Journal: Military Medical Research

Article Title: Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking.

doi: 10.1186/s40779-024-00520-z

Figure Lengend Snippet: Fig. 9 Hypothetical model illustrating that endophilin A2 (EndoA2) interacts with Piezo2 and KIF5B to form a complex to regulate the membrane trafficking of Piezo2 in sensory neurons and thus contributes to mechanical hypersensitivity

Article Snippet: DRG neurons from L4–L6 of mice were cultured for 2 d. Following fixation, DRG coverslips were treated with primary antibodies against Piezo2 (rabbit, 1:200, Novus, USA, NBP1-78,624) and NF200 (mouse, 1:200, Sigma, USA, N0142) and the appropriate secondary antibodies.

Techniques: Membrane

Journal: PLoS Genetics

Article Title: Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

doi: 10.1371/journal.pgen.1003518

Figure Lengend Snippet: A) Schematic diagram of the wild type mouse Hat1 locus (top), the Hat1 locus following integration of loxP sequences flanking intron three (middle) and the Hat1 locus following Cre mediated deletion of exon 3. Exons are represented by purple rectangles. Locations of probes and PCR primers are indicated. (B) Genomic DNA isolated from a parental (C57/bl6) mouse and mice generated from a cross between a chimeric mouse and a wild type mouse was digested with EcoRV and analyzed by Southern blot using the indicated probe. Mice 1, 4 and 6 are Hat1 flox/WT . C) A Hat1 flox/WT mouse was crossed with a mouse that ubiquitously expresses the cre recombinase. Genomic DNA was isolated from mice generated by this cross, digested with EcoRV and analyzed by Southern blot with the indicated probe. Mice 3, 5 and 9 are WT/KO/Cre. D) Table lists the number of expected, obtained and viable pups of the indicated genomes derived from matings of Hat1 +/− mice. E) PCR genotyping of a representative litter from a Hat1 +/− X Hat1 +/− mating. Primers P1 and P2 (see above) were used for amplification. Arrows indicate specific PCR products. F) Representative neonatal pups from Hat1 +/− X Hat1 +/− matings with the indicated genotype. F) Body weight of pups was measured immediately following birth. Data are derived from 10 pups of each genotype.

Article Snippet: Slides were also stained with primary antibodies against Hat1 (Abcam, 1/50 dilution), α-Ki67 (Novocastra, NCL-KI67-P) and anti-Cleaved Caspase 3 (Cell Signaling, #9661)Images were captured with a Zeiss AxioImager Z1 microscope.

Techniques: Isolation, Generated, Southern Blot, Derivative Assay, Amplification

Journal: PLoS Genetics

Article Title: Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

doi: 10.1371/journal.pgen.1003518

Figure Lengend Snippet: A) Histologic appearance of lungs from newborn pups obtained from a Hat1 +/−+ and Hat1 −/− mice. Staining was with hematoxylin-eosin; magnification 20×, (inlets ×40). The lungs of Hat1 −/− show less aeration, due to thickened mesenchyme resulting in death due to respiratory failure. B) Hat1 is highly expressed in lungs of Hat1 +/+ but not in Hat1 −/− mice; magnification 20×, (inlets ×40) C) Cleaved Caspase3 stained by IHC showed no difference between lungs of Hat1 ++ and Hat1 −/− mice; magnification 20×, (inlets ×40) D) Ki67 stained by IHC shows significantly higher proliferation rates in lungs of Hat1 −/− mice compared to controls; magnification 20×, (inlets ×40). Quantification was done by HistoQuest software.

Article Snippet: Slides were also stained with primary antibodies against Hat1 (Abcam, 1/50 dilution), α-Ki67 (Novocastra, NCL-KI67-P) and anti-Cleaved Caspase 3 (Cell Signaling, #9661)Images were captured with a Zeiss AxioImager Z1 microscope.

Techniques: Staining, Software

Journal: PLoS Genetics

Article Title: Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

doi: 10.1371/journal.pgen.1003518

Figure Lengend Snippet: A) Micro CT scans of the heads of neonates with the indicated genotype. Top row shows a dorsal view and bottom row shows a ventral view. Arrows indicate defects in the nasal cavity (top) and jaw structures (bottom). B) Micro CT scans of Hat1 +/+ and Hat1 −/− neonates (as indicated) showing a dorsal view of the entire animal. C) Acian blue and Alizarin red stained Hat1 +/+ and Hat1 −/− neonates. D) Hat1 +/+ and Hat1 −/− embryos (12.5 dpc) were stained with α-Hat1 antibodies. E) Cross section of the head and neck of 11.5 dpc Hat1 +/+ and (WT) and Hat1 −/− embryos stained with α-Hat1 antibody. 2.5× magnification.

Article Snippet: Slides were also stained with primary antibodies against Hat1 (Abcam, 1/50 dilution), α-Ki67 (Novocastra, NCL-KI67-P) and anti-Cleaved Caspase 3 (Cell Signaling, #9661)Images were captured with a Zeiss AxioImager Z1 microscope.

Techniques: Micro-CT, Staining

Journal: PLoS Genetics

Article Title: Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

doi: 10.1371/journal.pgen.1003518

Figure Lengend Snippet: A) Hat1 +/+ , Hat1 +/− and Hat1 −/− primary MEFs were genotyped by PCR as described in the legend to . Whole cell extracts from the indicated MEFs were analyzed by Western blots probed with the indicated antibodies. B) Equal numbers of primary MEFs of the indicated genotype were seeded at time zero. Cell numbers were counted at the indicated time points. C) Primary Hat1 +/+ and Hat1 −/− MEFs were stained with propidium iodide and analyzed by FACS. Fraction of cells in each phase of the cell cycle is indicated. D) Immortalized Hat1 +/+ and Hat1 −/− MEFs were grown under the indicated conditions. Plates were photographed after crystal violet staining.

Article Snippet: Slides were also stained with primary antibodies against Hat1 (Abcam, 1/50 dilution), α-Ki67 (Novocastra, NCL-KI67-P) and anti-Cleaved Caspase 3 (Cell Signaling, #9661)Images were captured with a Zeiss AxioImager Z1 microscope.

Techniques: Western Blot, Staining

Journal: PLoS Genetics

Article Title: Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

doi: 10.1371/journal.pgen.1003518

Figure Lengend Snippet: A) Hat1 +/+ and Hat1 −/− MEFs were stained with either DAPI or α-γH2AX antibodies as indicated (left). Visible γH2AX foci were counted in 12 cells of each genotype (right). B) Metaphase spreads from Hat1 +/+ and Hat1 −/− MEFs were analyzed for chromosome number and the presence of breaks and fusions. The percentage of cells containing the indicated chromosomal abnormality is given. The number of spreads analyzed was 56 (Hat1 +/+ ) and 112 (Hat1 −/− ). C) Metaphase spreads were generated from Hat1 +/+ and Hat1 −/− MEFs. Insets show enlarged views of selected abnormal chromosomes. Red arrows indicate examples of chromosome fusions and blue arrows indicate chromosome breaks.

Article Snippet: Slides were also stained with primary antibodies against Hat1 (Abcam, 1/50 dilution), α-Ki67 (Novocastra, NCL-KI67-P) and anti-Cleaved Caspase 3 (Cell Signaling, #9661)Images were captured with a Zeiss AxioImager Z1 microscope.

Techniques: Staining, Generated

Journal: PLoS Genetics

Article Title: Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

doi: 10.1371/journal.pgen.1003518

Figure Lengend Snippet: A and B) Hat1 +/+ and Hat1 −/− MEFs were pulse-labeled with EdU and chased with thymidine as schematically depicted at the top of each panel. Samples were isolated at the indicated time points and proteins associated with nascent DNA were resolved by SDS-PAGE following affinity purification of EdU-labeled DNA (iPond). The indicated amounts of the input fractions (prior to affinity purification) are on the left-hand side of each panel. Samples eluted from the affinity purification resin are on the right-hand side of each panel. In all cases, the respective Hat1 +/+ and Hat1 −/− samples were analyzed on the same gel (separated by a MW standard lane that has been removed). Western blots were probed with the antibodies indicated on the right. Numbers below each lane indicate the normalized intensity of the band as determined using Licor software. Each sample was normalized to the level of unmodified histone H3. The intensity observed in the 30′ pulse sample was arbitrarily set to 1.0. No Clck indicates control samples that were not biotin labeled.

Article Snippet: Slides were also stained with primary antibodies against Hat1 (Abcam, 1/50 dilution), α-Ki67 (Novocastra, NCL-KI67-P) and anti-Cleaved Caspase 3 (Cell Signaling, #9661)Images were captured with a Zeiss AxioImager Z1 microscope.

Techniques: Labeling, Isolation, SDS Page, Affinity Purification, Western Blot, Software

Journal: PLoS Genetics

Article Title: Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

doi: 10.1371/journal.pgen.1003518

Figure Lengend Snippet: Hat1 +/+ and Hat1 −/− MEFs were pulse-labeled with 3 H-lysine for 12 minutes. Histones were then isolated and resolved by Acid-Urea (AU) gel electrophoresis. Total protein was visualized with Coomassie blue staining and radio-labeled proteins visualized by fluorography (as indicated). The mobility of each histone is indicated. The brackets indicate the regions of mobility for the acetylated isoforms of histone H4 and histone H3.

Article Snippet: Slides were also stained with primary antibodies against Hat1 (Abcam, 1/50 dilution), α-Ki67 (Novocastra, NCL-KI67-P) and anti-Cleaved Caspase 3 (Cell Signaling, #9661)Images were captured with a Zeiss AxioImager Z1 microscope.

Techniques: Labeling, Isolation, Nucleic Acid Electrophoresis, Staining

Journal: Open Medicine

Article Title: Luteolin alleviates ulcerative colitis in rats via regulating immune response, oxidative stress, and metabolic profiling

doi: 10.1515/med-2023-0785

Figure Lengend Snippet: Effects of LUT on inflammation and immune response in UC rats. (a) Expression levels of TNF-α, CRP, IL-13, IL-33, and ST-2 were examined by ELISA. (b) Representative image of CD4+ T cell staining in the colon (100× magnification). (c) Representative image of CD8+ T cell staining (100× magnification). NC represents normal control. MOD represents model. MES represent mesalazine. LUT represents luteolin. There were 10, 8, 8, and 9 replicates in NC, MOD, MES, and LUT groups, respectively. ** P < 0.01 vs the NC group. # P < 0.05 and ## P < 0.01 vs the MOD group.

Article Snippet: The sections were blocked with 5% BSA and incubated with primary antibody against CD4 (1:2,000; Abcam, UK) and CD8 (1:2,000; Abcam, UK) overnight at 4°C.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Frontiers in Oncology

Article Title: Downregulation of TET1 Promotes Bladder Cancer Cell Proliferation and Invasion by Reducing DNA Hydroxymethylation of AJAP1

doi: 10.3389/fonc.2020.00667

Figure Lengend Snippet: Ten-eleven translocation 1 (TET1) inhibited urinary bladder cancer (UBC) progression by regulating β-catenin through mediating adherens junction-associated protein 1 (AJAP1) expression. (A) Western blot analysis showed the expression of β-catenin and the downstream targets (fibronectin and CD44) in empty vector (EV), TET1-CD, TET1-CD-mut-transfected T24 cells and shC, shTET1-transfected 5,637 cells (sh1 and sh2). (B) Validation of AJAP1 protein levels of groups after transfected with the siRNA to AJAP1 by Western blotting. (C,D) Cell viability and invasion ability of three groups of T24 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (C) and transwell invasion assay (D) . (E) The levels of β-catenin and the downstream targets of β-catenin were detected with Western blotting in TET1-CD overexpression T24 cells transiently transfected with siNC and siAJAP1. (F) Co-IP assays showed that endogenous protein of AJAP1 interacted with β-catenin in T24 cells. (G,H) Localization of β-catenin in TET1-CD overexpression T24 cells transiently transfected with siNC and siAJAP1, which were detected with Western blotting (G) on the fractionation of cytosol and nuclear compartments and immunofluorescence (H) with β-catenin antibodies (red), followed by the counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 20 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: T24 cells were transfected with siAJAP1 or siNC for 48 h, cells were seeded on glass slides, fixed in 4% paraformaldehyde, and blocked by 1% bovine serum albumin (BSA)/phosphate buffered saline (PBS), followed by permeabilization with 0.1% Triton X-100 for 30 min. Then cells were incubated overnight at 4°C with primary antibodies against with anti-β-catenin antibody (1:400, Cat# 610153; BD Biosciences, San Jose, CA).

Techniques: Translocation Assay, Expressing, Western Blot, Plasmid Preparation, Transfection, MTT Assay, Transwell Invasion Assay, Over Expression, Co-Immunoprecipitation Assay, Fractionation, Immunofluorescence

Journal: Frontiers in Oncology

Article Title: Downregulation of TET1 Promotes Bladder Cancer Cell Proliferation and Invasion by Reducing DNA Hydroxymethylation of AJAP1

doi: 10.3389/fonc.2020.00667

Figure Lengend Snippet: The downregulation of ten-eleven translocation 1 (TET1)/adherens junction-associated protein 1 (AJAP1) axis predicts worse clinical outcomes. (A) Representative images of the localization and expression of AJAP1, β-catenin, and TET1 in normal urothelium and urinary bladder cancer (UBC) tissues were presented by immunohistochemical (IHC) staining. Scale bar, 50 and 20 μm (inset). (B) The relationship between TET1 and AJAP1 protein expression was determined by Pearson correlation analysis. (C,D) The correlation of nuclear expression of β-catenin with TET1 (C) and AJAP1 (D) in UBC specimens were determined by Pearson correlation analysis. (E) Kaplan–Meier survival curve of UBC patients with high AJAP1 and low AJAP1 protein level. (F) The correlation between the combined expression of TET1 and AJAP1 with the overall survival of UBC patients were determined by Kaplan–Meier analysis. (G) A working model of TET1/AJAP1/β-catenin axis in UBC progression.

Article Snippet: T24 cells were transfected with siAJAP1 or siNC for 48 h, cells were seeded on glass slides, fixed in 4% paraformaldehyde, and blocked by 1% bovine serum albumin (BSA)/phosphate buffered saline (PBS), followed by permeabilization with 0.1% Triton X-100 for 30 min. Then cells were incubated overnight at 4°C with primary antibodies against with anti-β-catenin antibody (1:400, Cat# 610153; BD Biosciences, San Jose, CA).

Techniques: Translocation Assay, Expressing, Immunohistochemical staining, Immunohistochemistry