Journal: Cancer research

Article Title: Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin and Other Platinum Complexes

doi: 10.1158/0008-5472.CAN-14-0247

Figure Lengend Snippet: Comparison of the effect of DMSO on cisplatin, carboplatin and oxaliplatin-mediated DNA damage and cell death. Cellular DNA damage recognition caused by platinum drugs observed via confocal images (63 x) of DLD-1 cells exposed to platinum drugs as indicated, and stained with an antibody that recognizes phosphorylated (γ) H2A.X (A). Immunoblot (B) of protein lysate from DLD-1 cells treated as described for (A) and probed for expression of total PARP, cleaved PARP, cleaved caspase-3 and phosphor (γ) H2A.X, with β-actin probed as a control for protein loading.

Article Snippet: H2AX immunofluorescence staining Fixed DLD-1 cells were stained with Phospho-Histone H2A.X (Se139) (20E3) Rabbit mAb (Alexa Fluor 488 Conjugate; all components from Cell Signaling Technology, Danvers, MA) following the manufacturer's instructions, and as previously described ( 22 ).

Techniques: Staining, Western Blot, Expressing

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Hydrogen peroxide and hypoxia resulted in DNA damage, G2/M arrest and senescence in HK-2 cells. (A) Representative western blots analysis of γH2A.X (ser139), and bar graphs showed the fold changes compared to control group at different time points. (B) Flow cytometry analysis of cell cycle. * P < 0.05, **** P < 0.0001, compared with control group. (C) SA-β-gal staining analysis at different time points. (D) EdU incorporation analysis at 48 h. (E) Representative SA-β-gal staining at 48 h, Scale bars, 50 μm. (F) Representative EdU incorporation images at 48 h showed decreased proliferation rate and larger cell size in H 2 O 2 and hypoxia groups, Scale bars, 50 μm. n = 3–5/group. Data are means ± SD. SA-β-gal, Senescence Associated β-Galactosidase; EdU, 5-Ethynyl-2′-deoxyuridine.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Western Blot, Flow Cytometry, Staining

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) attenuated hydrogen peroxide and hypoxia induced injuries and senescence in HK-2 cells. (A) Cell viability percentage of HK-2 cells. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with H 2 O 2 or hypoxia group. (B) Quantification of NAD + levels in HK-2 cells. (C,D) Flow cytometry analysis of γH2A.X (ser139) in H 2 O 2 group, and bar graph showed decreased DNA damage in NMN-treated group. (E) Representative SA-β-gal staining images, Scale bars, 50 μm. (F) Bar graphs showing significantly decreased percentage of SA-β-gal positive cells in NMN-treated groups at a dose of 1 mM. (G,H) Western blots analysis showed reduction of collagen IV in NMN-treated groups, compared with H 2 O 2 or hypoxia group. n = 3–5/group. Data are means ± SD. NMN, Nicotinamide Mononucleotide.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Staining, Western Blot

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) administration before ischemia and during acute phase attenuated tubular DNA damage and cellular injury in uIRI mice at day 3 after surgery. (A) Schematic protocol of the experiments. (B) Representative images of HE (hematoxylin-eosin) staining and histologic analysis of tubular damage. Scale bars, 100 μm. (C) Quantification of damaged tubules of ischemic kidneys in (B) . (D) Representative images of in situ TUNEL assay. Scale bars, 50 μm. (E) Quantification of apopotic tubular cells in ischemic kidneys in (D) . (F) Western blots analysis of γH2A.X (ser139) showed decreased DNA damage in ischemic kidneys of NMN-treated mice compared with ischemic kidneys of PBS-treated mice. (G) Representative images of co-immunostaining of anti-Ki-67 and anti-p-H3 in kidney tissues. Scale bars, 50 μm. (H) Bar graphs showed no changes in number of Ki-67 positives tubular cells in NMN-treated mice, but a markedly reduction of tubular cells in G2/M phase compared with PBS-treated mice. n = 6–10/group. Data are means ± SD.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Staining, In Situ, TUNEL Assay, Western Blot, Immunostaining

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) administration before ischemia and during acute phase attenuated tubular senescence in uIRI mice at day 21 after surgery. (A) Representative western blots analysis of γH2A.X (ser139). (B) Bar graph showed reduction of DNA damage in NMN-treated group. (C,D) SA-β-gal staining analysis showed decreased senescent tubular cells in NMN-treated uIRI mice. Scale bars, 50 μm. (E) Relative expression of IL-6, IL-8, and TGF-β1 mRNAs after normalization with β-actin. n = 6–7/group. Data are means ± SD.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Western Blot, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) administration at recovery phase attenuated uIRI induced DNA damage, inflammation and fibrosis at day 21 after surgery. (A) Schematic protocol of the experiments. (B,C) Representative western blots analysis of γH2A.X (ser139), and bar graph showed reduction of DNA damage in NMN-treated group. (D) Representative images of SA-β-gal staining in both groups. Scale bars, 50 μm. (E) Quantification of expression of SA-β-gal in (D) . (F) Relative expression of IL-6, IL-8, and TGF-β1 mRNA after normalization with β-actin. (G,H) Representative sirius red staining images and semiquantitative analysis of the percentage of positive area. Scale bars, 100 μm. (I,J) Representative western blots and semiquantitative analysis of Collagen IV (Col IV), n = 6/group. Data are means ± SD.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Western Blot, Staining, Expressing

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: A Pt(IV)-conjugated brain penetrant macrocyclic peptide shows pre-clinical efficacy in glioblastoma

doi: 10.1016/j.jconrel.2022.10.051

Figure Lengend Snippet: Dose-regime and efficacy studies of Pt(IV)-M13 reveal DNA-damage effects on GBM tumors. a) Percentage of weight variation of naïve nude mice administered after platinum compound tail-vein injection at the indicated concentrations. Black arrows indicate days of injection (d1, d4, d9, d13, d17, d19). Mean and standard deviation are shown, n = 5 animals per group. b) Average clinical score from study shown in a). Mice were monitored every 2 days for symptomatic changes. Symptoms were classified from no symptoms (0), visible (2), to fatal (4). Eye redness, eye twitching and weight loss were the most visible effects. c) Efficacy study of Pt(IV)-M13 (5 mg/kg, 0.3 mM) compared to cisplatin MTD (5 mg/kg, 3 mM). d) Immunostaining of ϒH2AX (Ser139) using anti-rabbit Alexa Fluor 647, and nuclei with Hoechst 33342. Representative image of independent tissue triplicates from the efficacy study c). Scale bar, 10 μm. e) Area percentage of positive cells with ϒH2AX foci within the tumor core from study in d). Statistical significance for c) using log-rank test, *p = 0.020. Median survival for each group is shown. For e), mean and standard deviation are shown, n = 3. Two-way ANOVA test **p < 0.005, ***p = 0.0004.

Article Snippet: For DNA damage staining, tissue slides were incubated with rabbit or mouse anti-phospho-ser139 histone γH2AX antibody (Cell Signaling Technologies) at a dilution of 1:100.

Techniques: Injection, Standard Deviation, Immunostaining