Journal: Cells

Article Title: Histone Chaperone Nrp1 Mutation Affects the Acetylation of H3K56 in Tetrahymena thermophila

doi: 10.3390/cells11030408

Figure Lengend Snippet: Nrp1 mutation leads to reductions in H3K56ac and H4K5ac. ( A ) Indirect immunofluorescence localization of H3K56ac and H4K5ac. The cells were induced with 0.3 µg/mL Cd 2+ for 24, 48, and 96 h. The primary antibody was histone H3K56ac or H4K5ac rabbit polyclonal antibody, and the secondary antibody was FITC-conjugated goat anti-rabbit. White arrows indicate MIC, while white arrowheads indicate MAC. Scale bar, 10 µm. ( B ) Western blot analysis of H3K4me3, H3K56ac, and H4K5ac. Histone extracts prepared from WT and Nrp1 TrNLS1 cells at the vegetative growth stage were induced with 0.3 µg/mL Cd 2+ for 96 h. The extracted histone samples were separated by 15% SDS–PAGE. The gels were transferred to polyvinylidene difluoride membranes and probed by using anti-H3K4me3, H3K56ac, and H4K5ac antibodies. ( C ) The intensities of H3K4me3, H3K56ac, and H4K5ac were obtained by ImageJ software. The fluorescence of H3K4me3, H3K56ac, and H4K5ac in WT cells was arbitrarily set as 1, and the Nrp1 TrNLS1 mutation fluorescence was normalized to signal from WT cells.

Article Snippet: The membrane was blocked with 5% skim milk in TBST and incubated with the antibodies H3K4me3 (1:500 dilution; ab8580, abcam, Cambridge, UK), H4K5ac (1:300; bs-10721R, Bioss, Beijing, China), and H3K56ac (1:500; AB_2661786, USA) and HRP-conjugated secondary antibody (diluted 1:1000, A9169, Sigma, Santa Clara, CA, USA).

Techniques: Mutagenesis, Immunofluorescence, Western Blot, SDS Page, Software, Fluorescence

Journal: bioRxiv

Article Title: FLASH X-rays preserve neutrophil chromatin to boost antitumor immunity

doi: 10.64898/2026.10.11.758276

Figure Lengend Snippet: a , b , NETs were visualized by staining for MPO (magenta) and extruded DNA (SYTOX Green) at 4 h after the indicated treatments. Representative confocal images are shown in ( a ), and quantification of NETs is shown in ( b ). c , d , Western blot analysis of histone acetylation levels in mouse neutrophils following 8-Gy CONV or FLASH irradiation at the indicated times post-irradiation ( c ) and the corresponding quantification ( d ). e – h , Radiation-induced PARylation was inhibited by PJ34 pretreatment. Protein PARylation levels ( e ) and histone acetylation levels ( f ) were examined and quantified by Western blot immediately after 8-Gy irradiation. Representative confocal images of NETs at 4 h after 8-Gy CONV irradiation are shown in ( g ), and quantification of NETs is shown in ( h ). i – k , FLASH X-ray exposure was fractionated equally by introducing a 5-min interval (termed split-dose FLASH). Protein PARylation levels ( i ) and histone acetylation levels ( j ) were examined and quantified by Western blot immediately after FLASH irradiation (either continuous or fractionated) with or without PJ34 pretreatment, and NET formation was assessed by confocal microscopy at 4 h after 8-Gy FLASH irradiation ( k ). Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA ( b , e , f , h , i , j ) and two-way ANOVA followed by Šídák’s multiple comparisons test (for each time point between groups) ( d ). NI, Non-irradiated; TPI, time post-irradiation; im, immediately.

Article Snippet: The primary antibodies used were as follows: anti-Histone H3 (1:1,000, CST, 4499), anti-Acetyl-Histone H3 (1:2,000, Active Motif, 39140), anti-H3K9ac (1:2,000, Active Motif, 39918), anti-H3K27ac (1:2,000, Active Motif, 39134), anti-H3K14ac (1:2,000, Active Motif, 65702), anti-H3K56ac (1:2,000, Active Motif, 61061), anti-H4K5ac (1:2,000, Active Motif, 39700), anti-poly/mono-ADP ribose (1:1,000, CST, 89190), and anti-GAPDH (1:1,000, Beyotime Inc., AF0006).

Techniques: Staining, Confocal, Western Blot, Analysis, Irradiation, Microscopy