Journal: Genes & Development

Article Title: The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells

doi: 10.1101/gad.227496.113

Figure Lengend Snippet: Increased apoptosis in FL ΔTAD/ΔTAD LT-HSCs. ( A ) Flow cytometry analysis of E11.5 AGM regions and E11.5 FLs from +/+ and ΔTAD/ΔTAD embryos. Cells were gated on 7-AAD − TER − 119 − populations. Endothelial cells were identified as CD144 + , hematopoietic progenitors are CD45 + , and HSCs are CD45 + CD144 + . ( B ) Absolute numbers of cells gated as CD45 + CD144 + (as depicted in A ) in E11.5 AGM regions and E11.5 FLs from three independent experiments. ( C ) Cell cycle analysis of +/+ and ΔTAD/ΔTAD E14.5 FL SLAM-LSKs. Representative flow cytometry plot of the cell cycle by DAPI and Ki-67. ( D ) The bar graph represents percentages of SLAM-LSKs in each cell cycle stage from three independent experiments. ( E ) Increased apoptosis in ΔTAD/ΔTAD E14.5 FL SLAM-LSKs. Representative flow cytometry plots of Annexin V + cells from +/+ (dotted line) and ΔTAD/ΔTAD (bold gray line) E14.5 FL SLAM-LSKs. Annexin V expression on internal control Lin + cells of +/+ (solid black line) and ΔTAD/ΔTAD (light-gray shading) was used to determine the positive gate for Annexin V staining. ( F ) The bar graph represents the normalized percentage of Annexin V + 7-AAD − cells from E14.5 +/+ and ΔTAD/ΔTAD FL SLAM-LSKs ( n = 4). Values were determined by subtracting the mean percentage of +/+ Annexin V + Lin + cells (calculated as percent Annexin V + cells ± SEM, which was 1.600 ± 0.147; n = 4) from the mean percentage of Annexin V + +/+ SLAM-LSKs and by subtracting the mean percentage of ΔTAD/ΔTAD Annexin V + Lin + cells (4.025 ± 0.728; n = 4) from the mean percentage of Annexin V + ΔTAD/ΔTAD SLAM-LSKs.

Article Snippet: For SLAM-LSK staining, the antibodies used were CD45.2 FITC (BD, 104), Sca-1 (Ly6A/E) PerCP-Cy5.5 or FITC (eBioscience, D7), CD48 APC or phycoerythrin (PE) (eBioscience, HM48-1), CD150 PE-Cy7 (Biolegend, TC15-12F12.2), c-Kit APC-Cy7 (eBioscience, 2B8), and DAPI (BD).

Techniques: Flow Cytometry, Cell Cycle Assay, Expressing, Staining

Journal: Genes & Development

Article Title: The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells

doi: 10.1101/gad.227496.113

Figure Lengend Snippet: Competitive defects and reduced LT-HSC frequency in ΔTAD FL HSCs. ( A ) Schematic for noncompetitive E14.5 FL transplants. ( B ) Multilineage reconstitution of primary recipients by ΔTAD/ΔTAD E14.5 FL cells. Representative flow cytometry plots from the thymus and spleen of +/+ or ΔTAD/ΔTAD reconstituted recipients at 16 wk. ( C ) E14.5 FL cells (2 × 10 6 ) from B6 (CD45.2 + ) +/+, +/ΔTAD, or ΔTAD/ΔTAD embryos were transplanted into lethally irradiated SJL (CD45.1 + ) recipients. Bar graph represents mean reconstitution at 16 wk in BM, measured by the percentage of CD45.2 + cells. ( D ) E14.5 FL cells (1 × 10 6 ) from B6 (CD45.2 + ) +/ΔTAD or ΔTAD/ΔTAD embryos were transplanted in competition with 1 × 10 6 wild-type CD45.1 + E14.5 FL cells into lethally irradiated SJL CD45.1 recipients. The bar graph represents mean reconstitution at 16 wk in peripheral blood, measured by the percentage of CD45.2 cells. ( E ) SLAM-LSK gating strategy to identify LT-HSCs. ( F ) Percentage of LSK from +/+, +/ΔTAD, or ΔTAD/ΔTAD E14.5 FL cells. All cells were first gated on DAPI − CD45.2 + . ( G ) Number of LSKs ( left panel) and SLAM-LSKs ( right panel) per 10 6 cells from +/+, +/ΔTAD, or ΔTAD/ΔTAD E14.5 FLs.

Article Snippet: For SLAM-LSK staining, the antibodies used were CD45.2 FITC (BD, 104), Sca-1 (Ly6A/E) PerCP-Cy5.5 or FITC (eBioscience, D7), CD48 APC or phycoerythrin (PE) (eBioscience, HM48-1), CD150 PE-Cy7 (Biolegend, TC15-12F12.2), c-Kit APC-Cy7 (eBioscience, 2B8), and DAPI (BD).

Techniques: Flow Cytometry, Irradiation

Journal: Biology

Article Title: Free Fatty Acid Species Differentially Modulate the Inflammatory Gene Response in Primary Human Skeletal Myoblasts

doi: 10.3390/biology10121318

Figure Lengend Snippet: FFAs do not affect the desmin expression pattern in human primary myoblasts. ( a ) SkMs were treated with FAs conjugated with BSA or BSA alone (control) before DAPI and desmin staining. ( b ) Fluorescence images from 3 sets of experiments were analyzed, as described in the legend to . The number of DAPI-positive FFA-treated cells was compared to that of control cells, which was set to 1 (c, dashed gray line). The data are presented as the mean ± SD of cell number (dotted bars). Statistical significance was calculated using one-way ANOVA with Bonferroni’s multiple comparison test. p ≤ 0.05 (*). p ≤ 0.01 (**).

Article Snippet: After staining, cells were covered with a coverslip in mounting medium with DAPI (DK2488, Vector Laboratories).

Techniques: Expressing, Staining, Fluorescence