Journal: Science signaling

Article Title: CXCR4 signaling determines the fate of hematopoietic multipotent progenitors by stimulating mTOR activity and mitochondrial metabolism

doi: 10.1126/scisignal.adl5100

Figure Lengend Snippet: (A) Schematic diagram for the generation of CD45.2 + ->CD45.1 + short-term (3 weeks) and long-term (16 weeks) BM chimeras. (B) Absolute numbers of donor-derived CD45.2 + (WT, +/1013, or 1013/1013) MPP2, MPP3, and MPP4 recovered from the marrow of chimeras in CD45.1 + WT recipients 3 weeks or 16 weeks after marrow transplantation. n= 3 independent experiments for a total of 5 WT, 8 +/1013, and 8 1013/1013 donor mice (3-week chimeras) or 4 independent experiments for a total of 17 WT, 19 +/1013, and 15 1013/1013 donor mice (16-week chimeras). (C) Schematic diagram for the generation of CD45.1 + + CD45.2 + -> CD45.1 + /CD45.2 + competitive BM chimeras. (D) Relative contributions of CD45.2 + and CD45.1 + cells to donor-derived MPP2, MPP3, MPP4, and CLPs in the BM of CD45.1 + /CD45.2 + WT recipient mice 3 weeks after competitive BM transplantation. n=2 independent experiments for a total of 5 WT, 4 +/1013, and 4 1013/1013 donor mice. (E) Schematic diagram for the generation of CD45.1 + ->CD45.2 + short- and long-term BM chimeras. (F) Absolute numbers of donor CD45.1 + WT MPP2, MPP3, and MPP4 recovered from the marrow of chimeras in CD45.2 + (WT, +/1013, or 1013/1013) recipients 3 weeks or 16 weeks after marrow transplantation. n= 3 independent experiments for a total of 10 WT, 10 +/1013, and 5 1013/1013 recipient mice (3-week chimeras) or 4 independent experiments for a total of 12 WT, 11 +/1013, and 5 1013/1013 recipient mice (16-week chimeras). (G) Representative BM sections from WT and knockin mice stained to show laminin and nuclei (DAPI). MPP4 identified by RNA-scope and immunofluorescence staining ( fig. S3 , B and C ) are indicated with yellow circles. Spatial distribution of centroids of MPP4 cells plot as yellow circles within points of DAPI detected cells (blue points). Scale bar, 800 μm. (H) Proportions of MPP4 (c-Kit + Sca-1 + Flt3 + ) detected in the BM sections of WT and knockin mice after imaging ( fig. S3 , B and C ). n= 3 independent determinations for a total of 6 WT, 4 +/1013, and 4 1013/1013 mice. (I) Proportions of Dsred + cells within arteriolar cells (Lam + Sca-1 + ) in the BM sections of Cxcr4 +/+ Cxcl12 Dsred , Cxcr4 +/1013 Cxcl12 Dsred , and Cxcr4 1013/1013 Cxcl12 Dsred mice. n= 3 independent determinations for a total of 3 mice per group. (J) Spatial distance analysis between each MPP4 centroid and the centroid of the nearest arteriolar cell within representative BM sections from WT and knockin mice. Dots indicate individual MPP4 analyzed for one representative mouse per group with >140 cells analyzed. Quantifications are representative of 3 independent determinations per group. Mann–Whitney U test was used to assess statistical significance ( ££££ , P < 0.0001 between WT and 1013/1013 mice). (K) Representative histograms for intracellular antibody-based detection of phosphorylated STAT5 (p-STAT5) in sorted, untreated MPP4 from the marrow fraction of WT and knockin mice. Fluorescence in absence of the p-STAT5–specific antibody (dashed line) is shown. MFI values for p-STAT5 were determined by flow cytometry on sorted MPP4 from the marrow fraction of WT and knockin mice. n= 3 independent experiments for a total of 3 WT, 2 +/1013 and 3 1013/1013 mice. # P < 0.05; ## P < 0.005; ### P < 0.0005 for Kruskal–Wallis tests; * P < 0.05; ** P < 0.005; *** P < 0.0005 for Dunn’s test compared with WT, and § for Dunn’s test comparing +/1013 and 1013/1013; ££££ P < 0.0001 for Mann-Whitney’s test.

Article Snippet: Adult Boy/J (CD45.1) (Charles River) mice were used as transplantation recipients and BM donors.

Techniques: Derivative Assay, Transplantation Assay, Knock-In, Staining, RNAscope, Immunofluorescence, Imaging, MANN-WHITNEY, Fluorescence, Flow Cytometry

Journal: Science signaling

Article Title: CXCR4 signaling determines the fate of hematopoietic multipotent progenitors by stimulating mTOR activity and mitochondrial metabolism

doi: 10.1126/scisignal.adl5100

Figure Lengend Snippet: (A) RNAseq-based heatmap showing normalized dye intensity for the expression of genes involved in lymphocyte differentiation (GO:0030098) in WT, +/1013, and 1013/1013 MPP4. Data were generated from 2 (1013/1013 MPP4) or 3 (WT and +/1013 MPP4) independent experiments (9 mice each for WT and +/1013 MPP4, 6 mice for 1013/1013 MPP4). Three mice of the same genotype have been pooled to generate one replicate. (B) Biomark-based PCA of relative expression of selected genes involved in lympho-myeloid differentiation in MPP4. (C) The heatmap shows the relative quantification (RQ) for lymphoid transcripts normalized to Actb expression in each sample. The bar graph shows the RQ of the most decreased pro-lymphoid transcripts in MPP4. n = 4 WT, 3 +/1013, and 5 1013/1013 mice with samples from each mouse run in duplicate. (D) The heatmap shows the RQ for myeloid transcripts normalized to Actb expression in each sample. The bar graph shows the RQ of the most increased pro-GM transcripts in MPP4. n = 4 WT, 3 +/1013, and 5 1013/1013 mice with samples from each mouse run in duplicate. (E) Representative histograms for detection of Flt3 and Irf8 in MPP4. Fluorescence in absence of Irf8 antibody (dashed line) is shown. MFI values for surface Flt3 and proportions of Irf8 high cells were determined by flow cytometry in MPP4. n = 6 independent experiments for a total of 11 WT, 18 +/1013, and 12 1013/1013 mice (Flt3) or n= 3 independent experiments for a total of 6 mice per group (Irf8). (F) Representative flow cytometric analyses comparing the frequencies of B cells (B220 + CD11b − ) between WT and knockin MPP4 co-cultures. Proportions of B cells after co-culture between MPP4 and OP9/IL-7 stromal cells in the presence or absence of AMD3100 were determined after 7 days. n = 3 independent experiments for a total of 4 WT, 3 +/1013, and 4 1013/1013 mice. (G) Representative flow cytometric analyses comparing the frequencies of Lin − Sca-1 − c-Kit + [LK], Lin − Sca-1 low c-Kit low , Gr1 − CD11b − and Gr1 + CD11b + cells between WT and knockin MPP4 cultures at day 4 or day 7. Absolute numbers of GMPs were determined after 4 days of culture, whereas absolute numbers of Gr1 + CD11b + cells were assessed at day 7. Proportions of Lin − Sca-1 low c-Kit low cells were determined after 4 days of culture. n= 4 independent experiments for a total of 8 WT, 8 +/1013, and 7 1013/1013 mice (day 4) or n= 3 independent experiments for a total of 7 WT, 7 +/1013 and 6 1013/1013 mice (day 7). (H) Proportions of MPP4 and Flt3 low cells in WT and knockin MPP4 cultures after 4 days of differentiation in the presence or absence of AMD3100. n = 5 independent experiments for a total of 10 WT, 11 +/1013, and 8 1013/1013 mice (no AMD3100) or n= 3 independent experiments for a total of 4 WT, 4 +/1013, and 3 1013/1013 mice (with AMD3100). (I) Expression of Flt3 determined by flow cytometry on MPP4 after 4 days of culture in the presence or absence of AMD3100. n= 3 independent experiments for a total of 4 WT, 4 +/1013, and 3 1013/1013 mice. (J) Proportions of donor CD45.2 + myeloid or B cells recovered from the marrow of CD45.1 + recipients 2 weeks after the transplantation. n= 2 independent experiments for a total of 6 WT, 5 +/1013, and 5 1013/1013 donor mice. (K) Absolute numbers of GMPs were determined after 4 days of WT MPP4 culture in the presence or absence of Cxcl12 and AMD3100 as indicated. Absolute numbers of Gr1 + CD11b + cells were assessed at day 7. n = 3 independent experiments for a total of 3 mice per group (day 4) or n= 5 independent experiments for a total of 9 mice per group (day 7). # P < 0.05; ## P < 0.005 for Kruskal–Wallis tests; * P < 0.05; ** P < 0.005 for Dunn’s test compared with WT, and § for Dunn’s test comparing +/1013 and 1013/1013; £ P < 0.05; £££ P < 0.0005 for Mann-Whitney’s test.

Article Snippet: Adult Boy/J (CD45.1) (Charles River) mice were used as transplantation recipients and BM donors.

Techniques: RNA sequencing, Expressing, Generated, Quantitative Proteomics, Fluorescence, Flow Cytometry, Knock-In, Co-Culture Assay, Transplantation Assay

Journal: Science signaling

Article Title: CXCR4 signaling determines the fate of hematopoietic multipotent progenitors by stimulating mTOR activity and mitochondrial metabolism

doi: 10.1126/scisignal.adl5100

Figure Lengend Snippet: (A) Representative histograms for intracellular detection of phosphorylated mTOR (p-mTOR) and phosphorylated S6 (p-S6) in MPP4 of the indicated genotypes stimulated with Cxcl12. Fluorescence of WT MPP4 in the absence of p-mTOR or p-S6 antibody (dashed line), in the absence of Cxcl12 stimulation (gray histogram) or in the presence of Cxcl12 and AMD3100 (black histogram) are shown. MFI values for p-mTOR and p-S6 were determined by flow cytometry. n=3 independent experiments for a total of 4 (with AMD3100) or 5 (no AMD3100) WT, 6 +/1013, and 6 1013/1013 mice. (B) OXPHOS and glycolytic contents measured by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, were determined in freshly sorted WT and 1013/1013 MPP4 by Seahorse Mito Stress Test. n= 3 independent experiments for total of 9 WT, 9 +/1013, and 9 1013/1013 mice. Three mice of the same genotype were pooled for each independent experiment. Statistical significance compared with WT cells for each time point is shown. (C) Representative histograms and MFI values of TMRE and MTG staining in MPP4. n=3 independent experiments for a total of 10 WT, 10 +/1013, and 9 1013/1013 mice. (D) Representative images showing staining for TOMM20 and Hoechst 33342 in freshly isolated MPP4. Scale bar, 5 μm. n=3 independent determinations for a total of at least 50 cells analyzed per genotype. (E) MFI values of TOMM20 and number of mitochondrial fragments per WT MPP4 were determined using FIJI software. n=3 independent determinations for a total of at least 50 cells analyzed per condition (3 WT mice in total). (F) TMRE and MTG MFI values in WT MPP4 co-cultured with OP9/IL-7 cells. n= 3 independent experiments for a total of 3 mice per group. (G) Frequencies of TMRE high/+ fraction A (FR. A) and TMRE low/- fraction B (FR. B) within WT and knockin MPP4. n= 3 independent experiments for a total of 8 WT, 8 +/1013, and 6 1013/1013 mice. (H) Absolute numbers of GMPs were determined after 4 days of fraction A or fraction B MPP4 culture, whereas absolute numbers of mature myeloid cells were assessed at day 7. n= 3 independent experiments for a total of 6 WT, 6 +/1013, and 5 1013/1013 mice (day 4) or n= 3 independent experiments for a total of 7 WT, 10 +/1013, and 7 1013/1013 mice (day 7). * for Dunn’s test compared with WT cells, and £ for Mann-Whitney test comparing FR. A and FR. B. (I) Expression of pro-GM genes in single TMRE low (fraction B) MPP4 as determined by Biomark. Proportions of WT and 1013/1013 TMRE low MPP4 with detectable or undetectable expression of Mpo and Irf8 . n= 2 independent experiments with > 50 cells per condition analyzed (2 mice of each genotype in total). (J) Proportions of donor CD45.2 + (fraction A or B) myeloid or B cells recovered from the marrow of CD45.1 + WT recipients 2 weeks after the transplantation. n= 2 independent experiments for a total of 3 mice per group. (K) Absolute numbers of mature myeloid cells in WT or knockin MPP4 cultures in the presence or absence of Mito-q or CCCP were assessed at day 7. n= 3 independent experiments for a total of 3 mice per group. # P < 0.05; ## P < 0.005; ### P < 0.0005 for Kruskal–Wallis tests; * P < 0.05; ** P < 0.005; **** P < 0.0001 for Dunn’s test; £ P < 0.05; ££ P < 0.005; £££ P < 0.0005 for Mann-Whitney’s test.

Article Snippet: Adult Boy/J (CD45.1) (Charles River) mice were used as transplantation recipients and BM donors.

Techniques: Knock-In, Fluorescence, Flow Cytometry, Staining, Isolation, Software, Cell Culture, MANN-WHITNEY, Expressing, Transplantation Assay

Journal: Cell stem cell

Article Title: Asymmetrically Segregated Mitochondria Provide Cellular Memory of Hematopoietic Stem Cell Replicative History and Drive HSC Attrition

doi: 10.1016/j.stem.2020.01.016

Figure Lengend Snippet: Key Resources Table

Article Snippet: When BoyJ mice were used as recipients, cells were immuno-stained for CD45.1 PE/BV605 (clone-A20, catalogue number- 553776, lot number- 5230561 and for BV605 catalogue number- 563010, lot number- 7075978, both BD Bioscience) , CD45.2 Percpcy5.5/AF700/FITC (Clone –104, catalogue number – 552950, lot number – 6021537, for AF700 catalogue number- 560693, lot number – 7034806 and for FITC catalogue number- 553772, lot number – 5356548 both BD bioscience) or when NSG mice were used as recipients, H2Kd FITC/PE (clone- SF1-1.1, catalogue number- 553565, lot number – 0000069718 and for PE catalogue number- 553566, lot number – 6053774 both BD bioscience) pan CD45 APCCy7 (clone – 30-F11, catalogue number- 557659, lot number – 4341648, BD bioscience) to identify recipient versus donor population.

Techniques: Flow Cytometry, Labeling, Microscopy, Imaging, Transduction, Electron Microscopy, Staining, Modification, Transgenic Assay, Software