Journal: Regenerative biomaterials

Article Title: Fe-Zn alloy, a new biodegradable material capable of reducing ROS and inhibiting oxidative stress.

doi: 10.1093/rb/rbae002

Figure Lengend Snippet: Figure 5. Effects of different positions around alloy samples on ROS production, cell viability and DMA damage. (A) Schematic diagram of P1, P2 and P3 positions around the alloy (B–D) are ROS production, cell viability (living cell/green, dead cell/red) and DNA damage, respectively, at P1, P2 and P3 positions. DAPI (blue) was used for the identification of cell nuclei and c-H2AX fluorescence (green) for DNA damage.

Article Snippet: DNA damage assay kit (Beyotime Biotechnology, China) was selected to detect the DNA damage marker c-H2AX (phosphorylated H2AX) by immunofluorescence staining.

Techniques: Fluorescence

Journal: DNA repair

Article Title: Quantitative Analysis of ATM Phosphorylation in Lymphocytes

doi: 10.1016/j.dnarep.2019.06.002

Figure Lengend Snippet: H2AX activation in human PBMCs. (A) Comparison of H2AX and ATM activation in 41 volunteers following 2 Gy IR (Spearman correlation coefficient ρ = −0.09, p-value=0.58). (B) H2AX activation in females versus males (p = 0.042).

Article Snippet: Antibodies: phospho-S1981-ATM (clone EP1890Y, cat#:ab81292, Abeam); pan-ATM (clone 2C1, cat#GTX70103, GeneTex); phosphoserine-139 histone H2AX (clone 20E3, cat#9718, Cell Signaling); histone H2AX (clone 322105, cat#MAB3406, R&D Systems); IRDye-800CW (cat#926-32211, LI-COR Biotechnology-US); IRDye-680RD (cat#926-68070, LI-COR).

Techniques: Activation Assay

Journal: Frontiers in Oncology

Article Title: cGAS/STING-mediated upregulation of NKG2D ligands in LSCs contributes to enhanced sensitivity to NK cells

doi: 10.3389/fonc.2026.1764427

Figure Lengend Snippet: PARP1 is involved in the regulation of NKG2D ligand expression in LSCs. (A) WB was used to detect the knockdown efficiency of PRAP1 in LSCs. GAPDH was employed as an internal loading control. (B) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B in PARP1-stable knockdown or -scramble control LSCs were detected by flow cytometry (BD FACSCalibur). Mouse IgG-APC was used as isotype control. (C) Formation of γH2AX foci mediated by the pretreatment of 1 μM PARP1 inhibitor (PARPi) for 6 hours in LSCs was detected by immunofluorescence and observed in a fluorescence microscope (Zeiss). DMSO was used as vehicle control. Scale bars, 10 μm. (D) The protein expression level of γH2AX, H2AX, phosphorylated-ATM (Ser1981), ATM, and PARP1 in LSCs pretreated with 1μM PARPi or Vehicle for 6 hours were detected by WB. GAPDH was employed as an internal loading control. (E) Quantification of the WB shown in (D) . The Integrated Optical Density of bands was measured by the software ImageJ. Data was presented as mean ± SD from three experiments. (F) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B on the cell surface of LSCs treated with 1 μM PARPi or co-treated with 1 μM PARPi and 1 μM ATM inhibitor (ATMi) for 6 hours were measured by flow cytometry (BD FACSCalibur). DMSO was used as Vehicle control. Mouse IgG-APC was used as isotype control. ***p < 0.001. ns, no significance.

Article Snippet: Antibodies against PARP1 (Cat #: 66520-1-Ig, Proteintech), cGAS (Cat # : 79978, Cell signaling technology), STING (Ca # : 13647, Cell signaling technology), phospho-STING (Ser366) (Cat#:72650, Cell signaling technology), phospho-TBK1 (Ser172) (Cat#: 5483, Cell signaling technology), TBK1 (Cat#: 3504, Cell signaling technology), IRF3(Cat#: 4302, Cell signaling technology), phospho-IRF3 (Ser396) (Cat#: 4947, Cell signaling technology), ATM (Cat#: 27156-1-AP, Proteintech), phospho-ATM (Ser1981) (Cat#: 5883, Cell signaling technology), phospho-γH2AX (Ser139) (Cat#: 83307-2-RR, Proteintech), H2AX (Cat#: 39689, Proteintech), GAPDH (Cat#: 60004-1-Ig, Proteintech), α-Tubulin (Cat#: 14555-1-AP, Proteintech) were used as primary antibodies in the western blotting assay; Goat Anti-Mouse IgG antibody, HRP conjugated (Cat#: 2900264, Millipore), and Goat Anti-Rabbit IgG antibody, HRP conjugated (Cat#: 3256751, Millipore) were used as second antibodies.

Techniques: Expressing, Knockdown, Control, Flow Cytometry, Immunofluorescence, Fluorescence, Microscopy, Software

Journal: Scientific Reports

Article Title: Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose

doi: 10.1038/s41598-020-64672-3

Figure Lengend Snippet: Fractionated, compared to single exposure, radiation profoundly suppresses levels of KLF2 and KLF4. Representative (3–5 independent experiments) Western blot analysis and quantification of KLF2 (n = 5) and KLF4 (n = 3) levels in whole-cell lysates from nonirradiated (sham) and irradiated HUVECs 4 h and 24 h after exposure to ( a ) either five fractions of 2 Gy (5 × 2 Gy) or single exposure to 10 Gy and ( b ) either five fractions of 2.5 Gy (5 × 2.5 Gy) or single exposure to 12.5 Gy. Fractions delivered at 24-h intervals. β-actin served as a loading control. c Representative photomicrograph (20× magnification) showing immunofluorescence of KLF2 (red) in nonirradiated (sham) and irradiated HUVECs 4 h after exposure to either five fractions of 2 Gy or single exposure to 10 Gy (n = 3). Nuclei were stained with DAPI (blue). d Representative Western blot analysis and quantification of KLF2 (n = 3) and γ-H2AX (n = 3) phosphorylation levels in nonirradiated (0 h) and irradiated HUVECs at indicated time intervals after single exposure to 2 Gy and 5 Gy. β-actin served as a loading control. (n, number of independent experiments performed; a, significant statistical difference between nonirradiated and irradiated groups; b, significant statistical difference between fractionated irradiation and single exposure; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: Antibodies to TM (Cat # NBP1-95319), ICAM-1 (Cat # NBP2-22541), eNOS (Cat # NB300–500), E-selectin (Cat #NBP1-45545), and gamma H2AX (Cat # NB100-384) were procured from Novus Biologicals.

Techniques: Western Blot, Irradiation, Control, Immunofluorescence, Staining, Phospho-proteomics

Journal: PLoS ONE

Article Title: Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction

doi: 10.1371/journal.pone.0248941

Figure Lengend Snippet: (A, C, and E) RAD52 and γH2AX (B, D, and F) levels were probed in the three parental (black) and (BRCA1 or BRCA2 proficient, grey) cell lines indicated was assessed with Simple Western using lysate post 72 h treatment with mitoxantrone at doses near their EC 50 values (see Figs – ). Data and ANOVA statistical analyses are described in the material and methods section. Significance statistics are shown with single, double, or triple asterisks representing P<0.05, P<0.005, or P<0.0005, respectively.

Article Snippet: Primary antibody concentrations were optimized and were diluted as follows: Abcam anti-RAD52 antibody (ab18263, diluted 1:25), R&D Systems Human Phospho-histone H2AX (S139) antibody (AF2288, diluted 1:100), and Cell Signalling Technology Pan-Actin antibody (#4968, diluted 1:2500).

Techniques: Simple Western