Journal: Reproductive Sciences

Article Title: Conservative Hypomethylation of Mesenchymal Stem Cells and Their Secretome Restored the Follicular Development in Cisplatin-Induced Premature Ovarian Failure Mice

doi: 10.1007/s43032-023-01389-4

Figure Lengend Snippet: Phenotype characterization of partially hypomethylated MSCs. The expression of CD105, CD90, CD45, and CD34 was analyzed by flow cytometry for normally methylated cells ( A ), cells treated with 2.5 μM ( B ), or 5 μM ( C ) of 5-Aza-dC. No significant differences were observed in the expression of CD markers between DMSO-treated cells and cells treated with 5-Aza-dC ( D ). Bars represent the average (±SD) of three independent measurements, ns , not significant. Comparison between means was performed by one-way ANOVA test

Article Snippet: Phycoerthrin (PE)-conjugated mouse monoclonal antibodies of CD90, and CD34 or FITC-conjugated monoclonal antibodies of CD105 and CD45 were from (Biotechne R&D System, MN, USA).

Techniques: Expressing, Flow Cytometry, Methylation, Comparison

Journal: Fluids and Barriers of the CNS

Article Title: Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease

doi: 10.1186/s12987-023-00471-y

Figure Lengend Snippet: Expression and localization of marker proteins in hiPSC-derived brain cell types of the NVU. HiPSCs were derived from a late-onset Alzheimer disease patient (LOAD NVU model) and a healthy elderly control subject (CON NVU model). ( A ) Flow cytometry confirmed high expression of cell type-specific phenotypic markers in pericytes (PCs), astrocytes (ACs), neural stem cells (NSCs; all mean ± SD; n ≥ 3), and microglia-like cells (MGCs; mean ± SD; n = 2). Representative immunofluorescence images confirmed protein expression of ( B ) NG2 and αSMA in PCs, scale bar 200 μm, ( C ) EAAT1 and EAAT2 in ACs, scale bar 100 μm, ( D ) CD45 and AIF1 in MGCs, scale bar 100 μm, ( E ) PAX6, SOX1, and NES in NSCs, scale bar 100 μm, and ( F ) GLUT1/SLC2A1, ZO1, OCLN, and CLDN5 in BCECs, scale bar 100 μm. ( G ) Measurement of transendothelial electrical resistance (TEER) and sodium fluorescein (NaF) permeability to analyze the integrity of the blood-brain barrier in CON and LOAD. TEER ( left ) and permeability coefficient (PC NaF ) ( right ) values are shown for mono- and co-cultures consisting of BCECs with or without PCs, ACs, MGCs, or NSCs. Box (median and lower/upper quartile) and whisker (minimum/maximum) plots display n = 4–6 independent biological assays (no outliers), one-tailed t-test, *p < 0.05

Article Snippet: The following antibodies and dilutions were used: Mouse IgG 2a anti-human actin alpha 2, smooth muscle (ACTA2/αSMA; 1:100; #ab7817, Abcam), Goat IgG anti-human allograft inflammatory factor 1 (AIF1/IBA1; 1:200; #NB100-1028, BioTechne), Rat IgG 2b anti-human CD45 (1:200; #NB100-77417, BioTechne), Rabbit IgG anti-human claudin 5 (CLDN5; 1:100; #ab15106, Abcam), Mouse IgG 2a anti-human EAAT1/ GLAST (1:100; #sc-515839, Santa Cruz Biotechnology), Mouse IgG 2a anti-human EAAT2 (1:100; #sc-365634, Santa Cruz Biotechnology), Rabbit IgG anti-human solute carrier family 2 member 1 (SLC2A1/GLUT1; 1:200; #ab115730, Abcam), Mouse IgG 1 anti-human nestin (NES; 1:1000; #60091, Stemcell Technologies), Mouse IgG 1 anti-human chondroitin sulfate proteoglycan 4 (CSPG4/NG2; 1:100; # 83508, Abcam), Mouse IgG 1 anti-human occludin (OCLN; 1:200; #331500, Thermo Fisher Scientific), Rabbit IgG anti-human PAX6 (1:200; #ab5790, Abcam), Rabbit IgG anti-human SOX1 (1:200; #ab22572 Abcam), and Rabbit IgG anti-human tight junction protein 1 (TJP1/ZO1; 1:400; #21773-1-AP, Proteintech).

Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry, Immunofluorescence, Permeability, Whisker Assay, One-tailed Test

Journal: Cell reports

Article Title: Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

doi: 10.1016/j.celrep.2022.110791

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Accell Mouse Selenop (20363) siRNA - SMARTpool , Horizon Discovery Ltd. , E-041618-00-0020.

Techniques: Virus, Plasmid Preparation, shRNA, Selection, Recombinant, Enzyme-linked Immunosorbent Assay, Synthesized, Software, Functional Assay, Saline, Gel Extraction, Expressing, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Next-Generation Sequencing

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: Defect of HIRA complex results in H2A.Z decrease on genome wide. ( A ) Schematic diagram of genome editing in defective cell lines. Hira-, Ubn2- and H3.3-knockout cell lines and Ubn1/2 double mutants were generated using CRISPR-Cas9 system. For subsequent experiments, 6 × HA tag was added to the N-terminus of Srcap in R1 wild type and above defective cell lines. ( B ) Western blot respectively shows the proteins levels of Hira (a), Ubn1&Ubn2 (b), and H3.3 (c) in the defective cell lines. ( C ) Meta-analysis (left) and heatmap (right) show H2A.Z reads density around summit (upper) and TSS (below) in R1 WT , Hira-KO , Ubn2-KO/Ubn1-KD , Ubn1/2-DM and H3.3-KO cells within a ±3 kb window. ( D ) Venn diagram shows the overlap of H2A.Z down-regulated peaks between the above defective cell lines. ( E ) The reduced H2A.Z peaks in either Hira-KO or Ubn2-KO/Ubn1-KD cell lines are defined as HIRA complex regulated (HR) peaks (23 530 peaks). The 5426 HR peaks overlapping with Ubn1/2-DM cell line is defined as HIRA-regulated and H3.3-dependent (HIRA-H3.3D). The other 18 104 HR peaks are HIRA-regulated and H3.3-independent (HIRA-H3.3ID).

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Genome Wide, Knock-Out, Generated, CRISPR, Western Blot

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: HIRA collaborates with SRCAP to deposit H2A.Z on genome. ( A ) Meta-analysis (left) and heatmap (right) show Srcap reads density around summit (upper) and TSS (below) within a ±3 kb window. ( B ) Representative loci on Slc25a42 gene show the enrichment of H2A.Z and Srcap. ( C ) ChIP-qPCR validation of H2A.Z and Srcap enrichment at promoter of Slc35a42 gene. Error bars represent data from three independent experiments. ( D ) Venn diagram shows the overlap between H2A.Z, Hira and Srcap peaks. ( E ) Western blot shows the interaction among endogenous HA-Srcap, Hira and Ubn1 from nuclear lysates. Ino80, Daxx, H2A.Z and H3.3 are also detected. ( F ) Top panel showed schematic presentation of full length and truncation mutants of Hira subunit. Bottom panel showed the biochemical interactions between Hira truncations and SRCAP complex are analyzed by GST pull-down coupled with western blot analyses. ( G ) Heatmaps (upper) show H2A.Z reads density around H2A.Z peak summits (upper) in R1 WT , Hira-KO and Hira-truncation mutant expressing cells within a ±3 kb window. Representative loci (bottom) show the enrichment of H2A.Z. (H) ChIP-qPCR of H2A.Z, Srcap and Flag at representative genes Nrg2 and Spata4 in the rescued overexpressed cell lines. Error bars represent data from three independent experiments.

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Western Blot, Mutagenesis, Expressing

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

Article Title: Optical imaging of the whole-body to cellular biodistribution of clinical-stage PEG-b-pHPMA-based core-crosslinked polymeric micelles

doi: 10.1016/j.jconrel.2020.09.046

Figure Lengend Snippet: (a) Fluorescence microscopy images and endothelial cells in the spleen. The enlarged images show high association of CCPM with leukocytes and minor association with endothelial cells. Scale bar = 100 μm. (b) Representative image of leukocytes and myeloid cells, exemplifying that more than half of all splenic immune cells are myeloid cells and that CCPM strongly associate with these cells. Scale bar = 100 μm. (c) Fluorescence microscopy images of macrophages and endothelial cells in the spleen, showing strong CCPM uptake by macrophages. Scale bar = 100 μm. (d) Pie chart displaying the cellular composition of the spleen: 67% of the splenic mass is composed of leukocytes (33% are macrophages, 28% lymphocytes and 6% other myeloid cells), 1% of endothelial cells and 32% of CD45- splenocytes. As shown in bold next to each cell type, 11/33 macrophages, 6/28 lymphocytes and 5/32 CD45- splenocytes are CCPM positive. (e) Pie chart displaying the distribution of intracellular CCPM within different cell types in the spleen, showing that 50% is taken up by macrophages, 27% by lymphocytes and 23% by other cells.

Article Snippet: The slices were then incubated for 1 h with anti-CD45 antibody (1:100) (Bio-Techne, Germany) and anti-CD31 antibody (1:50) diluted in a PBS/azide solution added with 1% mouse serum (Sigma-Aldrich, Germany).

Techniques: Fluorescence, Microscopy