiometabolic proteins. ( A ) AMPK/SIRT 1 /PGC 1a signaling pathway. CR leads to activation of AMPK. AMPK regulates SIRT 1 by inducing NAD + . SIRT 1 activates PGC 1a by phosphorylation and deacetylation. AMPK and SIRT 1 synergistically inhibit PPARγ. CR increases SOD 2 expression in vivo and inhibits ROS. ( B ) Representative images of immunoblotting for p-AMPK, SIRT 1 , p-PGC 1a , PPAR-γ, and SOD 2 . ( C ) Quantitative results of p-AMPK protein. ( D ) Quantitative results of SIRT 1 protein. ( E ) Quantitative results of p-PGC 1a protein. ( F ) Quantitative results of PPAR-γ protein. ( G ) Quantitative results of SOD 2 protein. The westerns were cut prior to incubation with the primary antibodies, the full size blots can be seen in Supplementary Fig. . Western blotting showed protein abundance in each group, N = 5, y-AL group youth fed ad libitum group, a-AL aged fed ad libitum group, a-CR aged caloric restriction group. * P < 0.05 vs y-AL; # P < 0.05 vs a-AL; ‡ P < 0.05 vs sham operation.

Article Snippet: Rabbit antibodies P-AMPKα (thr172,2535), AMPKα (5831s), PPAPγ (2443s), and SOD 2 (13141), were purchased from Cell Signaling Technology (UK) rabbit anti-P-PGC 1a (ser571, AF6650), and PGC 1a (NFP1-04676) from Novus Biologics, and SIRT 1 monoclonal antibody (ab12193) from Abcam (USA).

Techniques: Expressing, Activation Assay, In Vivo, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Regulatory Mechanism between Ferritin and Mitochondrial Reactive Oxygen Species in Spinal Ligament-Derived Cells from Ossification of Posterior Longitudinal Ligament Patient

doi: 10.3390/ijms24032872

Figure Lengend Snippet: Assessment of mitochondrial mass, membrane potential (ΔΨM), and electron transport chain (ETC) protein complexes. ( a , left) Live cell images of mitochondria from Ctrl1 and OPLL4 stained using MitoTracker Green (MTG) and MitoTracker Deep Red (MTDR) for mitochondrial mass assessment. ( a , right) Mitochondrial mass was estimated by fluorescence using 200 nM MTG and 500 nM MTDR for 30 min at 37 °C. One dot implies the mean fluorescence of triplicate results. ( b ) Mitochondrial mass was estimated according to the mitochondrial outer membrane proteins TOM22 and VDAC. ( c ) For ΔΨ M evaluation, live cell images were obtained of Ctrl1 and OPLL4 cells stained with 100 nM tetramethylrhodamine methylester (TMRM) for 30 min. ( d ) The fluorescence levels for the Ctrl and OPLL groups were determined by flow cytometry. The TMRM fluorescence was decreased by CCCP. ( e ) The levels of the mitochondrial ETC complex I~V proteins were assessed by Western blotting. ( f ) Densitogram of the mitochondrial ETC complex normalized to GAPDH. ( g , left) Representative live cell images of Ctrl1 and OPLL4 were obtained after culturing the cells on cover slips for two days. Superoxide (O 2 •− ) levels were estimated with 5 μM MitoSox, and intracellular ROS (icROS) levels were estimated with 25 μM DCFDA. ( g , right) Superoxide (O 2 •− ) and icROS were measured for all Ctrl and OPLL groups. ( h , upper) Proteins related to the ROS scavenging system were evaluated. Catalase, SOD 1, SOD 2, GPX 1, and GPX 4 levels of the Ctrl and OPLL groups are shown. ( h , lower) Densitogram with GAPDH normalization showing that SOD1, SOD2, GPX1, and GPX4 levels were significantly decreased in the OPLL group. ( i ) Superoxide (O 2 •− ) indicated by MitoSox staining and apoptosis indicated by Annexin V staining were studied by flow cytometry. These cells were measured once. ( j ) The correlation of superoxide (O 2 •− ) and Annexin V fluorescence was measured simultaneously by flow cytometry. These cells were measured once. ( k ) Starvation of the Ctrl and OPLL groups for apoptosis evaluation using Annexin V by flow cytometry. These cells were measured in triplicate. * p < 0.05, ** 0.001 ≤ p ≤ 0.001, *** p < 0.001 within groups; # p < 0.05 between groups. Scale bars: white, 1000 μm.

Article Snippet: The following antibodies (ab) were used for Western blot analysis: anti-ferritin ab (ab75972; Abcam Inc, Boston, MA, USA); anti-ALP ab (Abcam, ab354); anti-VDAC ab (Abcam, ab34726); anti-TOM22 ab (Abcam, ab57523); anti-mitochondrial complex 1~5 ab cocktail (Abcam, 110413); anti-nestin ab (ThermoFisher, MA1-110); anti-GPX1 ab (3286; Cell Signaling Technologies, Danvers, MA, USA); anti-GPX4 ab (166120; Abcam); anti-SOD1 ab (ab16831; Abcam); anti-SOD2 ab (13141; Cell signaling); anti-tubulin ab (ab7291; Abcam); anti-rabbit IgG ab (7074s; Cell signaling); anti-mouse IgG ab (7076s; Cell Signaling); and goat anti-mouse IgG ab (35502; Thermo Fisher).

Techniques: Staining, Fluorescence, Flow Cytometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Investigating Therapeutic Effects of Indole Derivatives Targeting Inflammation and Oxidative Stress in Neurotoxin-Induced Cell and Mouse Models of Parkinson’s Disease

doi: 10.3390/ijms24032642

Figure Lengend Snippet: NC009-1 down-regulated neuroinflammation and up-regulated cellular redox signaling in MPTP-treated mice. ( A ) Expression levels of striatal IL-1β, IL-6, and TNF-α analyzed by ELISA ( n = 6). Expression levels of ( B ) NLRP3, CASP1, iNOS, and ( C ) SOD2, NRF2, and NQO1 in striatum analyzed by Western blot ( n = 6). GAPDH was included as a loading control. To normalize, the relative NLRP3, CASP1, iNOS, SOD2, NRF2, and NQO1 of control mice was set as 100%. p values: comparisons between MPTP and control ( # p < 0.05, ## p < 0.01, ### p < 0.001), or NC009-1-treated and untreated MPTP mice (* p < 0.05, ** p < 0.01).

Article Snippet: After blocking, the membrane was probed with dopamine transporter (DAT) (1:2000; Sigma-Aldrich #MAB369), NLRP3 (1:500; Cell Signaling #15101s), CASP1 (1:500; Cell Signaling #89332s), iNOS (1:500; Cell Signaling #2982), superoxide dismutase 2 (SOD2) (1:4000; Cell Signaling #13141), NFE2-like bZIP transcription factor 2 (NRF2) (1:1000; Sigma-Aldrich #ABE413), NAD(P)H dehydrogenase, quinone 1 (NQO1) (1:2000; Sigma-Aldrich # N5288), or GAPDH (1:1000; MDBio #30000002) primary antibody at room temperature 2 h or 4 °C overnight.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot