Journal: bioRxiv

Article Title: Exosomes are vehicles for the stress-regulated secretion of histones

doi: 10.1101/2024.04.08.588575

Figure Lengend Snippet: (A) Dot plots showing r epresentative IFCM data obtained from HeLa cell conditioned media (CM) and media controls stained with either AF488 labelled anti-H3 or FITC labelled anti-H4 antibodies or stained with a mixture of APC labelled anti-CD9/CD63/CD81 antibodies to detect total tetraspanin positive EVs. (B) Quantification of detected gateable events positive for single stained H3, H4, or tetraspanins (n=3) in CM samples collected at different time points from both control and H 2 O 2 treated cell cultures. Samples were diluted post staining as indicated on the y -axes. (C) H3 co-expression and (D) H4 co-expression, post double staining with anti-CD9, -CD63, or -CD81 antibodies on CM from 24 h H 2 O 2 treated samples. See also Fig. S15. Statistical comparisons by two-way ANOVA. ****, p < 0.0001, ***, p < 0.001, **, < 0.005. (E) Radial plot showing relative abundance of H3 post-translational modifications (PTMs) identified in EVs OptiPrep versus nuclear histones. (F) Representative examples of H3 PTMs that are selectively enriched in the nucleus, or in EVs. Lysine (K) trimethylation (Me3) results from the covalent attachment of three Me groups to a K residue and is therefore a relatively “slow” modification. H3 is poorly modified with Me3 in EVs relative to nucleus regardless of whether this PTM benchmarks actively transcribed chromatin (H3K4me3) or silenced heterochromatin (H3K9me3). H3K9 is relatively unmodified in EVs, and H3K79Me2 is an example of a modification that is enriched in EV histones. Phosphorylation of H3S10 is selectively enriched in EVs from LPS treated cells.

Article Snippet: The following antibodies were used: CD9-APC (Miltenyi Biotech, clone SN4, 130-128-037; final staining concentration 0.6 ng/μl), CD63-APC (Miltenyi Biotec, clone H5C6, 130-100-182; 0.6 ng/μL), CD81-APC (Beckman Coulter, clone JS64, A87789; 0.6 ng/μl), H3-AF488 (Invitrogen, clone 17H2L9, MA7-02023-A488; 2 ng/μL), and H4-FITC (Novus Biologicals; polyclonal, NB21-2044F; 2 ng/μL).

Techniques: Staining, Control, Expressing, Double Staining, Residue, Modification, Phospho-proteomics

Journal: Materials today. Bio

Article Title: Cyclic stretch induced epigenetic activation of periodontal ligament cells.

doi: 10.1016/j.mtbio.2024.101050

Figure Lengend Snippet: Fig. 8. Mechanical stretch regulates reactive oxygen species (ROS) resistance and lineage specification in periodontal ligament (PDL) cells. Mechanical stretch led to cell alignment via actin and tubulin reorganization, which increased Histone 3 acetylation levels. These epigenetic modifications enhanced gene transcription and provided ROS resistance by promoting an open chromatin state. Additionally, mechanical stretch affected lineage specification, inducing PDL cells into the osteoclast and osteoblast pathways. H3Ace; histone 3 Acetylation, H3K9me3; histone H3 lysine 9 trimethylation, Ac; acetylation, Me; methylation.

Article Snippet: The following primary antibodies were used: Rabbit Antiacetyl-Histone H3 Antibody (H3Ace; #06-599, Sigma-Aldrich), Mouse Histone H3 (Trimethyl Lys9) Antibody (H3K9me3; # NBP1-30141, Novus Biological, Centennial, CO), Rabbit Beta tubulin Antibody (#2146s, Cell signaling Technology, Danvers, MA, USA), Mouse Monoclonal Anti-Tubulin Acetylated Antibody (#T6793, SigmaAldrich), Mouse Lamin A/C (4C11) Antibody (#4777s, Cell Signaling Technology), Rabbit Phospho-Myosin Light Chain 2 (Ser19) Antibody (p-MLC2; #3671L, Cell Signaling Technology), Rabbit Vinculin Antibody (#ab129002, Abcam plc, Cambridge, UK), Mouse focal adhesion kinases (FAK) (D-1) Antibody (#sc-271126, SantaCruz Biotechnology Inc., Dallas, TX, USA).

Techniques: Methylation