Journal: Molecular cell

Article Title: BRCA1 mutational complementation induces synthetic viability

doi: 10.1016/j.molcel.2020.04.006

Figure Lengend Snippet: (A) The Brca1CC allele was generated using the indicated sgRNA sequence that targeted exon 13. DNA and amino acids deleted are shown (red). Below, electropherogram showing DNA sequences of Brca1+/+ and Brca1CC/CC mice, deleted base pairs (box) and deletion location (arrow) are indicated. (B) MEFs with the indicated genotypes were assessed for Brca1 protein expression by Western blotting. (C) MEFs with the indicated genotypes were assessed for RAD51 IRIF by immunofluorescence (IF). Nuclei with >5 foci were considered positive. Mean and S.E.M. are shown, n=3. Representative images, scale bar is 10 μm. (D) Summary of live pups born as well as live embryos at E16.5 generated from Brca1+/CC x Brca1+/CC intercross. (E) Representative photographs of E16.5 embryos, scale bar 0.5 cm; and 3-week old littermates, scale bar 2 cm. Hypopigmented fur area is indicated with an arrow. (F) Weights of individual mice at 3–4 weeks of age. (G) White blood cell (WBC), red blood cell (RBC) numbers from peripheral blood, and (H) mononuclear bone marrow (BM) cell numbers were measured in 3–4-week-old littermates with the indicated genotypes. Inset, representative H&E staining of BM, scale bar 100 μm. (I) Total number of long-term hematopoietic stem cells (LT-HSC) (Linlow; cKit+; Sca+; CD150+; CD48−; CD34−) analyzed by FACS in 3–4-week-old mice. See Fig. S1 for more information. (J) Representative H&E staining of testes and ovaries, scale bar 100 μm. (K) Representative images of T-ALL from a Brca1CC/CC mouse. H&E staining of thymic tumor and CD3+ staining of tumor cell infiltrates from the same mouse, scale bar 100 μm. Summary of Brca1CC/CC lifespans (days) and cause of death when known (inset). *p < 0.05 **p < 0.01, *** p < 0.001 (unpaired t-test).

Article Snippet: Cells were then resuspended in 0.5 ml of F-PBS, counted, and incubated on ice for 60 minutes with the following antibodies: CD3 (145–2C11 Biolegend), CD4 (RM4–5 eBiosience), CD8 (53–6.7 eBioscience), CD19 (6D5 Biolegend), B220 (RA3–6B2 Biolegend), Gr1 (RB6–8C5 Biolegend), Ter119 (Ter119 Biolegend), cKit (2B8 BD, Pharmingen), Sca-1 (D7, eBioscience), CD150 (TC15–12F12.2 Biolegend), CD34 ( RAM34 BD Pharmingen), FcgRII/III (93 Biolegend), CD48 (HM48–1 Biolegend).

Techniques: Generated, Sequencing, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Molecular cell

Article Title: BRCA1 mutational complementation induces synthetic viability

doi: 10.1016/j.molcel.2020.04.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were then resuspended in 0.5 ml of F-PBS, counted, and incubated on ice for 60 minutes with the following antibodies: CD3 (145–2C11 Biolegend), CD4 (RM4–5 eBiosience), CD8 (53–6.7 eBioscience), CD19 (6D5 Biolegend), B220 (RA3–6B2 Biolegend), Gr1 (RB6–8C5 Biolegend), Ter119 (Ter119 Biolegend), cKit (2B8 BD, Pharmingen), Sca-1 (D7, eBioscience), CD150 (TC15–12F12.2 Biolegend), CD34 ( RAM34 BD Pharmingen), FcgRII/III (93 Biolegend), CD48 (HM48–1 Biolegend).

Techniques: Recombinant, Plasmid Preparation, Staining, Mutagenesis, Modification, Software

Journal: Nature Communications

Article Title: Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo

doi: 10.1038/s41467-024-44913-z

Figure Lengend Snippet: a Three-dimensional (3D) wholemount immunostaining with αSMA, CD31 and NG2 of E10.5 (31–38 somite pairs (sp)) WT dorsal aorta; b NG2 and Runx1 expression on single plane wholemount WT E10.5 sections. NG2 + Runx1 + vSMCs (arrows), hemogenic endothelial cells (arrowheads) and intra-aortic hematopoietic clusters (IAHCs, stars) (Table ); c Representative example of flow cytometric analysis of NG2 + Runx1(GFP) + (green box) in E10.5 Runx1-IRES-GFP AGM and E10.5 WT control. d Percentages of NG2 + Runx1(GFP) + cells in E9 (21-25sp) body ( n = 6), E10/E10.5/E11 AGMs ( n = 8/7/7), N = 5, Kruskal-Wallis and Dunn’s post-hoc test. e Representative examples of wholemount 3D-images showing αSMA, CD31 and NG2 in E10.5 cKO dorsal aortae; f αSMA, Runx1 and CD31 immunofluorescence of E11 WT and cKO transversal frozen sections; n = WT/cKO: 2/2, N = 2. g cKit and CD31 wholemount 3D-images in E10.5 WT and cKO AGM; h Number of intra-aortic hematopoietic clusters (IAHCs) in E10.5 AGM; n = WT/KO: 5/4, N = 4. Number of colony forming unit-culture (CFU-C) in i E10.5 (31-38sp) AGM; n = WT/HET/KO: 14/10/5 embryos; N = 7 and j E11 (43–52sp) AGM; n = WT/HET/KO: 22/8/19 embryos; N = 11; one-way ANOVA and Tukey’s post-hoc test (Table ). k Percentages of donor cell chimerism 4-months post-transplantation of 6 E11 WT (NG2 +/+ ;Runx1 fl/+ or NG2 +/+ ;Runx1 fl/fl ) , 7 HET ( NG2-Cre;Runx1 fl/+ ) and 6 cKO AGMs ( NG2-Cre;Runx1 fl/fl ) into sub-lethally adult irradiated recipients (1xAGM cells transplanted/recipient; N = 4). Each dot represents one recipient. Mice are reconstituted when ≥5% donor cells are found in the host peripheral blood (dashed line) ; one-tailed Z score test for two population proportions (Tables and ). For wholemount staining in a , b , e , g : WT/cKO ( N = 6/4): αSMA ( n = 9/7), CD31 ( n = 10/7), cKit ( n = 3/2), NG2 ( n = 3/1) and WT Runx1 ( n = 4) in 3 distinct combinations (Table ). D = dorsal, V = ventral. N = number of independent experiments; n = number of biological samples (embryos). All data are presented as mean values ± SEM. Source data for d , h , i , j and k are provided as a file.

Article Snippet: As all E11 AGM HSPCs have been reported to express cKit , E11 AGM cells were also stained with cKit (1:500, BD Horizon, 562609) for further enrichment.

Techniques: Immunostaining, Expressing, Control, Immunofluorescence, Transplantation Assay, Irradiation, One-tailed Test, Staining