Journal: ACS Omega

Article Title: Ellagic Acid Induces DNA Damage and Apoptosis in Cancer Stem-like Cells and Overcomes Cisplatin Resistance

doi: 10.1021/acsomega.3c08819

Figure Lengend Snippet: EA treatment alters mitochondrial dynamics, leading to the accumulation of intracellular ROS and DNA damage. Western blot analysis of the protein levels of Mfn2, Drp1, total-ERK, and p-ERK in the (A) A2780 monolayer, (B) A2780 spheroids, and (C) SKOV3 spheroids cells; A549-CD133 + (D,E) and SKOV3- spheroids (F,G) were treated with different concentrations of EA (0, 5, 10, 25 μM) for 24 h. Intracellular ROS accumulation was determined using flow cytometry. Representative histograms and corresponding bar diagrams showing ROS level in CSLCs; A549-CD133 + (H) and SKOV3- spheroid (I) cells were treated with different doses of EA for 48 h. γH2AX accumulation was detected through flow cytometry to evaluate the extent of DNA damage induced by EA. Representative contour plots obtained from flow cytometry analysis showing the percentage of γH2AX positive cells. Data was reanalyzed using FlowJo software. In all graphs, the results are represented as mean ± SD of triplicate experiments. ** P < 0.01, and *** P < 0.001.

Article Snippet: Washing was done twice with 1× PBS and staining was performed using the γH2AX (Miltenyi Biotec cat. no. 130-130-829) antibody.

Techniques: Western Blot, Flow Cytometry, Software

Journal: ACS Omega

Article Title: Ellagic Acid Induces DNA Damage and Apoptosis in Cancer Stem-like Cells and Overcomes Cisplatin Resistance

doi: 10.1021/acsomega.3c08819

Figure Lengend Snippet: Combinatorial treatment of EA and cisplatin impairs DNA damage repair in lung and ovarian CSLCs. The DNA repair kinetics study depicts the percent DNA damage accumulation following DMSO, EA, cisplatin, and EA + cisplatin treatment for 12 h followed by 3 h damage recovery in (A) A549-CD133 + and (B) SKOV3 spheroid cells. (C) The immunofluorescence staining shows the increasing accumulation of DNA damage in DMSO, EA, cisplatin, and EA + cisplatin-treated cells. The corresponding graph represents the mean fluorescence intensity of accumulated γH2AX. N=3, Bar, SD; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Washing was done twice with 1× PBS and staining was performed using the γH2AX (Miltenyi Biotec cat. no. 130-130-829) antibody.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Nanoscale Advances

Article Title: A nano-integrated diagnostic and therapeutic platform with oxidation–reduction reactions in tumor microenvironments †

doi: 10.1039/c9na00786e

Figure Lengend Snippet: (a) Cytotoxicity of HCT-116 cells treated with MH, H 2 PtCl 6 , and MHP (MnO 2 /H 2 PtCl 6 = 50/17.5 μg mL −1 ) at different radiation doses at 0, 2, 4, and 8 Gy; (b) flow cytometry analysis; (c) quantitative analysis of apoptosis induced by MH or MHP (MnO 2 /H 2 PtCl 6 = 50/17.5 μg mL −1 ) combined with or without radiation (5 Gy); (d) immunofluorescence staining of γ-H2AX foci in HCT-116 cells treated with saline, MH, and MHP (MnO 2 /H 2 PtCl 6 = 50/17.5 μg mL −1 ), with or without radiation (5 Gy). Blue represents DAPI and red represents Cy3 double-strand breaks in DNA. Scale bar: 50 μm; (e) quantitative analysis of γ-H2AX foci (γ-H2AX foci/100 μm 2 ) for n > 100 cells in each treatment group. * P < 0.05; ** P < 0.01.

Article Snippet: Moreover, a cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan); γ-H2AX human anti-rabbit antibody was provided by Arigo Biolaboratories Inc. (Hsinchu, Taiwan); an Annexin V-FITC/PI apoptosis assay kit was purchased from TransDetect (Beijing, China); and Ki67 human anti-rabbit antibody was provided by Affinity Inc. (New York, NY, USA).

Techniques: Flow Cytometry, Immunofluorescence, Staining

Journal: Biology

Article Title: Observation of Histone H2AX Phosphorylation by Radiation-Induced Bystander Response Using Titanium Characteristic X-ray Microbeam

doi: 10.3390/biology12050734

Figure Lengend Snippet: Accumulation of γ-H2AX in Ti K X-ray microbeam-irradiated and non-irradiated HeLa cells. Three targeted cell nuclei were microbeam irradiated at 4 Gy. MBIR (microbeam irradiation) is a fluorescent image captured at the time of microbeam irradiation. Before irradiation, cell nuclei were stained with Hoechst 33258 solution for targeted irradiation of cell nuclei. The red squares are the irradiation positions set by the irradiation software. White dotted circles in the merged images indicate irradiated cell nuclei after fixation and immunofluorescent staining. Bars represent 30 μm.

Article Snippet: WI-38 cells were fixed as described previously [ ] and then incubated overnight at 4 °C with 1% BSA in T-PBS containing anti-γ-H2AX and anti-p53-binding protein 1 (53BP1) antibodies (PC712, Merck) diluted at 1:500.

Techniques: Irradiation, Staining, Software

Journal: Biology

Article Title: Observation of Histone H2AX Phosphorylation by Radiation-Induced Bystander Response Using Titanium Characteristic X-ray Microbeam

doi: 10.3390/biology12050734

Figure Lengend Snippet: Induction of bystander cells after Ti K X-ray microbeam irradiation of the nuclei of three cells in the center of the field of view at a dose of 4 Gy. Unirradiated HeLa cells with γ-H2AX fluorescence intensity higher than the average + σ of control cells were determined to be bystander cells. The error bars represent standard errors of the mean (SEM) based on the four to nine independent experiments. p -values (* p < 0.05, ** p < 0.01, *** p < 0.001) calculated with Tukey’s multiple comparison test.

Article Snippet: WI-38 cells were fixed as described previously [ ] and then incubated overnight at 4 °C with 1% BSA in T-PBS containing anti-γ-H2AX and anti-p53-binding protein 1 (53BP1) antibodies (PC712, Merck) diluted at 1:500.

Techniques: Irradiation, Fluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer

doi: 10.1016/j.apsb.2022.03.003

Figure Lengend Snippet: TBCE regulates chemosensitivity of HCC via affecting the cell cycle and cytoskeleton-related FAK phosphorylation pathway. (A, B) KEGG (A) and GSEA (B) analysis of DEGs showed that TBCE knockdown affects the cell cycle and G2/M checkpoint. (C) Heat map of cell cycle-related genes. (D, E) Representative flow cell cycle test chart (D) and statistical analysis (E) showed that TBCE knockdown significantly increased the DDP-induced G2/M block. (F) WB analysis of the FAK/AKT/ERK pathway showed that the activated FAK pathway in drug-resistant BEL-7402R cells and TBCE knockdown could block its phosphorylation activation. (G) TBCE silencing combined with DDP chemotherapy could significantly increase the activation of the caspase three apoptotic pathway and cause DNA damage ( γ H2AX). Data are presented as mean ± SD ( n = 3), ∗∗∗ P < 0.001 vs. indicated.

Article Snippet: Standard procedures were followed to conduct an IHC assay of TBCE (Abcam, ab224673, Cambridge, UK), γ H2AX (Servicebio, GB111841, Guangzhou, China), Ki67 (Servicebio, GB111499, Guangzhou, China), and cleaved-Caspase-3 (Servicebio, GB11532, Guangzhou, China) in the tumor sections.

Techniques: Phospho-proteomics, Knockdown, Blocking Assay, Activation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer

doi: 10.1016/j.apsb.2022.03.003

Figure Lengend Snippet: Endosomal-responsive NP-mediated TBCE knockdown increases chemosensitivity in subcutaneous tumor models. (A) Schematic illustration of BEL-7402R subcutaneous tumor models and the treatment process. (B, C) Tumor growth (B) and tumor weight (C) in different treatments of tumor-bearing mice. (D, E) Representative pictures of tumor-bearing mice (D) and tumor specimens (E). (F) The expression of TBCE, γ H2AX, Ki67, and cleaved caspase-3 determined by IHC in BEL-7402R tumors collected at the end point. Scale bar = 100 μm. Data are presented as mean ± SEM ( n = 5), ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. indicated.

Article Snippet: Standard procedures were followed to conduct an IHC assay of TBCE (Abcam, ab224673, Cambridge, UK), γ H2AX (Servicebio, GB111841, Guangzhou, China), Ki67 (Servicebio, GB111499, Guangzhou, China), and cleaved-Caspase-3 (Servicebio, GB11532, Guangzhou, China) in the tumor sections.

Techniques: Knockdown, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer

doi: 10.1016/j.apsb.2022.03.003

Figure Lengend Snippet: Endosomal responsive NP-mediated TBCE silencing enhances chemosensitivity in PDX tumor models. (A) Schematic illustration of PDX models and different treatments. The tumors of platinum-resistant HCC patients were cut into small, 1 mm 3 pieces, which were planted on the backs of first-generation NSG mice (P0). When the tumors grew to 1 cm 3 , the tumors were cut and passed down (P1). The procedure above was repeated to obtain the second and third generation of tumor-bearing mice. The third generation tumors were allowed to grow to about 200 mm 3 , and mice were treated with four rounds of PBS, NP(siTBCE), Free DDP, NP(siNC+DDP), or NP(siTBCE+DDP). (B, C) Relative tumor size (B) and tumor weight (C) of collected tumors. (D, E) Tumor specimens (D) and mice body weight (E) of different groups. (F) The expression of TBCE, γ H2AX, Ki67, and cleaved caspase-3 determined by IHC in PDX tumors collected at the end point (Day 14). Scale bar = 100 μm. Data are presented as mean ± SEM ( n = 5), n.s., P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . indicated.

Article Snippet: Standard procedures were followed to conduct an IHC assay of TBCE (Abcam, ab224673, Cambridge, UK), γ H2AX (Servicebio, GB111841, Guangzhou, China), Ki67 (Servicebio, GB111499, Guangzhou, China), and cleaved-Caspase-3 (Servicebio, GB11532, Guangzhou, China) in the tumor sections.

Techniques: Expressing