Journal: Nature Communications

Article Title: Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

doi: 10.1038/s41467-020-20259-0

Figure Lengend Snippet: a YB-1 was silenced in K562, K562R, and KBM5 T315I cells with a retroviral transduction system, and viable cells were counted in the transfected cell lines at different times ( n = 3 biologically independent samples per group). Data are mean ± s.d. p -values were analyzed by two-way analysis of variance (ANOVA). **** p < 0.0001. b DNA damage-related protein expression was measured by western blot after USP47 knockdown in the K562 and KBM5 T315I cells. c GFP + BM mononuclear cells were collected from Usp47 +/+ and Usp47 −/− CML mice, and the indicated proteins were examined by western blot. d Immunofluorescence staining of γH 2 AX foci in the USP47 or YB-1 knockdown K562 cells. Scale bars, 20 μm. e The number of AP sites (DNA damage quantification) was detected in genomic DNA after USP47 knockdown in the K562 and KBM5 T315I cells ( n = 3 biologically independent samples per group). Data are mean ± s.d. p -values were analyzed by one-way analysis of variance (ANOVA). *** p < 0.001. f Time-course analysis of the USP47 knockdown-induced DNA damage response in K562 cells and cleaved-PARP1 by western blot. g YB-1 , PCNA , and TOPO IIα mRNA levels after YB-1 knockdown at day 7 in K562 cells ( n = 3 biologically independent samples per group). Data are mean ± s.d. p- values were analyzed by one-way analysis of variance (ANOVA). * p < 0.05; ** p < 0.01; **** p < 0.0001. h DNA damage-related protein expression was measured by western blot after YB-1 knockdown in the K562 cells. i The time-course of DNA damage is shown by γH 2 AX expression after irradiation (IR, 3 Gy) in the control and YB-1 stably knockdown KBM5 T315I cells. Band intensity (γH 2 AX relative to β-actin) is shown by the histogram ( n = 3 biologically independent samples per group). Data are mean ± s.d. p- values were analyzed by two-way analysis of variance (ANOVA). *** p < 0.001; **** p < 0.0001. j Exogenous YB-1 protein was ex p ressed in the cell line with stably knocked down USP47 , the expression of γH 2 AX and exogenous YB-1 (GFP tag) was detected by western blot. k The number of AP sites was measured as described above ( n = 3 biologically independent samples per group). Data are mean ± s.d. p- values were analyzed by one-way analysis of variance (ANOVA). *** p < 0.001. l γH 2 AX expression in Usp47 −/− MEFs was measured by western blot after the reintroduction of YB-1. m , n USP47 was knocked down in primary CML CD34 + cells, then the cells were cultured in a stem cell colony formation medium. The colonies ( m ) were counted on day 14 ( n = 3 biologically independent samples per group). Data are mean ± s.d. p- values were analyzed by one-way analysis of variance (ANOVA). *** p < 0.001. Immunofluorescence staining of γH 2 AX foci ( n ) was performed ( n = 3 biologically independent samples per group). Scale bars, 20 μm. Source data are provided as a Source Data file.

Article Snippet: The number of apurinic/apyrimidinic sites (AP sites) was determined using the DNA Damage Quantification Kit (DK02, DOJINDO Laboratories, Kumamoto, Japan) according to the manufacturer’s instruction.

Techniques: Retroviral, Transduction, Transfection, Expressing, Western Blot, Knockdown, Immunofluorescence, Staining, Irradiation, Control, Stable Transfection, Cell Culture

Journal: Discover oncology

Article Title: Olaparib increases chemosensitivity by upregulating miR-125a-3p in ovarian cancer cells.

doi: 10.1007/s12672-025-02048-7

Figure Lengend Snippet: Fig. 4 Effects of miR-125a-3p overexpression induces cellular senescence and increases DNA damage. A The SPiDER-βGal staining results were detected by flow cytometry. Data represent the mean ± standard deviation (n = 3). B The effect of different expression induced DNA damage was detected by γ-H2AX staining (×200). C Using western blotting to detect the expression of the γ-H2AX protein

Article Snippet: The DNA damage (γ-H2AX) detection kit-green (G265) was purchased from Dojindo Laboratories, Inc.

Techniques: Over Expression, Staining, Flow Cytometry, Standard Deviation, Expressing, Western Blot

Journal: Discover oncology

Article Title: Olaparib increases chemosensitivity by upregulating miR-125a-3p in ovarian cancer cells.

doi: 10.1007/s12672-025-02048-7

Figure Lengend Snippet: Fig. 6 Proposed model summarizing olaparib increases chemosensitivity by upregulating miR-125a-3p. Upregulating the expression of miR-125a-3p, leading to cell senescence and G1 phase arrest of the cell cycle, then induces DNA damage and improving the therapeutic sensitivity of ovarian cancer cells

Article Snippet: The DNA damage (γ-H2AX) detection kit-green (G265) was purchased from Dojindo Laboratories, Inc.

Techniques: Expressing

Journal: Antioxidants (Basel, Switzerland)

Article Title: Oral Curcumin-Thioketal-Inulin Conjugate Micelles against Radiation-Induced Enteritis.

doi: 10.3390/antiox13040417

Figure Lengend Snippet: Figure 5. CTI protects intestinal cells from radiation-induced damage. (A) Cell viability of IEC- 6 cells after different treatments. (B) Flow cytometry plots of IEC-6 cells after FITC–PI staining. (C) Population of cell apoptosis after radiation. (D) Calcein AM–PI dyeing of IEC-6 cells after different treatments. (E) Representative photographs of colony numbers with different treatments. (F) Quantitative analysis of the percentage of colony numbers after different treatments. (G) ROS levels were measured with a fluorescence microplate reader. (H) Intracellular ROS measurement of L929 cells by DCFH-DA staining (scale bars, 100 µm). (I) DNA double-strand damage in IEC-6 cells after different treatments by γH2AX immunofluorescence assay. Data are presented as means ± SD, n = 3 (** p < 0.01, *** p < 0.001, n = 3).

Article Snippet: After attachment, cells were treated with 10 μg/mL of Cur or 100 μg/mL of CTI micelles for 4 h. Then, 24 h after irradiation, the cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS and treated with 0.2% Triton-X 100 for 15 min. After PBS washing, the cells were incubated with rabbit polyclonal γH2AX (phospho S139) primary antibody (dilution 1:1000; G266; Dojindo, Tokyo, Japan) for 1 h. After discarding the primary antibody, the cells were gently washed with PBS three times and incubated with goat anti-rabbit secondary antibody (G266; Dojindo) at room temperature for 1 h. The nuclei were counterstained with DAPI (C0065, Solarbio, Beijing, China).

Techniques: Flow Cytometry, Staining, Fluorescence, Immunofluorescence

Journal: Antioxidants

Article Title: The Exacerbation of Aging and Oxidative Stress in the Epididymis of Sod1 Null Mice

doi: 10.3390/antiox9020151

Figure Lengend Snippet: List of antibodies used for immunofluorescence experiments.

Article Snippet: 8-OHG (DNA/RNA) , None , Mouse Monoclonal (15A3) , 1/1000 , Novus Biologicals , NB110-96878.

Techniques: Immunofluorescence, Conjugation Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: Nrf2 Plays a Key Role in Erythropoiesis during Aging.

doi: 10.3390/antiox13040454

Figure Lengend Snippet: Figure 4. Sorted Nrf2−/−mouse erythroblasts display overactivation of the system and impaired autophagy with caspase-3 pro-apoptotic pathway activation. (a) Wb analysis with specific anti- phospho-NF-kB (p-NF-kB) and NF-kB in sorted erythroid precursors from bone marrow of 12-month- old wild-type (WT) and Nrf2−/−mice. Actin was used as protein loading control. One representative gel of the other four is shown (upper panel). Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (b) Wb analysis with specific anti-HSP70, ATF6 and GADD34 in sorted erythroid precursors as in a. Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (c) Caspase 3 activity determined by cleavage of a fluorescent substrate in sorted erythroid precursors from bone marrow of WT and Nrf2−/−mice. Data are presented as means ± SD * p < 0.05 compared to WT. (d) Wb analysis with specific anti-LC3 I/II, Atg4, Atg5, Rab5 and p62 in sorted erythroid precursors from bone marrow of 12-month-old WT and Nrf2−/−mice. Actin was used as protein loading control. Densitometric analysis of immunoblots is shown in the right panel. Data are presented as means ± SD (n = 4); * p < 0.05 compared to WT mice. (e) Rab5 immunostaining of sorted erythroid precursors from bone marrow of 12-month-old WT and Nrf2−/−mice. DAPI was used to stain nuclei. Large clusters of positive cells were measured using ImageJ. At least 40 cells were analyzed in 8 different fields of acquisition. Data are presented as median and minimum/maximum, with boxes indicating 25–75th percentiles; * p < 0.05 compared to WT mice.

Article Snippet: The following specific antibodies were used: anti-NFkB-phospho-S536 (3033 Cell Signaling Technology, Leiden, NL, USA); anti-NFkB p65 and anti-Atg5 (8242, 12994 Cell Signaling Technology, Leiden, NL, USA); anti-Nrf2-phospho-S40 (ab76026 Clone EP1809Y, Abcam, Cambridge, UK); anti-Nrf2 (ab62352 Abcam, Cambridge, UK); anti Gadd34, anti-Lamp-1, anti-SQSTM1/P62, anti-Rab5 and anti-LC3A/B (ab9869, ab24170, ab109012, ab109534, ab62721 Abcam, Cambridge, UK); anti-APG7 (Atg7) (3615 ProSci, Poway, CA, USA); anti-ATF6 (70B1413.1 Novus Biologicals, Centennial, CO, USA); antiCHOP (ma1-250 Thermo Fisher Scientific, Waltham, MA, USA); anti-Actin (SAB4301137 clone BIII-136; Sigma-Aldrich, Saint Louis, MO, USA) and anti-GAPDH (HPA040067 SigmaAldrich, Saint Louis, MO, USA) as loading controls.

Techniques: Activation Assay, Control, Western Blot, Activity Assay, Immunostaining, Staining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Nrf2 Plays a Key Role in Erythropoiesis during Aging.

doi: 10.3390/antiox13040454

Figure Lengend Snippet: Figure 7. Schematic diagram of the role of Nrf2 in erythropoiesis during aging and the protective effects of astaxanthin PLGA nanoparticles. Aging is associated with increased ROS production, which is limited by the activation of Nrf2. This results in the upregulation of ARE genes encoding for antioxidants and cytoprotective systems as well as by the activation of adaptative mechanisms such as the UPR system to face ER stress and autophagy to clear damaged proteins. The absence of Nrf2 (Nrf2−/−mice) negatively affects the antioxidant cell machinery, resulting in severe and sustained oxidation. Nrf2−/−mouse red cells display severe membrane oxidation, exposition of phosphatidylserine, membrane binding of hemichromes and reduced expression of antioxidants and cytoprotective systems such as Prdx2. Red cell membrane protein oxidation favors band 3 protein clusterization, which is recognized by the naturally occurring anti-band 3 IgG antibodies. Both mechanisms drive Nrf2−/−mouse red cells towards erythrophagocytosis by splenic macrophages. In erythroblasts lacking Nrf2, the prolonged and severe oxidation due to the downregulation of antioxidants and cytoprotective systems promotes intense ER stress with overactivation of the UPR system and autophagy. Although the persistence of oxidative stress promotes compensatory activation of NF-kB, this is insufficient to prevent the overwhelming of proteostasis with impairment autophagy and accumulation of Rab5. This drives Nrf2−/−erythroblasts towards apoptosis via the caspase-3 pathway, resulting in ineffective erythropoiesis. ATS-NPs act as efficient antioxidants preventing the deleterious effects of the absence of Nrf2 on erythropoiesis and red cells during aging. PS: phosphatidylserine; ER: endoplasmic reticulum; UPR: unfolded protein response; ARE-: antioxidant-related element; ROS: reactive oxygen species; Prdx2: peroxiredoxin-2; Atg: autophagy- related protein; GADD34: growth arrest and DNA damage-inducible protein 34; PLGA: poly(lactic- co-glycolic acid).

Article Snippet: The following specific antibodies were used: anti-NFkB-phospho-S536 (3033 Cell Signaling Technology, Leiden, NL, USA); anti-NFkB p65 and anti-Atg5 (8242, 12994 Cell Signaling Technology, Leiden, NL, USA); anti-Nrf2-phospho-S40 (ab76026 Clone EP1809Y, Abcam, Cambridge, UK); anti-Nrf2 (ab62352 Abcam, Cambridge, UK); anti Gadd34, anti-Lamp-1, anti-SQSTM1/P62, anti-Rab5 and anti-LC3A/B (ab9869, ab24170, ab109012, ab109534, ab62721 Abcam, Cambridge, UK); anti-APG7 (Atg7) (3615 ProSci, Poway, CA, USA); anti-ATF6 (70B1413.1 Novus Biologicals, Centennial, CO, USA); antiCHOP (ma1-250 Thermo Fisher Scientific, Waltham, MA, USA); anti-Actin (SAB4301137 clone BIII-136; Sigma-Aldrich, Saint Louis, MO, USA) and anti-GAPDH (HPA040067 SigmaAldrich, Saint Louis, MO, USA) as loading controls.

Techniques: Activation Assay, Membrane, Binding Assay, Expressing