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: International Journal of Molecular Sciences

Article Title: Exploring the Physical and Biological Aspects of BNCT with a Carboranylmethylbenzo[ b ]acridone Compound in U87 Glioblastoma Cells

doi: 10.3390/ijms232314929

Figure Lengend Snippet: DSB quantification using the ɣ-H2AX assay (up); ( A ) represents the nuclei stained by DAPI, ( B ) the foci, pink dots, identified by TXR. The number of MN per BN cell is also presented for CMBA (down) ( C ). The error bars represent the standard deviation of the three independent experiments. The images were obtained using 64× magnification.

Article Snippet: Then, cells were incubated with an anti-γ-H2AX primary antibody (mouse anti-γ-H2AX (ser139), Stressgen, bioreagents Corp., Canada) at 2 μg/mL for 1 h. After being washed twice with 1% BSA in PBS, cells were incubated with a FITC-conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 mg/mL for 1 h, followed by three washing steps with PBS.

Techniques: Staining, Standard Deviation

Journal: Brain Communications

Article Title: Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration

doi: 10.1093/braincomms/fcz005

Figure Lengend Snippet: Targeting the MRN complex protects against Alzheimer’s disease-relevant phenotypes. ( A ) Expression of Aβ 1-42 in clock neurons leads to loss of Per + cells. In nbs −/+ flies, loss of cells is partially prevented (Kruskal–Wallis with Dunn’s post hoc test; P -values and n are indicated). ( B ) The free-running circadian locomotor cycle of control flies is approximately 24 h but is considerably lengthened when Aβ 1-42 is expressed in clock neurons indicating weaker rhythmicity in the circadian circuitry. The increase is suppressed in nbs 1/+ flies (CLEAN spectral analysis; comparisons by ANOVA with Tukey’s post hoc test; P -values and n are indicated). ( C ) Exposure of rat hippocampal neurons to Aβ 1-42 oligomers in vitro generate double-strand breaks that can be visualized by staining with anti-γH2Ax (red). DNA is visualized with DAPI (blue). ( D ) Quantification of synapsin levels in hippocampal neurons by western blot. Exposure to Aβ 1-42 oligomers leads to loss of the pre-synaptic protein, synapsin, which is reversed by the Mre11 inhibitor, mirin (mean ± SEM; ANOVA with Tukey’s post hoc test; P -values and n are indicated). Scale bars in C = 10 µm.

Article Snippet: Primary antibodies used were: mouse anti-H2Ax pSer139 (γH2Ax; JBW301; 1:400 dilution; Merck) and rabbit anti-neurofilament 200 (1:400 dilution; Sigma); mouse anti-GAP43 (ThermoFisher; 1:400 dilution) was used to detect regenerating axons in the optic nerve and spinal cord.

Techniques: Expressing, In Vitro, Staining, Western Blot

Journal: Brain Communications

Article Title: Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration

doi: 10.1093/braincomms/fcz005

Figure Lengend Snippet: Inhibition of Mre11 prevents RGC apoptosis and stimulates neurite outgrowth after 4 days in culture in the presence of inhibitory CNS myelin extracts. ( A ) Western blot and ( B ) subsequent densitometry to show attenuated levels of anti-γH2Ax after treatment with mirin and KU-60019. ( C ) Mirin or KU-60019 significantly enhanced RGC survival. ( D ) Representative images from RGC treated with vehicle, CNTF (positive control), mirin and KU-60019. Mirin or KU-60019 treatment ( E ) increased the mean RGC neurite length and ( F ) % RGC with neurites. n = 3 wells/treatment, three independent repeats (total n = 9 wells/condition). AU = arbitrary units. Comparisons in B , C , E and F by one-way ANOVA with Dunnett’s post hoc test. Scale bars in D = 100 µm.

Article Snippet: Primary antibodies used were: mouse anti-H2Ax pSer139 (γH2Ax; JBW301; 1:400 dilution; Merck) and rabbit anti-neurofilament 200 (1:400 dilution; Sigma); mouse anti-GAP43 (ThermoFisher; 1:400 dilution) was used to detect regenerating axons in the optic nerve and spinal cord.

Techniques: Inhibition, Western Blot, Positive Control

Journal: Brain Communications

Article Title: Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration

doi: 10.1093/braincomms/fcz005

Figure Lengend Snippet: Inhibition of Mre11 and ATM prevents RGC apoptosis and stimulates axon regeneration at 24 days after optic nerve crush injury. ( A ) Western blot and ( B ) subsequent densitometry to show phosphorylation of H2Ax (γH2Ax) as a marker of DNA damage after optic nerve crush injury ( n = 6 retinae/time point, three independent repeats (total n = 18 retinae/time point)). ( C ) Western blot and ( D ) densitometry to show that mirin and KU-60019 significantly suppress optic nerve crush injury-induced γH2Ax levels ( n = 6 retinae/time point, three independent repeats (total n = 18 retinae/condition). ( E ) Representative images and ( F ) quantification of FluoroGold backfilled RGC in retinal wholemounts to demonstrate that mirin and KU-60019 significantly enhanced RGC survival at 24 days after optic nerve injury ( n = 6 retinae/time point, three independent repeats (total n = 18 retinae/condition). ( G ) Representative images and ( H ), quantification to show that mirin and KU-60019 significantly enhanced RGC axon regeneration as detected by GAP43 immunoreactivity ( n = 6 nerves/condition, three independent repeats (total n = 18 nerves/condition). AU = arbitrary units. Comparisons in B , D , F and H by one-way ANOVA with Dunnett’s post hoc test. Scale bars in E = 50µm and in G = 200 µm.

Article Snippet: Primary antibodies used were: mouse anti-H2Ax pSer139 (γH2Ax; JBW301; 1:400 dilution; Merck) and rabbit anti-neurofilament 200 (1:400 dilution; Sigma); mouse anti-GAP43 (ThermoFisher; 1:400 dilution) was used to detect regenerating axons in the optic nerve and spinal cord.

Techniques: Inhibition, Western Blot, Marker

Journal: Brain Communications

Article Title: Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration

doi: 10.1093/braincomms/fcz005

Figure Lengend Snippet: Inhibition of Mre11 and ATM reduces the number of DRGN with double-strand breaks and the levels of γH2Ax after DC injury in vivo . ( A ) Representative images from DC + vehicle, DC + mirin and DC+KU-60019-treated sections of rat DRGN ( n = 6 rats/group, three independent repeats; total n = 18 rats/group) demonstrating γH2Ax (green) localization in the nucleus of DRGN (red). Images are counterstained with DAPI (blue) to demarcate the nucleus. ( B ) Quantification of the frequency of γH2Ax + DRGN sorted by different soma size. ( C ) Quantification of the mean pixel intensity/DRGN in DC + vehicle, DC + mirin and DC+KU-60019-treated DRGN. ( D ) Western blot and ( E ) subsequent densitometry to demonstrate reduction of γH2Ax protein levels in DRGN after treatment with mirin and ATM inhibitors. AU = arbitrary units. Comparisons in C and E by one-way ANOVA with Dunnett’s post hoc test. n = 6 rats/treatment, three independent repeats (total n = 18 rats/treatment). Scale bars in A = 50 µm, insets in A = 10 µm.

Article Snippet: Primary antibodies used were: mouse anti-H2Ax pSer139 (γH2Ax; JBW301; 1:400 dilution; Merck) and rabbit anti-neurofilament 200 (1:400 dilution; Sigma); mouse anti-GAP43 (ThermoFisher; 1:400 dilution) was used to detect regenerating axons in the optic nerve and spinal cord.

Techniques: Inhibition, In Vivo, Western Blot