rabbit anti phosphorylated histone h3 ph3  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: The transcription factor Chronophage/BCL11A/B promotes intestinal stem cell proliferation and endocrine differentiation in the Drosophila intestine

doi: 10.1101/2024.08.05.606739

Figure Lengend Snippet: a-b’’’ cph-YFP CPTI-1740 ;esg-Gal4 ts ,UAS-mCherry >UAS-Notch RNAi shows Cph-YFP expression in Notch-RNAi induced tumours. Animals were dissected after 7 days of induction at 29°C and stained with the EE marker Prospero (Pros, greyscale). a-a’’’ shows the expression of Cph in cph-YFP CPTI-1740 ; esg-Gal4 ts > UAS-mCherry control guts. b-b’’’ Cph expression in esg-Gal4 ts > UAS-mCherry, UAS-Notch RNAi guts. Note the distinct overlap in expression of Cph and the ISC/EB-marker escargot ( esg ) in the tumours outlined in white. There are also tumour cells that are Pros + and Cph + (arrowhead). This experiment was carried out three independent times (compiled n-values are as follows : esg-Gal4 ts > UAS-mCherry n =27 , cph-YFP CPTI-1740 ;esg-Gal4 ts >UAS-Notch RNAi n= 80). c qRT-PCR of cph mRNA in esg-Gal4 ts >UAS-Notch RNAi 14 days after induction alongside esg-Gal4 ts > UAS-mCherry control flies. n =15 guts were dissected from each genotype and used for total RNA extraction and cDNA generation. Data show a 34-fold increase in cph expression in the esg-Gal4 ts > UAS-Notch RNAi flies compared to esg-Gal4 ts > UAS-mCherry control flies ( p =3.7e-4). d cph knockdown decreases number of mitotic cells in Notch RNAi guts. Quantification of mitosis in esg-Gal4 ts > UAS-mCherry control , cph RNAi , Notch RNAi and cph RNAi ;Notch RNAi flies. Females of the appropriate genotype were collected, induced at 29°C and dissected after 7 days. Guts were stained with anti-pH3S10 and counted along the length of the intestine. There was a significant decrease in the numbers of pH3 + cells in the cph RNAi ;Notch RNAi compared to the Notch RNAi guts ( p =2.0e-5). The bar chart was compiled from two biological repeats. cph RNAi ;Notch RNAi n =51, Notch RNAi n =23, Cph RNAi n = 14, esg-Gal4 ts > UAS-mCherry n = 15. e Percentage of the portion of gut taken up by tumours in cph RNAi ;Notch RNAi guts is significantly reduced compared to Notch RNAi ( p 1.3e-6). f Loss of cph significantly extends lifespan of Notch RNAi tumour-bearing flies ( p = 4.2e-5). Data in Kaplan-Meier curve is representative of 3 independent repeats using two independent cph RNAi lines (see Supplementary Figure 4).

Article Snippet: The following antibodies were used: rabbit anti-phosphorylated Histone H3 (PH3) (1:500; Cell Signalling Technology Cat# 9701), chicken anti-GFP (1:1000; ThermoFisher A10262), mouse anti-Nubbin/Pdm1 (1:10; DSHB Cat# Nub 2D4O), mouse anti-Prospero (1:50; DSHB Cat# MR1A) and mouse anti-Delta (1:100; DSHB Cat# c594.9b).

Techniques: Expressing, Staining, Marker, Control, Quantitative RT-PCR, RNA Extraction, Knockdown

Journal: Molecular Medicine

Article Title: Advanced glycation end product-modified low-density lipoprotein promotes pro-osteogenic reprogramming via RAGE/NF-κB pathway and exaggerates aortic valve calcification in hamsters

doi: 10.1186/s10020-024-00833-8

Figure Lengend Snippet: RAGE mediates the inflammatory and osteogenic responses as well as NF-κB p65 nuclear translocation induced by AGE-LDL. A Representative immunohistochemical image and semiquantitative analysis show that the levels of RAGE are higher in CAVD group than in the non-CAVD group (bar = 100 µm). B HAVICs were transfected with si-RAGE for 6 h prior to AGE-LDL treatment and immunoblotting analysis showed that the expression of ICAM-1, IL-6, ALP and NF-κB p65 was significantly inhibited. C ELISA showed that the significant increase IL-6 level induced by AGE-LDL treatment was attenuated by si-RAGE in culture media. D Representative images from immunofluorescence staining show that the silencing of RAGE reduces AGE-LDL induced NF-κB p65 translocation to nucleus. n = 5. & P < 0.05. vs. non-CAVD. * P < 0.05. vs. untreated control, $ P < 0.05. vs. si-NC group, # P < 0.05. vs. AGE-LDL + si-NC group alone. The data are presented as the means ± SEM. RAGE, Receptor for advanced glycation end product; si-NC, negative control small interfering RNA; other abbreviations as in Figs. –

Article Snippet: Antibodies against phosphorylated NF-κB (3033, Rabbit mAb), and total NF-κB (8242, Rabbit mAb) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Translocation Assay, Immunohistochemical staining, Transfection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Control, Negative Control, Small Interfering RNA

Journal: Molecular Medicine

Article Title: Advanced glycation end product-modified low-density lipoprotein promotes pro-osteogenic reprogramming via RAGE/NF-κB pathway and exaggerates aortic valve calcification in hamsters

doi: 10.1186/s10020-024-00833-8

Figure Lengend Snippet: The NF-κB signaling is involved in mediating the AGE-LDL induced inflammatory and osteogenic responses in HAVICs. A HAVICs were treated with AGE-LDL for 0–24 h and the phosphorylation of NF-κB were detected by immunoblotting. B Pretreatment of HAVICs with Bay11-7082 prior to AGE-LDL stimulation showed a corresponding decrease in ICAM-1, IL-6 and ALP levels. C ELISA showed that the significant increase IL-6 level induced by AGE-LDL treatment was attenuated by Bay11-7082 in culture media. D Pretreatment of HAVICs with NF-κB si-RNA prior to AGE-LDL stimulation showed a corresponding decrease in ICAM-1, IL-6 and ALP levels. E ELISA showed that the significant increase IL-6 level induced by AGE-LDL treatment was attenuated by Bay11-7082 in culture media. F Pretreatment of HAVICs with Bay11-7082 prior to AGE-LDL stimulation showed that AGE-LDL-induced nuclear translocation of NF-κB p65 was abolished significantly inhibited. (bar = 50 μm). G Pretreatment of HAVICs with Bay11-7082 prior to AGE-LDL stimulation and stained by Alizarin red S staining showed that AGE-LDL-induced calcium deposit formation was significantly reduced. (bar = 200 µm). n = 3. * P < 0.05. vs. untreated control, # P < 0.05. vs. DMSO group, & P < 0.05. vs. DMSO + AGE-LDL. The data are presented as the means ± SEM

Article Snippet: Antibodies against phosphorylated NF-κB (3033, Rabbit mAb), and total NF-κB (8242, Rabbit mAb) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Translocation Assay, Staining, Control

Journal: Molecular Medicine

Article Title: Advanced glycation end product-modified low-density lipoprotein promotes pro-osteogenic reprogramming via RAGE/NF-κB pathway and exaggerates aortic valve calcification in hamsters

doi: 10.1186/s10020-024-00833-8

Figure Lengend Snippet: IL-37 suppressed the expression of inflammatory mediators by inhibiting the activation of NF-κB p65. A Semi-quantitative analysis of immunohistochemical images showed that patients in the non-CAVD group had higher levels of IL-37 in the aortic valve tissue compared to the CAVD group (bar = 100 µm). B PCR analysis show that human calcified aortic valve expresses lower levels of IL-37 mRNA. C Immunoblotting was used to detect the expression of H3k27me3 and IL-37 after treating HAVICs with AGE-LDL (100 μg/ml) for 48 h. D CHIP-qPCR analysis show that AGE-LDL treatment increases the enrichment of H3K27me3 in IL-37 promoter region. E IL-37 (1.0 ng/ml) treated HAVICs 1 h prior to AGE-LDL (100 μg/ml) treatment for 48 h. Immunoblotting analysis of ICAM-1, IL-6 and ALP levels. F ELISA showed that the significant increase IL-6 level induced by AGE-LDL treatment was attenuated by IL-37 in culture media. G AGE-LDL (100 μg/ml) treated HAVICs for 12 h in the presence or absence of IL-37 (1.0 ng/ml). Immunoblotting analysis shows an increase in phosphorylated NF-κB p65, indicating an elevation in NF-KB phosphorylation modifications. H Immunofluorescence images of NF-κB p65 nuclear translocation (bar = 50 μm). The immunofluorescence results indicate that NF-κB p65 translocates to the nucleus after stimulation with AGE-LDL in HAVICs, suggesting activation of the non-phosphorylated form of NF-κB p65. I Alizarin red S staining of calcium deposition (bar = 200 µm). n = 5. * P < 0.05. vs. untreated control, # P < 0.05. vs. PBS, % P < 0.05. vs. IgG, & P < 0.05. vs. PBS + AGE-LDL. The data are presented as the means ± SEM. IL-37 = interleukin-37; other abbreviations as in Figs. –

Article Snippet: Antibodies against phosphorylated NF-κB (3033, Rabbit mAb), and total NF-κB (8242, Rabbit mAb) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Activation Assay, Immunohistochemical staining, Western Blot, ChIP-qPCR, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Immunofluorescence, Translocation Assay, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Deficiency of mastl , a mitotic regulator, results in cell detachment from developing tissues of zebrafish embryos

doi: 10.3389/fcell.2024.1375655

Figure Lengend Snippet: mastl kt441b homozygotes exhibit mitotic defects. (A) Phosphorylation of Histone H3 Ser10 (pHH3) in whole-mount embryos at the 6-somite stage (ss) and the 14 ss. At the 6 ss, no difference in phosphorylation was detectable among embryos containing siblings and mastl kt441b homozygotes. At the 14 ss, the number of pHH3-positive cells was increased in mastl kt441b homozygous embryos. For 14 ss embryos, 10 embryos of each phenotype were genotyped. (B) Time from prometaphase to the onset of anaphase of CNH cells. The duration was measured by time-lapse imaging of CNH cells with labeling of nuclei using Histone H2A-mChrery. The percentage of cells in which the duration of this process exceeded 30 min is indicated in dark gray. siblings: n = 10, mastl kt441b homozygotes: n = 18. (C) Classification of abnormalities of mitotic cells in the tail bud of siblings and mastl kt441b homozygous embryos. Embryos were labeled with Histone H2A-mCherry and sparsely labeled with membrane-Venus. All membrane-Venus labeled cells with condensed chromosomes within the first 30 min were tracked until the end of time-lapse imaging (2–3.5 h) or as long as we could track them (siblings: n = 50 cells, mast kt441b homozygous: n = 66 cells, from 3 embryos each). We classified tracked cells as follows. normal: normal cell division, missegregation: chromosomes were scattered around an elongated spindle, and cytokinesis was completed to pinch off some of the chromosomes, no segregation: cytokinesis was completed without obvious chromosome segregation, prolonged metaphase: metaphase was prolonged for more than 60 min without chromosome segregation or cytokinesis; decondensation: decondensation of chromosomes without segregation. cell death: change to hypercondensed nucleus. Time lapse images of representative cells for each type are shown in (D) . (D) Time lapse images of representative cells for each type shown in (C) . Green indicates membrane-Venus, while magenta indicates Histone H2A-mCherry. Mitotic cell types were classified as described in (C) . White arrows: mother cells with condensed chromosomes; Yellow arrows: daughter cells with condensed chromosomes; Orange arrows: hypercondensed chromosomes. Since the state of chromosome condensation was sometimes unclear, we carefully judged it referring the orthogonal views. Scale bar, 20 μm.

Article Snippet: Permeabilization was done with 0.5% Triton X-100 at room temperature for 1.5 h or acetone at −20°C for 5 min (for anti-phosphorylated Histone H3 Ser 10 antibody) and then incubated with 1/500 diluted rabbit anti-Kaede antibody (MBL; PM012) or 1/1000 diluted rabbit anti-phosphorylated Histone H3 Ser10 antibody (Millipore; 06–570) overnight at 4 °C.

Techniques: Phospho-proteomics, Imaging, Labeling, Membrane