Journal: bioRxiv

Article Title: Chromatin accessibility dynamics of neurogenic niche cells reveal a reversible decline in neural stem cell migration during aging

doi: 10.1101/2021.03.29.437585

Figure Lengend Snippet: a) Heatmap showing accessibility levels of differential ATAC-seq peaks associated with the “Cell Adhesion” GO category (GO:0007155) that change with age in qNSCs (left) and aNSCs (right) respectively. Selected gene names with associated differentially accessible peaks are displayed. TMM-normalized read counts (by EdgeR), scaled row-wise. b) Genome browser (IGV) view of chromatin accessibility signal tracks from representative RPKM-normalized libraries of quiescent and activated NSCs isolated from young and old brains illustrating differential chromatin peaks that change with age in young quiescent NSCs (left) and old activated NSCs (right) respectively. Black arrows represent sites of differentially accessible peaks that change with age in qNSCs (left) and aNSCs (right) respectively. Cdhr3 , Cadherin Related Family Member 3. Pcdh18 , Protocadherin 18. Scale bar, 5kb. c) Enrichment of top-ranked transcription factor motifs by TF-MoDISco within differentially accessible ATAC-seq peaks that change with age in qNSCs (left) and aNSCs (right). Red box indicates motif associated with NFI family. d) Violin plots of the average distribution of single cell cumulative expression profiles of genes within the “Cell Adhesion” GO category (GO:0007155) for young and old qNSCs/astrocytes (left) and aNSCs/NPCs (right) from scRNA-seq data . Each overlaid dot represents the cumulative expression of cell adhesion genes in a single cell. P -values were calculated using a two-tailed Mann-Whitney test. e) Schematic illustrating the general protocol for experiments involving cultured qNSCs and aNSCs/NPCs. Young (3-5 months) and old (20-24 months) C57BL/6 SVZs were microdissected and tissues were processed to enable NSC culture. Quiescence was induced with growth factors bFGF and BMP4 for 5-10 days, and activation was maintained with bFGF and EGF for 1-2 days prior to downstream experiments (ATAC-seq library generation, confocal microscopy, and live-cell migration tracking). f) Representative images of immunofluorescence staining of a young (3 months) qNSC (left) and aNSC/NPC (right) cultured on Poly-D-Lysine. Blue, DAPI (nuclei); green, phalloidin (F-actin). Scale bar, 50 μm. g,h) Selected GO terms for genes associated with differentially accessible ATAC-seq peaks (FDR<0.05) that change with age in cultured qNSCs (g) and aNSCs/NPCs (h) generated by EnrichR and ranked by P -value. Red boxes indicate GO terms associated with cell adhesion and cell migration. i) Representative images of a young (3-4 months) aNSC/NPC in the presence of cell-permeant red fluorescent nucleic acid stain Syto64 taken at t = 0, 10, 15, and 20 hours after sorting onto Poly-D-Lysine. The Incucyte system was used for 20x imaging and migration tracks were determined using Imaris (v9.3.0). Color gradient bar represents the passage of time from 0 (blue) to 20 (red) hours. Scale bar, 50 μm. j) Migration speed of young (3-4 months) and old (23-24 months) qNSCs on Poly-D-Lysine. Each dot represents the average velocity of a single cell over a 20-hour period ( n = 6 young male mice, and n = 4 old male mice) (combined over 2 experiments). Data are mean ± SEM. P -values were calculated using a two-tailed Mann-Whitney test. k) Migration speed of young (3-4 months) and old (21-24 months) aNSCs/NPCs on Poly-D-Lysine. Each dot represents the average velocity of a single cell over a 20-hour period ( n = 9 young male mice, and n = 7 old male mice) (combined over 3 experiments). Data are mean ± SEM. P -values were calculated using a two-tailed Mann-Whitney test. l) Representative images of young (top) and old (bottom) aNSC/NPC invasion through Matrigel taken 0, 24, and 48 hours after seeding aNSCs/NPCs. The dotted white outline represents the outermost extent of invasion and the inner dotted white line represents the initial extent of the cells after seeding (t = 0 hours). Scale bar, 800 μm. m) Migration distance timecourse of young (3-4 months) and old (21-24 months) aNSC/NPC invasion through Matrigel over 48 hours with 12-hour intervals. ( n = 7 young male mice, and n = 10 old male mice) (combined over 2 experiments). For each biological replicate, 1-4 technical replicates were evaluated, and migration distance was averaged at each timepoint. Data are mean ± SEM. P -values were calculated at each timepoint using a two-tailed Mann-Whitney test.

Article Snippet: The media was then replaced with complete quiescent media supplemented with 10 nM Syto64 (Invitrogen, S11346, Lot #8344573) prior to imaging.

Techniques: Isolation, Expressing, Two Tailed Test, MANN-WHITNEY, Cell Culture, Activation Assay, Confocal Microscopy, Migration, Immunofluorescence, Staining, Generated, Imaging

Journal: bioRxiv

Article Title: Chromatin accessibility dynamics of neurogenic niche cells reveal a reversible decline in neural stem cell migration during aging

doi: 10.1101/2021.03.29.437585

Figure Lengend Snippet: a) Top 10 canonical pathways enriched for genes associated with differentially accessible peaks that open with age in freshly isolated aNSCs (FDR<0.05) generated by Ingenuity Pathway Analysis (IPA) and ranked by P -value. ATAC-seq peaks were annotated with their nearest gene using ChIPSeeker (v1.18.0) and P -values were calculated using Fisher’s Exact Test by IPA. b) Diagram of the G α 12/13 signaling pathway with selected targets including ROCK and downstream biological processes regulated by ROCK (adapted from the IPA canonical pathway diagram “G α 12/13 Signaling” and previous work ). RhoA, Ras homolog family member A. ROCK, Rho-associated coiled-coiled kinase. PI3K, phosphoinositide 3-kinase. AKT, AKT serine/threonine kinase 1. NF- κ B, Nuclear factor-kappa B. JNK, c-Jun N-terminal kinase. c) Representative images of old (21 months) cultured aNSCs/NPCs treated with H 2 O vehicle (top) or with 10 μM Y-27632 (bottom) seeded on RGD molecular tension sensors taken with phase contrast (left) and FRET donor/acceptor fluorescence wavelengths to generate a high-resolution traction map of FRET efficiency (right). A low FRET efficiency indicates high force (blue color) and a high FRET efficiency indicates low force (yellow/green color). Black dotted lines around the adhered cell bodies were generated using phase contrast images and overlaid onto FRET images. Scale bar, 10 μm. d) Truncated violin plot of average force per sensor (pN) in individual focal adhesions exhibited by young (3 months) and old (21 months) cultured aNSCs/NPCs treated with H 2 O vehicle or with 10 μM Y-27632 determined using RGD molecular tension sensors ( n = 85 young aNSCs/NPCs, n = 54 young aNSCs/NPCs + 10 μM Y-27632, n = 90 old aNSCs/NPCs, and n = 74 old aNSCs/NPCs +10 μM Y-27632) (combined over 2 separate experiments). For cells exhibiting focal adhesion patterns, each dot represents the average force sensor reading among focal adhesions for a single cell. For cells without any focal adhesions, each dot represents the average force sensor reading underneath the cell body. P -values were calculated using a two-tailed Mann-Whitney test. e) Truncated violin plot of adhesion area under focal adhesions of young (3 months) and old (21 months) aNSCs/NPCs treated with H 2 O vehicle or with 10 μM Y-27632 determined using RGD molecular tension sensors ( n = 85 young aNSCs/NPCs, n = 54 young aNSCs/NPCs + 10 μM Y-27632, n = 90 old aNSCs/NPCs, and n = 74 old aNSCs/NPCs +10 μM Y-27632) (combined over 2 separate experiments). Each dot represents the adhesion area of force-producing focal adhesions for a single cell. P -values were calculated using a two-tailed Mann-Whitney test. f) Representative images of immunofluorescence staining of an old (21-23 months old) aNSC/NPC treated with H 2 O vehicle (top) or with 10 μM Y-27632 (bottom) cultured on Poly-D-Lysine. Blue, DAPI (nuclei); green, phalloidin (F-actin); white, high-intensity phalloidin mask (bundled actin). Scale bar, 50 μm. g) Fluorescence intensity of bundled actin in old (21-23 months) aNSCs/NPCs treated with H 2 O vehicle or with 10 μM Y-27632 cultured on Poly-D-Lysine, normalized to the mean levels of the untreated condition. Each gray dot represents bundled actin levels in a single cell and each colored dot represents the average cellular bundled actin level per animal ( n = 6 old cultures for both untreated and treated conditions) (combined over 2 experiments). Data are mean ± SEM. P -values were calculated using a two-tailed Mann-Whitney test comparing sample means between conditions. h) Representative images of old (21-24 months) cultured aNSCs/NPCs 12 hours after sorting onto Poly-D-Lysine-coated migration plates treated with H 2 O vehicle (top) or 10μM Y-27632 (bottom). Images were taken with the Incucyte S3 system with phase contrast and RFP imaging overlaid. Live cells were stained with Syto64, a cell-permeant red fluorescent nucleic acid stain, for migration tracking. Outlined inset (top-right) displays a representative magnified cell. Scale bar, 50 μm. i) Migration speed of young (3-4 months) and old (21-24 months) aNSCs/NPCs cultured on Poly-D-Lysine treated with H 2 O vehicle or with 10 μM Y-27632. Each dot represents the average velocity of a single cell over a 20-hour period ( n = 9 young control cultures, n = 9 young treatment cultures, n = 7 old control cultures, n = 9 old treated cultures) (combined over 3 experiments). Data are mean ± SEM. P -values were calculated using a two-tailed Mann-Whitney test. j) Distance migrated through Matrigel after 48 hours by young (3-4 months) and old (21-23 months) cultured aNSCs/NPCs treated with H 2 O vehicle or with 10 μM Y-27632. Each dot represents the distance migrated by a single biological replicate after 48 hours ( n = 6 young cultures for treated and untreated conditions, n = 6 old cultures for treated and untreated conditions) (combined over two experiments). For each biological replicate, 1-4 technical replicates were evaluated, and migration distance was averaged. P -values were calculated using a two-tailed Mann-Whitney test.

Article Snippet: The media was then replaced with complete quiescent media supplemented with 10 nM Syto64 (Invitrogen, S11346, Lot #8344573) prior to imaging.

Techniques: Isolation, Generated, Cell Culture, Fluorescence, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Staining, Migration, Imaging

Journal: Cytotechnology

Article Title: In vitro induction of quiescence in isolated primary human myoblasts

doi: 10.1007/s10616-019-00365-8

Figure Lengend Snippet: Expression of myogenic and cell cycle regulatory factors PHMs. Myoblasts isolated from muscle biopsies (KH3, KH1 and S6.3) were cultured in proliferating media prior to induction of quiescence in Ham’s F-10 Nutrient Mixture Medium with KnockOut™ Serum Replacement (KOSR). Cells in induced quiescence were harvested and the expression of proliferating marker (Ki67), myogenic marker (Myf5) and cell cycle inhibitor (p21) was quantified using quantitative polymerase chain reaction (PCR). Results represent mean ± SEM (n = 4). **p < 0.005 comparing differences between donors. Results indicate fold change—quiescence/proliferation. Dash line indicate no fold change = 1

Article Snippet: The resulting media was termed quiescence-induction media and contained Ham’s F-10 Nutrient Mixture Medium (Sigma-Aldrich, N6908), supplemented with 20% (v/v) KnockOut™ Serum Replacement (KOSR) (Life Technologies, 10828-010) and 1% (v/v) penicillin/streptomycin (Life Technologies, 15140-122) Cells were cultured for 10 days in quiescence-induction media, with daily media changes, to clear pro-proliferative secretions, in order to induce cellular quiescence.

Techniques: Expressing, Isolation, Cell Culture, Knock-Out, Marker, Real-time Polymerase Chain Reaction

Journal: Cytotechnology

Article Title: In vitro induction of quiescence in isolated primary human myoblasts

doi: 10.1007/s10616-019-00365-8

Figure Lengend Snippet: Effect of proliferation media and quiescence-inducing media on cell cycle dynamics of PHMs. Myoblasts isolated from muscle biopsies (KH3, KH1 and S6.3) were cultured in proliferation media prior to induction of quiescence in Ham’s F-10 Nutrient Mixture Medium with KnockOut™ Serum Replacement (KOSR). Propidium iodide staining of myoblast nuclei was analysed for cell cycle proportions using ModfitLT 3.0 software. The G0/G1 proportion corresponds to single copies of DNA (n); S corresponds to a range of n–2n and G2 corresponding to diploid nuclei (2n). Results represent mean ± SEM (n = 4 replicates for each). *p < 0.05 comparing differences between donors while #p < 0.05 comparing quiescent versus corresponding cell cycle proportion in proliferating cells

Article Snippet: The resulting media was termed quiescence-induction media and contained Ham’s F-10 Nutrient Mixture Medium (Sigma-Aldrich, N6908), supplemented with 20% (v/v) KnockOut™ Serum Replacement (KOSR) (Life Technologies, 10828-010) and 1% (v/v) penicillin/streptomycin (Life Technologies, 15140-122) Cells were cultured for 10 days in quiescence-induction media, with daily media changes, to clear pro-proliferative secretions, in order to induce cellular quiescence.

Techniques: Isolation, Cell Culture, Knock-Out, Staining, Software