Journal: Oncology Letters

Article Title: STAT3 is a key molecule in the oncogenic behavior of diffuse intrinsic pontine glioma

doi: 10.3892/ol.2020.11699

Figure Lengend Snippet: STAT3 inhibition sensitizes human diffuse intrinsic pontine glioma cells to radiation by interfering with DNA damage repair. SF8628 cells were treated with control vehicle (DMSO) or AG490. SF8628 cells were transfected with shCtrl or shSTAT3. (A and B) CCK-8 assays of control cells and STAT3-inhibited cells at 0 and 24 h after radiation treatment. Cell viability was analyzed using a CCK-8 assay, and absorbance was measured at 420 nm. *P<0.05 using one-way ANOVA. Data are presented as the mean ± SD. (C and D) Analysis of DNA damage repair after treatment with 4 Gy radiation by visualizing the double-strand marker γH2AX. The panel shows representative images of γH2AX (green), DAPI-stained nuclei (blue) and merged images of SF8628 cells without irradiation and at 1, 4 and 24 h after radiation. Scale bar, 1 µm. STAT3, signal transducer and activator of transcription 3; shCtrl, control short hairpin RNA; shSTAT3, STAT3 short hairpin RNA; CCK-8, Cell Counting Kit-8; γH2AX, phosphorylated H2A X variant histone; DAPI, 4′6′-diamidio-2-phenoylindole.

Article Snippet: After blocking with 5% skim milk diluted in 0.05% Triton X-100 for 30 min at room temperature, the slides were washed for 5 min in TTBS and incubated with a primary rabbit anti-γH2AX antibody (clone 2577; cat. no. 2577; Cell Signaling Technology, Inc.) at a 1:200 dilution in TTBS for 2 h at room temperature in a humidified chamber.

Techniques: Inhibition, Transfection, CCK-8 Assay, Marker, Staining, Irradiation, shRNA, Cell Counting, Variant Assay

Journal: Scientific Reports

Article Title: Toxoflavin analog D43 exerts antiproliferative effects on breast cancer by inducing ROS-mediated apoptosis and DNA damage

doi: 10.1038/s41598-024-53843-1

Figure Lengend Snippet: D43 induces DNA damage by increasing the level of ROS in TNBC cells. ( A ) RNA sequencing of HCC1806 cells treated with D43 (1 μM) for 24 h revealed a KEGG enrichment scatterplot. ( B ) D43 caused DNA damage in a concentration-dependent manner. Western blot assays were performed to detect the expression of ATR, p-CHK1, CHK1, and γH2AX in MDA-MB-231 and HCC1806 cells treated with D43 at different concentrations for 48 h with tubulin as a loading control. ( C ) D43 caused DNA damage in a time-dependent manner. After treatment of D43 with a time gradient (0, 6, 12, 24, 36, 48 h), the above proteins were also detected by Western blotting. The samples derive from the same experiment and that blots were processed in parallel. Original blots are presented in . ( D ) D43 stably increased the ROS level in TNBC cells. MDA-MB-231 and HCC1806 cells treated with a D43 concentration gradient for 48 h were labeled with the fluorescent probe DCFH-DA, and ROS levels were detected by flow cytometry. ( E ) Statistics of relative ROS levels. ( F ) D43 was able to activate the NRF2-HO-1 signaling axis in response to oxidative stress in a dose-dependent manner. Western blot assays were performed to detect the expression of NRF2 and HO-1 after treatment with D43 at different concentrations for 48 h. ( G ) D43 treatment resulted in a higher number of γ-H2AX foci. MDA-MB-231 and HCC1806 cells were treated with a D43 concentration gradient for 24 h, and the expression of γ-H2AX was detected by immunofluorescence staining and recorded by high-resolution fluorescence microscopy. ( H ) Four samples were taken from each treatment group to count the proportion of positive cells with more than 10 foci in the cells. Data are representative of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001; ns, not significant.

Article Snippet: They were permeabilized with 0.2% Triton X-100 for 5 min and blocked with PBS containing 3% BSA for 1 h. The fixed cells were washed with PBS and then incubated with a primary anti-phospho-histone H2AX-Ser139 antibody (AP0687, ABclonal) in 3% BSA overnight at 4 °C, followed by incubation with Alexa Fluor 594 (Invitrogen) for 1 h and mounting with mounting medium containing DAPI.

Techniques: RNA Sequencing, Concentration Assay, Western Blot, Expressing, Control, Stable Transfection, Labeling, Flow Cytometry, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Toxoflavin analog D43 exerts antiproliferative effects on breast cancer by inducing ROS-mediated apoptosis and DNA damage

doi: 10.1038/s41598-024-53843-1

Figure Lengend Snippet: NAC rescued D43-induced DNA damage and ROS upregulation. ( A ) NAC partially alleviated the cell death induced by D43 in HCC1806 and MDA-MB-231 cells. After pretreatment with NAC (1 mM) for 6 h, MDA-MB-231 (0.5 μM) and HCC1806 (1 μM) cells were treated with D43 for 48 h, and cell viability was verified by SRB. ( B ) Cell proliferation was verified by colony formation assay after treatment with NAC and D43, respectively, or in combination. ( C ) Western blot analysis showed that pretreatment with NAC (1 mM, 6 h) antagonized D43-induced increase of γH2AX protein levels. The samples derive from the same experiment and that blots were processed in parallel. Original blots are presented in . ( D ) NAC effectively antagonized the D43-induced upregulation of ROS. MDA-MB-231 and HCC1806 cells were treated with D43 or NAC for 48 h and labeled with the fluorescent probe DCFH-DA, and ROS levels were measured by flow cytometry. ( E ) Statistics of relative ROS levels. ( F ) NAC antagonized the upregulation of γ-H2AX foci formation induced by D43. After NAC pretreatment for 6 h, MDA-MB-231 (0.5 μM) and HCC1806 (1 μM) cells were treated with D43 for 24 h, and the expression of γ-H2AX was detected by immunofluorescence staining and recorded by high-resolution fluorescence microscopy. ( G ) Four samples were taken from each treatment group to count the proportion of positive cells with more than 10 foci in the cells. Data are representative of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001; ns, not significant.

Article Snippet: They were permeabilized with 0.2% Triton X-100 for 5 min and blocked with PBS containing 3% BSA for 1 h. The fixed cells were washed with PBS and then incubated with a primary anti-phospho-histone H2AX-Ser139 antibody (AP0687, ABclonal) in 3% BSA overnight at 4 °C, followed by incubation with Alexa Fluor 594 (Invitrogen) for 1 h and mounting with mounting medium containing DAPI.

Techniques: Colony Assay, Western Blot, Labeling, Flow Cytometry, Expressing, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Toxoflavin analog D43 exerts antiproliferative effects on breast cancer by inducing ROS-mediated apoptosis and DNA damage

doi: 10.1038/s41598-024-53843-1

Figure Lengend Snippet: D43 inhibits the growth of patient-derived TNBC organoids and xenograft tumors. ( A ) Tissue information of TNBC patients sampled from Yunnan Cancer Hospital. ( B ) Treatment with D43 compromised the structural integrity of TNBC PDOs. Morphological records of breast tumor organoids at different D43 treatment concentrations for different days. The red arrow points to the disintegrating or completely disintegrating TNBC PDOs. ( C ) D43 inhibited the ATP activity of TNBC PDOs. An ATPase activity kit was used to detect the changes in the viability of TNBC PDO cells treated with different D43 concentrations for 5 days. ( D ) D43 had a significant inhibitory effect on the growth of TNBC PDOs. The area of PDOs was calculated by ImageJ, and the growth curve was drawn. ( E ) Schematic diagram of subcutaneous tumor model construction in nude mice and administration time and frequency. ( F ) D43 significantly suppressed tumor growth in nude mice. After 15 days of continuous administration, the mice were sacrificed by cervical dislocation, and tumors were removed and photographed. ( G ) The tumor weights of control (DMSO) and experimental (5 mg/kg) mice are shown. ( H ) The tumor volume was weighed every 3 days after the beginning of the drug administration treatment and is shown as a line chart. ( I ) Changes in body weight of mice before and after D43 administration. ( J – L ) Immunohistochemistry was used to detect the expression of Ki-67 and γH2AX in tumor tissues, and γH2AX immunoreactivity was scored semiquantitatively. *p < 0.05, **p < 0.01 and ***p < 0.001; ns, not significant.

Article Snippet: They were permeabilized with 0.2% Triton X-100 for 5 min and blocked with PBS containing 3% BSA for 1 h. The fixed cells were washed with PBS and then incubated with a primary anti-phospho-histone H2AX-Ser139 antibody (AP0687, ABclonal) in 3% BSA overnight at 4 °C, followed by incubation with Alexa Fluor 594 (Invitrogen) for 1 h and mounting with mounting medium containing DAPI.

Techniques: Derivative Assay, Activity Assay, Control, Immunohistochemistry, Expressing

Journal: Acta Pharmacologica Sinica

Article Title: Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

doi: 10.1038/aps.2014.106

Figure Lengend Snippet: Nutlin-3 causes DNA DSB damage in SMMC-7721 cells. (A) Nutlin-3 increased γH2AX expression level. β-Actin served as the loading control. (B) The relative expression levels of γH2AX. The value represents the mean±SD derived from triplicate tests. bP<0.05 vs NC group. (C) Representative images of γH2AX foci formation (1000×). SMMC-7721 cells were treated with 0.1% DMSO, Etoposide (40 μmol/L), or Nutlin-3 (10 μmol/L) for 48 h and analyzed for γH2AX (red). Nuclei were counter-stained with DAPI (blue). The images were merged using Image-Pro plus 6.0. (D) Statistical analysis of the number of γH2AX foci. Data are shown as the mean±SD (n=3). bP<0.05 vs NC group.

Article Snippet: The primary antibodies were rabbit anti-γH2AX polyclonal antibody (bs-3185R, Bioss, diluted 1:200), mouse anti-IFI16 monoclonal antibody (ab50004, Abcam, diluted 1:1000), rabbit anti-H2b polyclonal antibody (BS1657, Bioworld, diluted 1:500), and β-actin antibody (BA2305, Boster, diluted 1:500).

Techniques: Expressing, Derivative Assay, Staining