Journal: Journal of Biological Chemistry

Article Title: Effect of Human Carbonic Anhydrase II on the Activity of the Human Electrogenic Na/HCO3 Cotransporter NBCe1-A in Xenopus Oocytes

doi: 10.1074/jbc.m602181200

Figure Lengend Snippet: FIGURE 6. Immunolocalization of EGFP and CA II domains in oocytes expressing EGFP-e1-CAII or EGFP- e1. Three days after injection of cRNA encoding EGFP-e1-CAII or EGFP-e1, oocytes were prepared for immu- nocytochemistryasindicatedunder“ExperimentalProcedures.”Preparationswereincubatedovernightsimul- taneously with mouse monoclonal anti-EGFP and with rabbit polyclonal anti-human CA II. The secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 594-conjugated goat anti-rab- bit. A-C, Alexa Fluor 488-labeled images were taken with excitation/emission filters set at band pass 488 nm/long pass 505 nm to detect EGFP signals. D-F, Alexa Fluor 594-labeled images were taken with excitation/ emission filters set at band pass 543 nm/long pass 560 nm to detect CA II signals. These images show that EGFP-e1-CAIIandEGFP-e1arecorrectlytargetedtotheplasmamembrane.G-I,mergesoftheimagesinthefirst and second columns. G shows that the EGFP staining (green) of EGFP-e1-CAII overlapped with the CA II staining (red). J–L, Nomarski images taken simultaneously with either the EGFP or CA II images. Bar 50 m.

Article Snippet: Slides were blocked by washing (once for 15 min) in TBS 0.1% BSA 10% normal goat serum (Vector Laboratories, Burlingame, CA).We incubated the preparations with mouse monoclonal anti-EGFP (BD Biosciences; 1:100 in TBS 0.1% BSA 10% goat serum) and rabbit polyclonal anti-human CA II (Rockland, Gilbertville, PA; 1:1000 in TBS 0.1% BSA 10% goat serum) overnight.

Techniques: Expressing, Injection, Labeling, Staining

Journal: bioRxiv

Article Title: Lymph node resident memory T cells retain effector capabilities by evading lung resident memory dysfunction

doi: 10.64898/2026.02.03.703569

Figure Lengend Snippet: (A) Naive CD90.1+ OT-I T cells were transferred into naive B6 mice before intranasal infection with influenza expressing SIINFEKL (PR8-OVA). Expression of CD69 and CD103 was measured 21 days post infection on extravascular cells. (B) Quantification of CD69 + CD103 + OT-I T cells. (C) Naïve CD90.1 + OT-I Nur77-GFP T cells were transferred into mice one day before intranasal PR8-OVA infection. Nur77-GFP expression was evaluated 3-14 days later by extravascular OT-I T cells. (D)Experimental setup for (E-G). Flow cytometry plot (E) of CD90.1 + OT-I T cells isolated from indicated locations. (F-G) Longitudinal labeling of cells and quantification. (H) Experimental setup for (I-J). (I) Labeling after intravenous and intraperitoneal injection of OT-I T cells or naïve endogenous T cells. (J) Expression of CD69 and CD103. Naïve= CD8 + CD90.1 - CD44 low . Each point represents a mouse and N=2-5 mice per experiment. Data are representative (A) or are pooled from at least two experiments (n=2–4/group), except for day 113 in (B), which reflect a single experiment.

Article Snippet: Measurements were obtained in Flow Cytometry (FACS) Staining Buffer (Rockland, MB-086-0500) at room temperature for about 30 minutes per sample and channels were flushed with buffer in between samples.

Techniques: Infection, Expressing, Flow Cytometry, Isolation, Labeling, Injection

Journal: bioRxiv

Article Title: Lymph node resident memory T cells retain effector capabilities by evading lung resident memory dysfunction

doi: 10.64898/2026.02.03.703569

Figure Lengend Snippet: (A)Chromatin accessibility with gene expression (right) and linked chromatin peaks (bottom) at the Gzmb locus. (B) Protein expression of GranzymeB as measured by flow cytometry. (C-D) Same as (A-B) but for the Pdcd1 locus. Protein expression on OT-I T cells is 49 days post infection where each dot represents one mouse. (J and L) Data are from one experiment and representative of at least two experiments with N=3. (E-F) Surface expression of PD-1 and TIM3 at 7 days post infection. (G) Transcription factor motif footprint analysis from dataset. (H) Inferred eRegulons identified by SCENIC+ across memory T cells after influenza infection. (I-K) Legend (left) for network plots illustrating inferred transcription factor relationships to target genes. Transcription factor and gene relationship is denoted by color of line represents activation (red) or repression (blue).

Article Snippet: Measurements were obtained in Flow Cytometry (FACS) Staining Buffer (Rockland, MB-086-0500) at room temperature for about 30 minutes per sample and channels were flushed with buffer in between samples.

Techniques: Gene Expression, Expressing, Flow Cytometry, Infection, Activation Assay

Journal: Cell stem cell

Article Title: Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration

doi: 10.1016/j.stem.2019.11.004

Figure Lengend Snippet: Identification of Hic1 as an enriched transcript in MPs. A, schematic overview of the strategy used to purify MPs from TA muscle. Numbers in parentheses indicate percent of the total mononuclear fraction from 3 independent isolations (see Figure S1A for markers and sorting gates). B, heat map from RNA-seq analysis of the various fractions indicated in A. Select genes associated with various cell types within muscle are shown in the right panel, with Hic1 showing enrichment in the Lin− LY6A+ fraction. C, Hic1 transcript abundance in the different fractions from the RNA-seq analyses (n = 3–4, data represent the mean ± SD). D, representative image of anti-HIC1 staining of 8 wk adult TA muscle counterstained with Laminin, LY6A or CD31. White arrowheads, HIC1+ cells. E, schematic representation of the Hic1 null allele that expresses nuclear LacZ (see Figure S1E for Hic1 knock-in allele description). F, the distribution of Hic1+ cells was evaluated in representative whole-mount (upper left panel) and in situ X-gal stained sections of TA muscles and the myotendinous junction (bottom series) from Hic1+/nLacZ mice. PLN, phalloidin+ muscle fibre. G, Hic1+ cells were enumerated from stained samples in F (n = 5, data represent the mean ± SD). MF, myofibre; TN, tendon.

Article Snippet: To isolate single nuclei, ~ 100,000 fresh FACS purified Hic1 ; tdTomato reporter + cells were collected as per scRNA-seq sorting conditions and lysed according to the described protocol (10x Genomics, https://support.10xgenomics.com/single-cell-atac ) for 5 min.

Techniques: RNA Sequencing, Staining, Knock-In, In Situ, Muscles

Journal: Cell stem cell

Article Title: Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration

doi: 10.1016/j.stem.2019.11.004

Figure Lengend Snippet: HIC1 regulates MP quiescence: deletion of Hic1 leads to an increase in MP number and an activated MP-like phenotype. A, chronological series of representative X-gal stained muscle sections following TA muscle injury in Hic1nLacZ/+ mice. Arrowheads, centrally located nuclei in newly regenerated myofibres. B, enumeration of DAPI nuclei per high powered field (hpf) from A. UI, uninjured control; I, injured. C, quantification of X-gal stained Hic1+ cells from A (data represent the mean ± SD, n = 3–4). D, representative whole-mount images of X-gal stained myofibres 5 d post-TAM treatment from 9 wk old male mice. E, representative histological images of X-gal stained cells from Hic1-deleted and non-deleted TA muscles at 8 months post-TAM. F, enumeration of X-gal stained cells from TA muscles (n = 3–5, data represent the mean ± SD, one-way ANOVA Bonferroni post-test, ***p<0.001 relative to 9 wk Hic1+/nLacZ baseline). G, FCM analysis of Lin−LY6A+ MPs in TA muscle from Hic1f/f and Hic1-deleted mice 10 d post-TAM injection (see Figure S2E for sorting gates and markers) (n = 8, data represent the mean ± SD, unpaired t-test ***p<0.001). H, FCM analysis of EdU incorporation in Lin−LY6A+ MPs from wild-type and Hic1-deleted mice (n ≥ 4). Mice received TAM at 2 m of age and 3 consecutive daily doses (0.5 mg/mouse) of EdU starting on day 5 post-TAM and were collected for analysis 24 h after the last EdU injection (n = 4–7, data represent the mean ± SD, unpaired t-test *p<0.05). I, heatmap of differentially expressed genes (Cuffdiff, q< 0.05) from RNA-seq analysis of sorted MPs from 2 m TA muscles, (n = 3). J, pairwise Venn diagram plots of overlapping gene expression between the indicated datasets. The 1 d p.i. dataset was generated using a comparison of 0 d MPs to 1 d p.i. as described in Figure 5A. K, IPA interrogation of transcriptomic data, from data shown in I (top panel) and L (bottom panel). L, heatmap of differentially expressed genes (Cuffdiff, q< 0.05) from RNA-seq analysis of whole 18 m TA muscles (TAM at 2 m) (n = 7–9). M, pairwise Venn diagram plots of overlapping gene expression between samples described in H and K.

Article Snippet: To isolate single nuclei, ~ 100,000 fresh FACS purified Hic1 ; tdTomato reporter + cells were collected as per scRNA-seq sorting conditions and lysed according to the described protocol (10x Genomics, https://support.10xgenomics.com/single-cell-atac ) for 5 min.

Techniques: Staining, Control, Muscles, Injection, RNA Sequencing, Gene Expression, Generated, Comparison

Journal: Cell stem cell

Article Title: Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration

doi: 10.1016/j.stem.2019.11.004

Figure Lengend Snippet: The activated MP phenotype displays stage-specific activities indicative of a coordinated response to injury. A, RNA-seq analysis of tdTomato+ enriched MPs at various time points p.i. Select gene profiles of identified cellular programs are shown. Cxcl5, cytokines; Mki67, cell cycle; Postn, provisional matrix; Lamc1, basement membrane. B, heatmap from popRNA-seq of differentially-expressed cytokine genes after injury. C, representative image of anti-CXCL5 staining in control and NTX-injured TA muscles from Hic1;tdTomato reporter mice. Graph inset, quantification of CXCL5+tdTomato+ cells p.i. (n ≥ 3, data reflect the mean ± SD). D, scRNA-seq t-SNE clustering of cells at the indicated time points p.i. E, heat-map of scRNA-seq hierarchically clustered genes. The vertical colored lines reflect the programs indicated in A. T, tenogenic cluster; P, pericytic cluster. F, t-SNE plots of scRNA-seq data for Pdgfra and Cxcl5. The pericytic and tendogenic lineages are indicated by salmon and green dashed lines, respectively. G, analysis of cytokine gene chromatin accessibility and gene expression using scATAC-seq and scRNA-seq, respectively. Genome browser tracks displaying the promoter sum signal around the indicated gene loci from the 4 defined clusters from scATAC-seq (D0) are shown along with the corresponding tSNE plot of the scRNA-seq expression data. H, heatmap from popRNA-seq highlighting genes associated with cell proliferation. I, t-SNE plots of clusters expressing Mki67 at different times p.i. J, analysis of EdU+, tdTomato+ cells at 0 and 4 d p.i. including representative images of stained sections (see Figure S5C for sorting gates and markers) (n ≥ 3, data reflect the mean ± SD, unpaired t-test ***p<0.001). K, multiple genes associated with provisional ECM production and turnover are coordinately expressed starting at D1 through to D14 (see Figure S5E for additional genes). L, distribution of Postn and Lox transcripts in t-SNE derived clusters of single cells (scRNA-seq). M, genome browser tracks showing the promoter sum signal around the Lox and Postn gene loci within the 4 different clusters (scATAC-seq) from tdTomato+ cells at D0. N, representative images of IF staining of 0, 4 and 14 d p.i. TA muscles from Hic1; tdTomato reporter mice with anti-POSTN.

Article Snippet: To isolate single nuclei, ~ 100,000 fresh FACS purified Hic1 ; tdTomato reporter + cells were collected as per scRNA-seq sorting conditions and lysed according to the described protocol (10x Genomics, https://support.10xgenomics.com/single-cell-atac ) for 5 min.

Techniques: RNA Sequencing, Membrane, Staining, Control, Muscles, Gene Expression, Expressing, Derivative Assay

Journal: Cell stem cell

Article Title: Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration

doi: 10.1016/j.stem.2019.11.004

Figure Lengend Snippet: Generation and use of a Hic1CreERT2 knock-in allele to characterize Hic1+ MPs. A, overview of the Hic1CreERT2 knock-in allele and associated lineage-tracing strategy (see Figure S3A for description of Hic1 knock-in allele). B, FCM analysis of enzymatically dissociated TA muscles from Hic1CreERT2; Rosa26LSL-tdTomato mice post-TAM (n ≥ 4, data reflect the mean ± SD). C, t-SNE plot of scRNA-seq data from enriched tdTomato+ MPs 10 d post-TAM induction. D, heat-map of scRNA-seq data showing enriched genes in the 4 different clusters. E, violin plots showing expression of select lineage-associated genes from the scRNA-seq data. F, tSNE plot of scATAC-seq from enriched tdTomato+ MPs using a similar TAM injection regime as in D. The clusters indicative of the 4 populations were colored using the same convention as in C–E. G, genome browser tracks displaying the promoter sum signal around the indicated gene loci from the 4 defined clusters. H, representative transverse and longitudinal sections of the TA muscle at 5 d post-TAM, counterstained as indicated.

Article Snippet: To isolate single nuclei, ~ 100,000 fresh FACS purified Hic1 ; tdTomato reporter + cells were collected as per scRNA-seq sorting conditions and lysed according to the described protocol (10x Genomics, https://support.10xgenomics.com/single-cell-atac ) for 5 min.

Techniques: Knock-In, Muscles, Expressing, Injection

Journal: Cell stem cell

Article Title: Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration

doi: 10.1016/j.stem.2019.11.004

Figure Lengend Snippet: Hic1-tracked MPs directly contribute to multiple mesenchymal cell types within the regenerated muscle. A, FCM analysis of LY6A expression in tdTomato+ cells 14 d p.i. shows a significant increase in LY6A+tdTomato+ cells. The dashed line represents the undamaged sample at 0 d (n ≥ 3, data reflect the mean ± SD, one-way ANOVA Bonferroni post-test, *** p<0.001). B, popRNA-seq analysis of tdTomato+ enriched fraction from D14 TA muscle p.i. shows enrichment of transcripts associated with tendon (Scx, Mkx, Tnmd and Kera) and the MTJ (Col22a1). C, tSNE overview of Hic1;tdTomato sub-populations and cluster-marker genes. D, UMAP (left panel) with cells colored by library ID indicative of the time of collection (see Figure S6A for UMAP colored by cluster). Cell partitions identified through Monocle3 and the representative markers (Pdgfra, Ly6a, Rgs5 and Tnmd) used to identify the cell types. E, UMAP plot (left panel) of pseudotime trajectories for the FAP subpopulation. The right panels contain plots of the individual time points (library ID). F-H, replicate of what is shown in D for the pericytic (F), tenogenic (G) and myotenogenic (H) populations.

Article Snippet: To isolate single nuclei, ~ 100,000 fresh FACS purified Hic1 ; tdTomato reporter + cells were collected as per scRNA-seq sorting conditions and lysed according to the described protocol (10x Genomics, https://support.10xgenomics.com/single-cell-atac ) for 5 min.

Techniques: Expressing, Marker

Journal: Cell stem cell

Article Title: Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration

doi: 10.1016/j.stem.2019.11.004

Figure Lengend Snippet: Hic1-marked MPs directly contribute to regeneration of the myotendinous junction. A, t-SNE plots from scRNAseq-analyses of Hic1;tdTomato sorted MPs. Tendon-expressed genes are shown from 2,173 and 3,527 profiled cells from D0 and D14, respectively. B, violin plots of gene expression for the clusters shown in Figure 3E. C, representative images of anti-COL22a1 stained sections from uninjured and 14 d p.i. TA muscles (white arrowheads, tdTomato+ cells embedded in COL22a1 rich matrix). D, visualization of GFP (Scx-GFP) and tdTomato expression in representative images from within MTJ regions that were identified by anti-COL22A1 staining. White arrowheads indicate Scx-GFP single positive cells and white arrows denote GFP+tdTomato+ cells. Blue arrowheads indicate GFP+tdTomato+ tenocytes. E, FCM analysis of GFP+tdTomato+ cells prior to injury and at 28 d p.i. (n ≥ 3, data reflect the mean ± SD, unpaired t-test). F, overarching schematic of Hic1+ MP participation in muscle regeneration.

Article Snippet: To isolate single nuclei, ~ 100,000 fresh FACS purified Hic1 ; tdTomato reporter + cells were collected as per scRNA-seq sorting conditions and lysed according to the described protocol (10x Genomics, https://support.10xgenomics.com/single-cell-atac ) for 5 min.

Techniques: Gene Expression, Staining, Muscles, Expressing

Journal: Cell stem cell

Article Title: Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration

doi: 10.1016/j.stem.2019.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To isolate single nuclei, ~ 100,000 fresh FACS purified Hic1 ; tdTomato reporter + cells were collected as per scRNA-seq sorting conditions and lysed according to the described protocol (10x Genomics, https://support.10xgenomics.com/single-cell-atac ) for 5 min.

Techniques: Recombinant, Reverse Transcription, Imaging, Wound Healing Assay, Multiplex Assay, Gene Expression, Software