Journal: eLife
Article Title: Activity-dependent modulation of synapse-regulating genes in astrocytes
doi: 10.7554/eLife.70514
Figure Lengend Snippet: ( A ) Analysis of cell-specific genes in HA-pulldown/input samples (log2) from RNA sequencing demonstrates enrichment for astrocyte genes and depletion of other cells in all samples (N = 3 at postnatal day [P]7, 4 at P14, 5 at P28, 3 at P120; for statistical comparisons, 3xP120 samples published in were added to increase the power of the analysis). ( B–D ) Immunostaining for the HA tag and cell-specific markers to determine cell-type expression of tagged ribosomes in P28 visual cortex. ( D ) Representative images, left panels: cell marker; middle panels: HA; right panels: merge with DAPI to mark nuclei. ( B, C ) Quantification of ( D ). ( B ) Quantification of colocalization of HA with each cell-specific marker, expressed as % of marker + cells, demonstrates that majority of HA+ cells are astrocytes. ( C ) Quantification of number of astrocytes (s100β+) that are also HA+ demonstrates that majority of astrocytes express HA-tagged ribosomes. N = 4 mice S100β, NEUN, IBA1; 3 mice MOG; 2 mice NG2. Bar graphs mean ± s.e.m. Scale bars = 20 µm. ( E ) Heatmaps of top 20 most astrocyte-enriched genes (highest astro/IN ratio; FPKM > 100) at each time point, as well as those in the top 20 at all time points, represented as fold change (FC) of astrocyte/input (log2). ( F ) Venn diagram showing overlap in astrocyte-enriched genes at each age. ( G ) Gene Ontology (GO) terms analysis with String db of Biological Process (BP) in astrocyte-enriched genes at each time point. Venn diagram showing overlap in GO terms at each age.
Article Snippet: The following antibodies were used: Chk anti-GFP (Millipore #06-896, 1:500), Rb anti-SOX9 (Abcam #ab185966, 1:2000), Rb anti-ALDH1L1 (Abcam #ab-87117, 1:500), Rb anti-HA (CST #3724), Rb anti-S100β (Abcam #ab52642, 1:100), Ms anti-NEUN (Millipore #MAB377 1:100), Rb anti-NG2 (Millipore # Ab5320), Rb anti-MOG (Proteintech # 12690-1-ap), Rb anti-IBA1 (Wako #016-20001), Gp anti-VGLUT1 (Millipore #AB5905, 1:2000), Gp anti-VGLUT2 (Millipore #AB2251 1:3000, 1:5000), Rb anti-GLUA1 (Millipore #AB1504, 1:400), Rb anti-GLUA2 (Millipore #AB1768-I, 1:400), and Ms anti-Bassoon (Enzo #VAMP500, 1:500).
Techniques: RNA Sequencing Assay, Immunostaining, Expressing, Marker
![( A ) Analysis of cell-specific genes in HA-pulldown/input samples (log2) from RNA sequencing demonstrates enrichment for astrocyte genes and depletion of other cells in all samples (N = 3 at postnatal day [P]7, 4 at P14, 5 at P28, 3 at P120; for statistical comparisons, 3xP120 samples published in were added to increase the power of the analysis). ( B–D ) Immunostaining for the HA tag and cell-specific markers to determine cell-type expression of tagged ribosomes in P28 visual cortex. ( D ) Representative images, left panels: cell marker; middle panels: HA; right panels: merge with DAPI to mark nuclei. ( B, C ) Quantification of ( D ). ( B ) Quantification of colocalization of HA with each cell-specific marker, expressed as % of marker + cells, demonstrates that majority of HA+ cells are astrocytes. ( C ) Quantification of number of astrocytes (s100β+) that are also HA+ demonstrates that majority of astrocytes express HA-tagged ribosomes. N = 4 mice S100β, NEUN, IBA1; 3 mice MOG; 2 mice <t>NG2.</t> Bar graphs mean ± s.e.m. Scale bars = 20 µm. ( E ) Heatmaps of top 20 most astrocyte-enriched genes (highest astro/IN ratio; FPKM > 100) at each time point, as well as those in the top 20 at all time points, represented as fold change (FC) of astrocyte/input (log2). ( F ) Venn diagram showing overlap in astrocyte-enriched genes at each age. ( G ) Gene Ontology (GO) terms analysis with String db of Biological Process (BP) in astrocyte-enriched genes at each time point. Venn diagram showing overlap in GO terms at each age.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7060/pmc08497060/pmc08497060__elife-70514-fig1-figsupp1.jpg)
