Journal: bioRxiv

Article Title: NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence

doi: 10.1101/212829

Figure Lengend Snippet: NOTCH1 signalling and JAG1 expression can non-autonomously repress SAHFs in adjacent cells. (a) Schematic showing experimental setup. IMR90 cells expressing either Doxycycline (DOX)-in-ducible FLAG-N1ICD or ER:RAS were co-cultured on 4OHT for 6 days with/without DOX. (b) Quantifi-cation of SAHF positive red cells for the experiment outlined in a. Alone, mono-cultured IMR90 ER:HRAS G12V cells; iN1ICD, DOX-inducible N1ICD. (c) Representative images of co-cultures indicated. Insets are unmerged DAPI images of indicated cells (arrows) (d) Schematic showing experimental setup. IMR90 ER:HRAS G12V cells were co-cultured with RPE1 cells stably expressing either mVenus or JAG1-mVenus for 6 days with/without 4OHT. (e) Quantification of SAHF positive red cells for the experiment outlined in d. (f) Representative images of co-cultures indicated. Insets are unmerged DAPI images of indicated cells (arrows) (b, c) . Lines indicate the mean value of individual replicates. n=3 biologically independent replicates for all conditions; Statistical significance calculated using one-way ANOVA with Tukey’s correction for multiple comparisons; *P ≤ 0.05, **P ≤ 0.01, *P ≤ 0.001.

Article Snippet: Cells were stained with anti-JAG1-APC (FAB1726A, R&D systems) or isotype control antibody before analysis on a FACSCalibur flow cytometer (Becton Dickenson).

Techniques: Expressing, Cell Culture, Stable Transfection

Journal: bioRxiv

Article Title: NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence

doi: 10.1101/212829

Figure Lengend Snippet: Ectopic JAG1 expressing RPE1 cells require cell-cell contact to repress SAHFs in RIS cells (a) RPE1 JAGGED1-mVenus cells analysed for cell surface expression of JAG1 by flow cytometry (n=1). (b) Quantification of SAHF positive IMR90 ER:HRAS G12V cells cultured in a transwell dish with the indicated RPE1 cells for 6 days +4OHT (n=3)

Article Snippet: Cells were stained with anti-JAG1-APC (FAB1726A, R&D systems) or isotype control antibody before analysis on a FACSCalibur flow cytometer (Becton Dickenson).

Techniques: Expressing, Flow Cytometry, Cell Culture

Journal: bioRxiv

Article Title: NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence

doi: 10.1101/212829

Figure Lengend Snippet: NOTCH1 signalling represses SAHFs partially by repressing HMGA proteins. (a, b) qRT-PCR (n=6) (a) and immunoblotting (b) for the indicated mRNA and proteins in IMR90 ER:HRAS G12V cells stably infected with control vector or FLAG-N1ICD ± 4OHT for 6 days. (c, d) qRT-PCR (n=4) (c) and immunoblotting (d) of IMR90 cells expressing doxycycline (DOX)-inducible FLAG-N1ICD (iN1ICD) and infected with a control vector or dnMAML1 ± DOX for 3 days. (e) Quantification of SAHFs in IMR90 cells expressing ER:HRAS G12V , iN1ICD, and EGFP or EGFP-HMGA1 fusion ± 4OHT for 6 days and ± DOX for 3 days. (f) qRT-PCR for the indicated mRNA in IMR90 cells co-cultured with RPE1 mVenus or JAG1-mVenus cells and sorted using MACS (n=3). Statistical significance calculated using one-way ANOVA with Tukey’s correction for multiple comparisons (a, c) or paired t-test (e, f) . *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Cells were stained with anti-JAG1-APC (FAB1726A, R&D systems) or isotype control antibody before analysis on a FACSCalibur flow cytometer (Becton Dickenson).

Techniques: Quantitative RT-PCR, Western Blot, Stable Transfection, Infection, Plasmid Preparation, Expressing, Cell Culture

Journal: bioRxiv

Article Title: NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence

doi: 10.1101/212829

Figure Lengend Snippet: Tumour cells can repress SAHFs and RIS specific NFRs in adjacent RIS fibroblasts. (a) Normalised RNA expression values from the Cancer Cell Line Encyclopaedia (CCLE) and immunoblotting of JAG1 in the mour cell lines indicated. (b) Quantification of SAHFs in red cells of co-culture between IMR90 ER:HRAS G12V RFP1 cells and tumour cell lines indicated for 6 days + 4OHT ± DAPT. (c, d) Experimental setup (c) and T-PCR (d) of mRNA isolated from flow sorted IMR90 ER:HRAS G12V mRFP1 cells cultured with tumour cell lines OHT for 6 days relative to cells cultured alone. n=3 biologically independent replicates. Statistical significance lculated using one-way ANOVA with Tukey’s correction for multiple comparisons; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ (e) Number of ‘RIS specific NFRs’, identified in , that are lost when IMR90 ER:HRAS G12V mRFP1 cells e cultured with the tumour cell lines indicated before flow sorting as in described in c. (f) Representative genome owser images showing normalised ATAC-seq coverage files around an altered region in the cell conditions dicated and described in e. Note that ‘+MCF7’, ‘+A549’ and ‘+Hep3B’ denotes that RIS cells have been previously co-cultured with these cell lines for 6 days prior to flow sorting.

Article Snippet: Cells were stained with anti-JAG1-APC (FAB1726A, R&D systems) or isotype control antibody before analysis on a FACSCalibur flow cytometer (Becton Dickenson).

Techniques: RNA Expression, Western Blot, Co-Culture Assay, Isolation, Cell Culture

Journal: bioRxiv

Article Title: NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence

doi: 10.1101/212829

Figure Lengend Snippet: NOTCH1 signalling mediates non-cell autonomous regulation of chromatin structure at the microscopic and nucleosome scale. Lateral induction of NOTCH1 activity in a signal-receiving cell by JAG1 on the surface of an adjacent cell (including cancer cells) can drive NIS. NIS cells form ‘NIS unique NFRs’, which tend to be relatively GC rich, and microscopically ‘smoothened’ chromatin. In the context of RIS, non-cell autonomous activation of NOTCH1 signalling can repress the formation of AT-rich ‘RIS unique NFRs’ at the nucleosome level and SAHFs at the microscopic level. Mechanistically, N1ICD represses HMGA1, which is responsible for SAHF formation and at least partially for the formation of ‘RIS-unique NFRs’.

Article Snippet: Cells were stained with anti-JAG1-APC (FAB1726A, R&D systems) or isotype control antibody before analysis on a FACSCalibur flow cytometer (Becton Dickenson).

Techniques: Activity Assay, Activation Assay

Journal: International Journal of Oral Science

Article Title: Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

doi: 10.1038/s41368-021-00140-6

Figure Lengend Snippet: Cellular composition switch of freshly isolated human dental pulp stem cells (hDPSCs) upon in vitro culture. a UMAP visualization of monolayer cultured cells (red dots) compared to the freshly isolated cells (blue dots). The purple dash circles highlighted the most overlapped zone in both conditions. b UMAP visualization of 14 color-coded clusters of integrated freshly isolated and 10-day in vitro cultured hDPSCs. Four major cell types are identified, including dental pulp cells, endothelia cells, immune cells, and glial cells. Cluster 4 cells present in both datasets. c Differentially expressed genes in cluster 4 from fresh and cultured datasets were listed in the blue and pink circles, respectively. The overlapped gray region highlights the genes with similar expression levels in cluster 4 from both datasets. Expression dynamics along the pseudotime of selected genes from left panel was plotted by Monocle3. d The distribution (left) and quantification (right) of selected stem cell surface makers genes (MCAM, JAG1, and PDGFRA) expression in both cultured and fresh hDPSCs. Cells with low and high expression were marked with gray and purple color, respectively

Article Snippet: Each of the following conjugated anti-human monoclonal antibodies were incubated with the cell suspensions at 37 °C for 30 min in the dark: MCAM/PE (BD 550315), JAG1/APC (R&D FAB1277A) and PDGFRa/BB700 (BD 746023).

Techniques: Isolation, In Vitro, Cell Culture, Expressing

Journal: International Journal of Oral Science

Article Title: Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

doi: 10.1038/s41368-021-00140-6

Figure Lengend Snippet: Distribution of MCAM(+)JAG1(+)PDGFRA(−) cells in the extracted human third molar. a Immunofluorescence double staining of endothelial cells markers CD31 (red) with MCAM (green, top) and JAG1 (green, bottom), respectively. b Immunofluorescence double staining of MCAM (red) and JAG1 (green) c Immunofluorescence double staining of PDGFRA (red) with MCAM (green) and JAG1 (green), respectively. The odontoblast layer was indicated by the dash lines, and the magnified area (dotted boxes) was shown in yellow dotted boxes (the middle panels). Nucleus were labeled with DAPI (blue). White arrowheads indicated characteristic blood vessel structures. Scale bars = 50 μm

Article Snippet: Each of the following conjugated anti-human monoclonal antibodies were incubated with the cell suspensions at 37 °C for 30 min in the dark: MCAM/PE (BD 550315), JAG1/APC (R&D FAB1277A) and PDGFRa/BB700 (BD 746023).

Techniques: Immunofluorescence, Double Staining, Labeling

Journal: International Journal of Oral Science

Article Title: Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

doi: 10.1038/s41368-021-00140-6

Figure Lengend Snippet: Flow-cytometry analysis of MCAM( + )JAG1( + ) PDGFRA(−) cells in human dental pulps from young, aged, healthy, and dental caries samples. a Representative graphs of dental pulp cell suspensions gained from young, following exclusion of PDGFRA(+) cells (purple boxes) and selection of double positive of JAG1 and MCAM cells (blue boxes). b Representative graphs of dental pulp cell suspensions gained from aged samples. c The percentage of MCAM(+)JAG1(+) PDGFRA(−) cells (blue numbers) in young/aged groups showed no significant differences by two-tailed t test. d Representative graphs of dental pulp cell suspensions gained from healthy samples. e Representative graphs of dental pulp cell suspensions gained from dental caries samples. f The percentage of MCAM(+)JAG1(+)PDGFRA(−) cells (blue numbers) in healthy and dental caries groups shows no significant differences by two-tailed t test

Article Snippet: Each of the following conjugated anti-human monoclonal antibodies were incubated with the cell suspensions at 37 °C for 30 min in the dark: MCAM/PE (BD 550315), JAG1/APC (R&D FAB1277A) and PDGFRa/BB700 (BD 746023).

Techniques: Flow Cytometry, Selection, Two Tailed Test

Journal: International Journal of Oral Science

Article Title: Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

doi: 10.1038/s41368-021-00140-6

Figure Lengend Snippet: In vitro cellular performance of MCAM( + )JAG1( + ) PDGFRA(−) hDPSCs. a MCAM(+)JAG1(+) PDGFRA(−) and PDGFRA(+) hDPSCs were sorted by FACS. b Contrast microscopic images of cellular spheres (diameter exceeds 25 μm) formed by MCAM(+)JAG1(+) PDGFRA(−) and PDGFRA(+) hDPSCs, Scale bar = 100 μm; and the quantification of the spheres from each group formed after 7 days. * P < 0.05. c In vitro proliferation of MCAM(+)JAG1(+)PDGFRA(−) and PDGFRA(+) hDPSCs during 6-day culture. * P < 0.05. d Osteogenic marker genes expressions of RUNX2, COL1A1 , and BSP of MCAM(+)JAG1(+)PDGFRA(−) and PDGFRA(+) hDPSCs after 14-day culture. e Western blot and quantitative analysis of RUNX2 expression in protein level after 7 days induction. f Alizarin Red S staining and quantification of the mineralized matrix obtained from MCAM(+)JAG1(+)PDGFRA(−) and PDGFRA(+) hDPSCs after 21 day culture in osteogenic medium

Article Snippet: Each of the following conjugated anti-human monoclonal antibodies were incubated with the cell suspensions at 37 °C for 30 min in the dark: MCAM/PE (BD 550315), JAG1/APC (R&D FAB1277A) and PDGFRa/BB700 (BD 746023).

Techniques: In Vitro, Marker, Western Blot, Expressing, Staining

Journal: International Journal of Oral Science

Article Title: Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

doi: 10.1038/s41368-021-00140-6

Figure Lengend Snippet: In vitro cellular performance of MCAM(+)JAG1(+) PDGFRA(−) hDPSCs. a Chondrogenic marker gene expression of SOX9, ACAN, and COL2A1 of MCAM(+)JAG1(+)PDGFRA(−) and PDGFRA(+) hDPSCs after 14-day culture. b Western blot and quantitative analysis of SOX9 expression in protein level after 7 days induction. c Alcian blue staining and quantification of the cultured pellets obtained from MCAM(+)JAG1(+)PDGFRA(−) and PDGFRA(+) hDPSCs after 28 days induction. d Adipogenic marker gene expression of PPARG, FABP4 , and CEBPA of MCAM(+)JAG1(+)PDGFRA(−) and PDGFRA(+) hDPSCs after 14-day culture. e Western blot and quantitative analysis of PPARG expression in protein level after 14 days induction. f Oil red O staining and quantification of the adipocytes obtained from MCAM(+)JAG1(+)PDGFRA(−) and PDGFRA(+) hDPSCs after 28 days induction. GAPDH was used as the normalization control in qPCR. The levels of ACTIN are used as loading control in western blot. Bars represent mean ± SD values. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Each of the following conjugated anti-human monoclonal antibodies were incubated with the cell suspensions at 37 °C for 30 min in the dark: MCAM/PE (BD 550315), JAG1/APC (R&D FAB1277A) and PDGFRa/BB700 (BD 746023).

Techniques: In Vitro, Marker, Expressing, Western Blot, Staining, Cell Culture

Journal: International Journal of Oral Science

Article Title: Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

doi: 10.1038/s41368-021-00140-6

Figure Lengend Snippet: Osteogenic differentiation of MCAM( + )JAG1( + )PDGFRA(−) and PDGFRA( + ) hDPSCs in vivo. a Representative images of HE staining from cell-laden constructs after 4 weeks subcutaneous implantation. S scaffolds, B newly-formed bone tissues. Scale bars = 50 μm. b Representative images of immunohistochemical staining of human mitochondria. Scale bars = 100 μm. c Representative images of immunohistochemical staining of human specific human specific osteocalcin (OCN). Scale bars = 100 μm

Article Snippet: Each of the following conjugated anti-human monoclonal antibodies were incubated with the cell suspensions at 37 °C for 30 min in the dark: MCAM/PE (BD 550315), JAG1/APC (R&D FAB1277A) and PDGFRa/BB700 (BD 746023).

Techniques: In Vivo, Staining, Construct, Immunohistochemical staining

Journal: International Journal of Oral Science

Article Title: Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

doi: 10.1038/s41368-021-00140-6

Figure Lengend Snippet: Adipogenic differentiation of MCAM( + )JAG1( + )PDGFRA(−) and PDGFRA( + ) hDPSCs in vivo. a Representative images of HE staining from cell-laden constructs when transplanted into immunocompromised mice for 4 weeks. A higher magnification emphasizing the yellow dotted portions. Scale bars = 200 μm. b Representative images of immunohistochemical staining of human specific mitochondria. Scale bars = 100 μm

Article Snippet: Each of the following conjugated anti-human monoclonal antibodies were incubated with the cell suspensions at 37 °C for 30 min in the dark: MCAM/PE (BD 550315), JAG1/APC (R&D FAB1277A) and PDGFRa/BB700 (BD 746023).

Techniques: In Vivo, Staining, Construct, Immunohistochemical staining

Journal: Molecular Neurodegeneration

Article Title: Tolerogenic bone marrow-derived dendritic cells induce neuroprotective regulatory T cells in a model of Parkinson’s disease

doi: 10.1186/s13024-018-0255-7

Figure Lengend Snippet: Surface expression on BMDCs . GM-CSF-generated BMDCs were cultured in media alone or with 20 ng/ml GM-CSF for 2 days prior to stimulation with 30 μg/ml N-α-Syn for 1 day. Treatment groups were as follows: (1) media-cultured, unstimulated BMDCs; (2) GM-CSF-cultured, unstimulated BMDCs; (3) media-cultured, N-α-Syn-stimulated BMDCs; and (4) GM-CSF-cultured, N-α-Syn-stimulated BMDCs. Cells were harvested and reacted with antibodies to detect expression of CD11c, CD11b, MHC II, CD86, OX40L, Jag-1, CD39 and CD73, then evaluated by flow cytometric analysis. a Cells were gated by forward scatter area vs height to include only single cells and CD11b+CD11c+ BMDC populations were identified. Percentages of cells expressing CD11b or CD11c were determined and the mean percentages of single cells positive for each marker are shown within the bars. ( b and c ) BMDCs were gated to include the CD11b+CD11c+ cell population and the geometric mean fluorescent intensity (MFI) was determined for expression of MHC II, CD86, OX40L, Jag-1, CD39, and CD73. b Overlays of representative histograms are shown for BMDCs treated with media (red), GM-CSF (blue), N-α-Syn (orange), or GM-CSF + N-α-Syn (green). c Histograms represent the means ± SEM for 7 replicates from each treatment group. The means were compared by one-way ANOVA and Newman-Keuls post-hoc test whereby p ≤ .0.05 compared to BMDCs treated with a media, b GM-CSF, or c N-α-Syn

Article Snippet: BMDCs were stained with the following mixture of AlexaFluor 488-anti-CD11c, PECy7-anti-CD11b, PE-anti-Jagged-1, APC-anti-OX40L, AlexaFluor 700-anti-MHC II, eFluor 450-anti-CD86, eFluor 710-anti-CD39 (eBioscience, San Diego, CA) and APC-Vio 770-anti-CD73 (Miltenyi Biotec, Auburn, CA) for 30 min at 4 °C.

Techniques: Expressing, Generated, Cell Culture, Marker

Journal: Immunology

Article Title: Notch signalling suppresses regulatory T‐cell function in murine experimental autoimmune uveitis

doi: 10.1111/imm.12663

Figure Lengend Snippet: Notch signalling inhibits expression of immunoregulatory molecules in infiltrating regulatory T (Treg) cells. Infiltrating Treg cells were cultured for 24 hr the same way as described in Fig. ​Fig.4(c).4(c). (a–e) Expression of indicated cytokines and programmed cell death protein 1 (PD‐1) in infiltrating Treg cells after culture was determined by real‐time PCR. (f–h) Expression of interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4) and IL‐17a in cultured Treg cells. Fresh, freshly sorted Treg cells. Alone, Treg cells alone. Ligand, Treg cells cultured with immobilized Jagged‐1 (JAG1) and Delta‐like ligand 1 (DLL1). Ligand+Abs, Treg cells cultured with immobilized JAG1 and DLL1 in the presence of neutralizing antibodies against JAG1 and DLL1. *P < 0·05; **P < 0·01; ***P < 0·001.

Article Snippet: APC anti‐T‐cell receptor‐ β (H57‐597), PE‐Cy7 anti‐CD154 (MR1), APC anti‐Helios (22F6), PE‐Cy5 anti‐CD25 (FC), PE anti‐Notch‐1 (HMN1‐12), PE anti‐Notch‐3 (HMN3‐133), and PE anti‐JAG1 (HMJ1‐29) were purchased from Biolegend (San Diego, CA, USA). eFluor450 anti‐CD137 (17B5) and eFluor450 anti‐PD‐1 (J43) were purchased from eBioscience (San Diego, CA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Immunology

Article Title: Notch signalling suppresses regulatory T‐cell function in murine experimental autoimmune uveitis

doi: 10.1111/imm.12663

Figure Lengend Snippet: Notch‐1 is crucial for regulatory T (Treg) cell function in experimental autoimmune uveitis (EAU). (a) Knockdown of Notch‐1 or Notch‐2 in Treg cells. Upper panel: representative Immunoblot image. Lower panel: statistical analysis for Notch‐1 and Notch‐2. (b) Experimental design for (c–e). (c) Representative dot plots of donor‐derived Treg cells in uveitic eyes. (d) Expression of Foxp3 and programmed cell death protein 1 (PD‐1) in donor‐derived Treg cells in uveitic eyes. Left panel: representative histograms. Right panel: statistical analysis. (e) mRNA levels of transforming growth factor‐β (TGF‐β), interleukin‐12a (IL‐12a), IL‐27b and interferon‐γ (IFN‐γ) in donor‐derived Treg cells in uveitic eyes. L‐SC, Treg cells transduced with L‐SC. L‐sh‐N‐1, Treg cells transduced with L‐sh‐N‐1. L‐sh‐N‐2, Treg cells transduced with L‐sh‐N‐2. *P < 0·05; **P < 0·01; ***P < 0·001. n = 3 or n = 5 per group. Each circle represents a mouse.

Article Snippet: APC anti‐T‐cell receptor‐ β (H57‐597), PE‐Cy7 anti‐CD154 (MR1), APC anti‐Helios (22F6), PE‐Cy5 anti‐CD25 (FC), PE anti‐Notch‐1 (HMN1‐12), PE anti‐Notch‐3 (HMN3‐133), and PE anti‐JAG1 (HMJ1‐29) were purchased from Biolegend (San Diego, CA, USA). eFluor450 anti‐CD137 (17B5) and eFluor450 anti‐PD‐1 (J43) were purchased from eBioscience (San Diego, CA, USA).

Techniques: Cell Function Assay, Western Blot, Derivative Assay, Expressing, Transduction

Journal: Development (Cambridge, England)

Article Title: Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling

doi: 10.1242/dev.173328

Figure Lengend Snippet: Isolation of distinct mesenchymal cell populations from forelimb buds at E10.5-E10.75 and E11.5. (A) FACS strategy to isolate the different cell populations from lineage-negative (Lin − ) mesenchymal cells of mouse forelimb buds (35-38 somites, E10.5-E10.75, first panel). S9 + Pα hi (violet): Sox9 -EGFP-positive cells corresponding predominantly to osteo-chondrogenic progenitors (OCPs) express high levels of PDGFRα. In addition, three mesenchymal populations of Sox9 -EGFP-negative cells were isolated: S9 − SCA-1 + (green), S9 − JAG1 + (red) and S9 − Pα hi (blue) cells. (B) Box and whisker plot showing the abundance (%) of the different cell populations within the Lin − mesenchymal cells. The midline represents the median, the upper and lower limits of the box indicate the first and third quartiles, and the whiskers indicate the lowest and highest values, respectively. (C) Relative Col2a1 expression levels in S9 + Pα hi OCPs (E10.5-E10.75) and S9 + Pα hi Col2a1 + chondroblasts (CBs, E11.5). (D) CB were isolated from Lin − forelimb bud mesenchymal cells at E11.5 as S9 + Pα hi cells that express Col2a1 : S9 + Pα hi Col2a1 + CBs (orange, see C for Col2a1 expression). Representative FACS experiments are shown and the same gates were used for all analyses.

Article Snippet: After the initial gating, the lineage-negative (Lin − ) cells were separated into different populations using the following antibodies: anti-PDGFRα (CD140a; clone APA5: BV421-conjugated, Biolegend); anti-JAG1 (clone HMJ1-29: PE-conjugated, Biolegend) and anti-SCA-1 (clone D7: APC-conjugated, eBioscience).

Techniques: Isolation, Whisker Assay, Expressing

Journal: Development (Cambridge, England)

Article Title: Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling

doi: 10.1242/dev.173328

Figure Lengend Snippet: Comparative transcriptome analysis identifies two early LMP populations. (A) Principal components analysis (PCA) of RNA-seq metadata from the five different forelimb bud mesenchymal cell populations identified. Three biological replicates were sequenced for all populations, with the exception of S9 − SCA-1 + cells, which yielded only two samples of sufficient sequencing quality. (B) Heatmap showing the relative expression levels of key genes involved in myoblast migration and differentiation. Analysis shows that the S9 − SCA-1 + progenitors express the highest levels of myoblast-specific markers in comparison with the other populations ( Tables S1 and S2 ). Higher than average expression, orange-red; lower than average, blue; average, white. (C-F) Global gene ontology (GO) enrichment analysis of the genes whose expression is higher in the cell population of interest than all other populations ( Tables S2-S6 ). (C) S9 − JAG1 + LMPs, (D) S9 − Pα hi LMPs, (E) S9 + Pα hi OCPs (all E10.5-E10.75) and (F) S9 + Pα hi Col2a1 + chondroblasts (E11.5). The x -axis shows the -log 10 of the false discovery rate (FDR). Asterisks indicate chondrogenesis- and limb-related GO terms.

Article Snippet: After the initial gating, the lineage-negative (Lin − ) cells were separated into different populations using the following antibodies: anti-PDGFRα (CD140a; clone APA5: BV421-conjugated, Biolegend); anti-JAG1 (clone HMJ1-29: PE-conjugated, Biolegend) and anti-SCA-1 (clone D7: APC-conjugated, eBioscience).

Techniques: RNA Sequencing Assay, Sequencing, Expressing, Migration