Journal: Cancers

Article Title: Blockade of IL-10 Signaling Ensures Mifamurtide Efficacy in Metastatic Osteosarcoma

doi: 10.3390/cancers15194744

Figure Lengend Snippet: Mifamurtide is unable to exert its antitumoral efficacy in osteosarcoma cell lines. Mifamurtide effect on MG--63, HOS and 143-B OS cells in co-culture with macrophages after 24 h of treatment. ( A ) Representative Annexin V/PI staining plots and relative quantification. ( B ) FACS analysis and relative quantification of live cells count. ( C ) Effect of mifamurtide treatment on oxygen reactive species production (ROS) with MitoSOX TM Red assay. Percentage of ROS + cells are shown. ( D ) Detection of Intracellular ROS generation using the DCFH-DA assay. Fluorescence intensity of ROS production is shown. ( E ) Elisa quantification of SOD-2 and ( F ) Catalase protein expression. Significance was calculated by unpaired Student t -test analysis. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTRL.

Article Snippet: In particular, the Annexin V-FITC Kit (#130-092-052, Miltenyi Biotec ® ) was used for the identification and enumeration of apoptotic/dead cells.

Techniques: Co-Culture Assay, Staining, Quantitative Proteomics, DCFH-DA Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Pharmaceutics

Article Title: Biomineralized MnO 2 Nanoparticle-Constituted Hydrogels Promote Spinal Cord Injury Repair by Modulating Redox Microenvironment and Inhibiting Ferroptosis

doi: 10.3390/pharmaceutics16081057

Figure Lengend Snippet: Effects of CGPG-HFn@MnO 2 /AS on H 2 O 2 -treated PC12 cells. ( A ) Cell viability was detected using the CCK-8 assay kit. ( B ) Cellular iron levels were detected using a kit. ( C ) Cellular GSH levels were detected using a kit. ( D ) Cellular MDA levels were detected using a kit. ( E ) Cellular SOD activity was detected using a kit. ( F , G ) Cellular ROS levels were detected using the fluorescent probe DCFH-DA. (Scale bar of G : 500 μm) ( H – M ) The protein levels of SIRT1, XCT, GPX4, 4-HNE, and TFR1 were detected by western blot assay. G1: control group. G2: H 2 O 2 group. G3: HFn@MnO 2 /AS group. G4:CGPG group. G5: CGPG-HFn@MnO 2 /AS. ( N , O ) Cell migration was evaluated using the wound healing assay. (Scale bar of N : 400 μm)Statistical analyses were performed using a one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test (n ≥ 3). # p < 0.05 and ## p < 0.01 vs. the control group; * p < 0.05 and ** p < 0.01 vs. the H 2 O 2 group.

Article Snippet: Then, 20 μM of H 2 O 2 peroxidized medium was used to stimulate the cells for 1 h. The culture medium was subsequently replaced with different formulations and incubated for another 24 h. The expression of ROS in the cells was detected using a DCFH-DA staining kit (Beyotime, Shanghai, China) and observed using a laser scanning confocal microscope (CLSM, Leica TCS SP8, Wetzlar, Germany).

Techniques: CCK-8 Assay, Activity Assay, Western Blot, Control, Migration, Wound Healing Assay

Journal: Development (Cambridge, England)

Article Title: Heme-deficient primitive red blood cells induce HSPC ferroptosis by altering iron homeostasis during zebrafish embryogenesis

doi: 10.1242/dev.201690

Figure Lengend Snippet: The HSPC ROS level is elevated in alas2 and alad mutants. (A) Schematic of biochemical assay of oxidative stress biomarkers (ROS, CAT, SOD, GSH and MDA) in zebrafish dissected trunk regions at 36 hpf. (B) Confocal imaging showing the co-localization of kdrl :GFP + and MitoSOX + cells in the VDA of control, alas2 −/− and alad −/− at 36 hpf. The dorsal aorta (DA) regions are denoted by white dashed lines, and the kdrl + /MitoSOX + cells are denoted by white arrowheads. (C) Quantification of kdrl + /MitoSOX + cells in B. n =3 experimental replicates. (D) Quantification of mean fluorescence intensity (MFI) of mitochondrial ROS level in HSPCs of control, alas2 −/− and alad −/− measured by MitoSOX staining. n =3 experimental replicates. (E) Expression of the HSPC marker runx1 in control, alas2 −/− and alad −/− with or without NAC treatment at 36 hpf examined by WISH. The AGM regions for marker gene-positive cell counting are denoted by red arrowheads. (F) Quantification of the runx1 -positive HSPCs in E. n =3 experimental replicates. (G) Expression of the HSPC marker runx1 in control, alas2 −/− and alad −/− with or without mitoTempo treatment at 36 hpf examined by WISH. The AGM regions for marker gene-positive cell counting are denoted by red arrowheads. (H) Quantification of the runx1 -positive HSPCs in G. n =3 experimental replicates. (I) Confocal imaging shows the kdrl + / cmyb + HSPCs in control, alas2 −/− and alad −/− with or without mitoTempo treatment at 36 hpf. (J) Quantification of the HSPCs in I.; n =3 experimental replicates. Number of samples are indicated. Data are mean±s.d. *** P <0.001 (one-way ANOVA, Tukey's multiple comparisons in C,D,F,H,J). n.s., not significant. Scale bars: 100 μm.

Article Snippet: All the commercially available kits were obtained from Nanjing Jiancheng Bioengineering Institute, including the ROS Assay Kit (chemical fluorescence method, DCFH-DA staining), SOD assay kit (hydroxylamine method), CAT assay kit (visible light), GSH assay kit (colorimetric method), MDA assay kit (TBA method) and total protein quantitative assay kit (Coomassie Brilliant Blue method).

Techniques: Imaging, Control, Fluorescence, Staining, Expressing, Marker, Cell Counting