Journal: Blood

Article Title: Metabolic Adaptation of Regenerative Hematopoiesis Depends on Docking-Independent Mitochondrial Connexin-43

doi: 10.1182/blood.2024028079

Figure Lengend Snippet: (A) Gene Ontology (GO) analysis for significantly altered genes-cellular component in WT and Cx43 Δ/Δ HSC. Numbers in each column denote the number of differentially regulated genes associated with a GO term. (B) Representative immunofluorescent images of activated AMP- activated protein kinase phosphorylated at residue Thr172 (AMPKα1, 2-Thr172, green) and mitochondria (Tomm20, red) in WT and Cx43 Δ/Δ BM HSC sorted from primary murine bone marrow. Nuclei were counterstained with DAPI (blue). Scale bars, 2 µm and 0.5 µm. (C) Quantification of AMPKα1, 2-Thr172 in WT and Cx43 Δ/Δ BM HSC. Dots depict individual cells. (D) Quantification of percentage of AMPKα1, 2-Thr172 (green) co-localization with mitochondria (Tomm20, red) in WT and Cx43 Δ/Δ BM HSC. Dots show individual cells. (E) Proximity ligation assay (PLA; left) and the quantification analysis (right) demonstrating interactions between Tomm20 and AMPKα1, 2-Thr172 (red) in WT and Cx43 Δ/Δ HSC. Nuclei were counterstained with DAPI (blue). Scale bar, 2 µm. Dots depict individual cells. (F) HSC-enriched LSK BM cells were sorted from WT and hematopoietic Cx43 Δ/Δ chimeric mice (2 months post transplantation) and mitochondrial OCR was measured by Seahorse flux analyzer using sequential injections of oligomycin (O), FCCP (F), and Rotenone/ Antimycin A (R/AA). (G) Quantification summary of mitochondrial OCR in WT and Cx43 Δ/Δ HSC/MPP. All data points show a pool of 2 mice HSC/MPP and three experimental replicates were performed. (H, I) Extracellular acidification rates (ECAR) measured by Seahorse glycolytic rate assay in WT and Cx43 Δ/Δ BM HSC/MPP following sequential injections of Glucose, oligomycin (O), and 2-deoxy-glucose (2DG). All data points show a pool of 2 mice HSC/MPP. (J) ADP/ATP ratio in WT and Cx43 Δ/Δ HSC. Data point depict individual experiments. Data represented as mean ± SEM. All immunofluorescence quantification data are pool of 2–3 independent experiments. p values were generated by unpaired, two-tailed t test. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Dots depict individual cells. (D) Left: Representative immunofluorescence image showing mitochondria (green) and Parkin (red) staining in WT and Cx43 Δ/Δ dividing HSC. (i) Imaris reconstructed mitochondrial surface (green) and Parkin spots was used to define inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) Parkin to mitochondria surface.

Techniques: Residue, Proximity Ligation Assay, Transplantation Assay, Immunofluorescence, Generated, Two Tailed Test

Journal: Blood

Article Title: Metabolic Adaptation of Regenerative Hematopoiesis Depends on Docking-Independent Mitochondrial Connexin-43

doi: 10.1182/blood.2024028079

Figure Lengend Snippet: (A) Schema representing isolation of functional mitochondria from WT and Cx43 Δ/Δ HSC/MPPs and WB and proteomics analysis were performed. (B) Representative Western Blot depicting expression of Cx43 in mitochondria isolated from WT and Cx43 Δ/Δ HSC/MPPs. Tomm20 was used as a mitochondrion loading control. (C) Left: Representative PLA of Tomm20 and Cx43 (red) in WT and Cx43 Δ/Δ HSC. Nuclei were counterstained with DAPI (blue). Scale bar, 5 µm. Right: Quantification of number of Tomm20/Cx43 PLA signals. Dots show individual cells. (D) Left: Representative image of WT and Cx43 Δ/Δ HSC depicting PLA between CoxVb and Cx43 (red). Nuclei were counterstained with DAPI (blue). Scale bar, 5 µm. Right: Quantification of number of CoxVb and Cx43 PLA signals. Dots show individual cells. (E) Functional mitochondria were isolated from WT and Cx43 Δ/Δ mito-Dendra2 + BM HSC/MPP and differential protein expression analysis were performed. GO pathways enrichment analysis exhibiting top differentially regulated mitochondrial pathways. Numbers in each column denote the number of differentially expressed mitochondrial proteins within each GO process. List of mitochondrial protein obtained from mouse MitoCarta3.0 database (Broad Institute mouse list). n = 3 ind. biol. rep. (F) Left: Representative western blots depicting expression of NADH dehydrogenase beta sub-complex subunit 8 of Complex I (NDUFB8), succinate dehydrogenase subunit B of Complex II (SDHB), cytochrome b-c1 complex subunit 2 of Complex III (UQCRC2), cytochrome c oxidase subunit 1 of Complex IV (MTCO1), and ATP synthase subunit alpha of Complex V (ATP5A) in WT and Cx43 Δ/Δ HSC/MPPs. GAPDH was used as an internal loading control. The blots are shown in two different exposures to highlight the difference in respective complex proteins. Right: Relative expression of mitochondria complex proteins (CI-CV) vs GAPDH in WT and Cx43 Δ/Δ HSC. Dots depict individual experiments. (G) Differential levels of TCA cycle metabolites following pairwise comparison of mitochondria isolated from WT and Cx43 Δ/Δ HSC/MPP. n = 3 ind. biol. rep. (H) Relative levels of TCA cycle metabolites (α-Ketoglutarate; αKG, succinate, and fumarate) in WT and Cx43 Δ/Δ BM HSC/MPP derived mitochondria. n = 3 ind. biol. rep. (I) OGDH activity and (J) , ratio of succinate and fumarate to αKG in HSC-enriched LSK BM cells sorted from WT and hematopoietic Cx43 Δ/Δ mice. n = 3–4 ind. biol. rep. (K) Left: Representative image depicting PLA between OGDH and Cx43 (red) in WT and Cx43 Δ/Δ HSC. Nuclei were counterstained with DAPI (blue). Scale bar, 3 µm. Right: Quantification of number of OGDH/Cx43 (red) PLA signals. Dots show individual cells. (L) Left: Representative immunofluorescent image depicting staining of OGDH (red) and Dendra2 (green) in WT and Cx43 Δ/Δ BM HSC. Nuclei were counterstained with DAPI (blue). Scale bars, 2 µm. Right: Quantification of OGDH intensity. Dots show individual cells. Data represented as mean ± SEM. All immunofluorescence quantification data are pool of 2–3 independent experiments. p values were generated by unpaired, two-tailed t test. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Dots depict individual cells. (D) Left: Representative immunofluorescence image showing mitochondria (green) and Parkin (red) staining in WT and Cx43 Δ/Δ dividing HSC. (i) Imaris reconstructed mitochondrial surface (green) and Parkin spots was used to define inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) Parkin to mitochondria surface.

Techniques: Isolation, Functional Assay, Western Blot, Expressing, Control, Comparison, Derivative Assay, Activity Assay, Staining, Immunofluorescence, Generated, Two Tailed Test

Journal: Blood

Article Title: Metabolic Adaptation of Regenerative Hematopoiesis Depends on Docking-Independent Mitochondrial Connexin-43

doi: 10.1182/blood.2024028079

Figure Lengend Snippet: (A) Representative images showing paired daughter cell assay of nuclear Myc distribution in WT and Cx43 Δ/Δ HSC. (i) Low Myc expression in paired daughter cells represents symmetric self-renewal (SD), (ii) high and low Myc expression between the 2 daughters represents an asymmetric division (AD), whereas (iii) high Myc expression in both cells is indicative of symmetric commitment (SC). (B) Pie charts depicting relative HSC fate distribution in WT and Cx43 Δ/Δ paired daughter assay. 98–101 cell pairs analyzed from three independent experiments. (C-H) WT and Cx43 Δ/Δ HSC were co-cultured overnight with Dendra2 + mitochondria isolated from WT and Cx43 Δ/Δ mito-Dendra2 + LSK cells and analyzed. (C) Schematic representing Dendra2 + mitochondria loading in WT and Cx43 Δ/Δ HSC. (D-E) Representative immunofluorescence staining of Myc and the corresponding HSC fate determination (D) , and its quantification (E) in WT and Cx43 Δ/Δ paired daughter HSC loaded with WT or Cx43 Δ/Δ mito- Dendra2 + HSC/MPP isolated mitochondria. 49–51 paired cells were analyzed from three independent experiments. *p<0.05 WT HSC WT mito vs Cx43 Δ/Δ HSC Cx43Δ/Δ mito ; $ p<0.05 Cx43 Δ/Δ HSC Cx43Δ/Δ mito vs Cx43 Δ/Δ HSC WT mito . (F-G) Representative immunofluorescence depicting OGDH (red) staining (F) and quantification (G) in WT and Cx43 Δ/Δ BM HSC loaded with WT or Cx43 Δ/Δ Dendra2 + mitochondria. Nuclei were counterstained with DAPI (blue). Scale bars, 2 µm. Dots show individual cells. (H) ATP levels in WT and Cx43 Δ/Δ BM HSC supplemented with WT or Cx43 Δ/Δ mito-Dendra2 + mitochondria. Data point depict individual experiments. (I) Schematic illustration of serial competitive repopulation assay. WT and Cx43 Δ/Δ BM HSC loaded with mitochondria isolated from WT or Cx43 Δ/Δ mito-Dendra2 + HSC/MPP were transplanted into lethally irradiated congenic CD45.1 + B6.SJL- Ptprc a Pepc b /BoyJ mice. On day 17 post-transplant BM cells were isolated from primary transplant mice (CD45.2 + , pool of 5 mice), mixed with congenic CD45.1 + B6.SJL- Ptprc a Pepc b /BoyJ competitor cells in 1:1 ratio and transplanted into lethally irradiated B6.SJL- Ptprc a Pepc b /BoyJ recipient mice for secondary transplantation followed by tertiary transplant and analyzed as indicated. (J-K) Peripheral blood chimerism for donor derived CD45.2 + cells (J) and count of peripheral blood CD45.2 + leukocyte and CD11b + cells (K) in primary, secondary, and tertiary recipients (4–5 mice/group, per time point, and two independent experiments were performed). (L) BM counts of LT-HSC and LSK in primary, secondary, and tertiary transplant mice (4–5 mice/group, per time point, and two independent experiments were performed). Data represented as mean ± SEM. Statistical significance was assessed using unpaired, two-tailed t test except in panel B and E where chi-square test and panel G where One-way ANOVA with Tukey’s multiple comparisons test was used. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Dots depict individual cells. (D) Left: Representative immunofluorescence image showing mitochondria (green) and Parkin (red) staining in WT and Cx43 Δ/Δ dividing HSC. (i) Imaris reconstructed mitochondrial surface (green) and Parkin spots was used to define inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) Parkin to mitochondria surface.

Techniques: Expressing, Cell Culture, Isolation, Immunofluorescence, Staining, Irradiation, Transplantation Assay, Derivative Assay, Two Tailed Test

Journal: Blood

Article Title: Metabolic Adaptation of Regenerative Hematopoiesis Depends on Docking-Independent Mitochondrial Connexin-43

doi: 10.1182/blood.2024028079

Figure Lengend Snippet: (A and B) Representative images of mitochondrial ultrastructure from WT and Cx43 Δ/Δ quiescent (0 h) and dividing (48 h) BM HSC revealed by transmission electron microscopy (TEM). The boxed areas a, b, c, and d in panel I were magnified in panel II. The mitochondria indicated in panel II (boxed areas i, ii, iii, and iv) were further magnified in panel III and mitochondrial ultrastructure were analyzed. Green triangles, healthy mitochondria with dense matrix and preserved cristae; Cyan*, mitochondria with disorganized/concentric cristae; Red*, damaged mitochondria. Scale bars, 500 nm. (A) . Quantification of mitochondria length (in µm), circularity, and mitochondrial fate in WT and Cx43 Δ/Δ quiescent (0h) and dividing (48h) HSC (B) . Dots depict individual cells. (C) Left: Representative immunofluorescence image of Dendra2 + mitochondria (green) and Pink1 (red) in WT and Cx43 Δ/Δ dividing HSC. Nuclei were counterstained with DAPI (blue). (i) Imaris surface-building algorithm was used to reconstruct mitochondrial surface (green) and Imaris spot-building algorithm was used to define Pink1 inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) of mitochondria surface. Scale bars, 2 µm and 0.5 µm. Right: Quantification of Pink1 colocalization to Dendra2 + mitochondria (mito-Pink1) in WT and Cx43 Δ/Δ dividing HSC. Dots depict individual cells. (D) Left: Representative immunofluorescence image showing mitochondria (green) and Parkin (red) staining in WT and Cx43 Δ/Δ dividing HSC. (i) Imaris reconstructed mitochondrial surface (green) and Parkin spots was used to define inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) Parkin to mitochondria surface. Scale bars, 2 µm and 0.5 µm. Nuclei were counterstained with DAPI (blue). Right: Quantification analysis depicting Parkin colocalization with mitochondria (mito- Parkin). Dots show individual cells. (E) Left: Representative image of Dendra2 + mitochondria (green), LC3 puncta (red), and LAMP2 (purple) in WT and Cx43 Δ/Δ HSC cultured for 48h in presence of SCF (50 ng/ml) and TPO (50ng/ml). Nuclei were counterstained with DAPI (blue). (i) Reconstructed mitochondrial surface is depicted in green. Reconstructed big red and violet spots depict LC3 and lamp2 inside mitochondria surface, respectively. Reconstructed small pink and lavender spots show LC3 and lamp2 outside mitochondria surface, respectively. Scale bars, 2 µm and 0.5 µm. Right: Quantification of Dendra2 + mitochondria related flux of LC3 puncta (mito-LC3) and LAMP2 (mito-Lamp2). Dots depict individual cells. (F-G) Representative flow cytometry histogram overlay (left) and the quantification (right) demonstrate mitochondrial calcium levels as assessed by Rhod-2 AM staining in WT and Cx43 Δ/Δ (F) quiescent (0h) and (G) dividing (48h) HSC. Dots show independent experiment. (H-I) Flow cytometry histogram overlay (left) and the quantification (right) depict intracellular calcium levels as measured by Fluo-4 AM in WT and Cx43 Δ/Δ (H) quiescent (0h) and (I) dividing (48h) HSC. Dots show independent experiment. (J-K) Kinetic plot (J) and quantification (K) of mitochondrial Ca 2+ (Rhod-2 AM intensity) in WT and Cx43 Δ/Δ steady-state HSC following treatment with EGTA, TG, and Ru265. n=3 independent biological replicates. (L) Mitochondrial calcium levels in WT and Cx43 Δ/Δ HSC loaded with Dendra2 + mitochondria isolated from WT or Cx43 Δ/Δ mito-Dendra2 + HSC/MPP. Data normalized with mitochondrial calcium levels in WT HSC supplemented with WT mitochondria and expressed as fold change. n=3 independent experiments. Data represented as mean ± SEM. TEM and immunofluorescence quantification data are pool of 2–3 independent experiments. p values were generated by unpaired, two-tailed t test except in figure B and L where One way ANOVA, Tukey’s multiple comparisons test was used. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Dots depict individual cells. (D) Left: Representative immunofluorescence image showing mitochondria (green) and Parkin (red) staining in WT and Cx43 Δ/Δ dividing HSC. (i) Imaris reconstructed mitochondrial surface (green) and Parkin spots was used to define inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) Parkin to mitochondria surface.

Techniques: Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Cell Culture, Flow Cytometry, Isolation, Generated, Two Tailed Test

Journal: Blood

Article Title: Metabolic Adaptation of Regenerative Hematopoiesis Depends on Docking-Independent Mitochondrial Connexin-43

doi: 10.1182/blood.2024028079

Figure Lengend Snippet: Representative images from WT and Cx43 Δ/Δ dividing HSC depicting the following: (A-B) Time-lapse microscopy of Dendra2 + mitochondria before (0 min) and after (0.5–30 min) photoconversion. Subsets of Dendra2 + mitochondria (white boxed area) were photoconverted by 405 nm and both photoactivated (red fluorescence, rD2) and non-photoactivated (green fluorescence, gD2) mitochondria were tracked over time. (B) The intensity profile of red (rD2) and green (gD2) signals along lines from A (lower panel) are provided in B. The line analysis demonstrates mitochondrial fusion (co-localization of rD2 with adjoining gD2 mitochondria over time) in WT HSC. rD2 mitochondria does not overlap with adjoining gD2 mitochondria in Cx43 Δ/Δ HSC over time. (C) Each color-coded surface represents individual photoconverted mitochondria distribution over a time of 30 min. White arrow represents start (arrow tail; 0.5 min) vs end (arrowhead; 30 min) of photoconverted mitochondrial tracking. (D) Quantification of photoconverted mitochondria distribution over time (0.5–30 min) in WT and Cx43 Δ/Δ HSC. n=3–4 independent experiment. (E-G) Dendra2 + mitochondria (green) and Mfn2 (red) staining (E) , quantification of Mfn2 intensity (F) , and co-localization of Mfn2 with mitochondria (Mito-Mfn2) (G) . Nuclei were counterstained with DAPI (blue). (i) Imaris surface-building algorithm was used to reconstruct mitochondrial surface (green) and spot-building algorithm was used to define Mfn2 inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) mitochondria surface. Dots depict individual cells. (H-I) Dendra2 + mitochondria (green) and Opa1 (red) staining (H) , and quantification of total Opa1 spots (I) . Nuclei were counterstained with DAPI (blue). Dots depict individual cells. (J) Quantification of the percentage of HSC with polarized Opa1. A reference point was positioned to the center of the nucleus (DAPI) and distribution of Imaris reconstructed Opa1 spots (red) were analyzed. For each data point 10–12 HSC were scored to calculate the percentage of Opa1 polarized distribution (n=3 independent experiment). (K) Dendra2 + mitochondria (green), S616-phosphorylated (active) dynamin-related protein 1; pDrp1 (S616) (red), and total Drp1 (purple) staining. Nuclei were counterstained with DAPI (blue). (i) Reconstructed mitochondrial surface were shown in green. Reconstructed big red and violet spots depict pDrp1 (S616) and total Drp1 inside mitochondria surface, respectively. Reconstructed small pink and yellow spots depict pDrp1 (S616) and total Drp1 outside mitochondria surface, respectively. (L-M) Quantification of pDrp1 (S616) (L) , and co-localization of pDrp1 (S616) with mitochondria (Mito-pDrp1; S616) (M) . Dots depict individual cells. (N-O) Quantification of total Drp1 (N) , and co-localization of total Drp1 with mitochondria (Mito-total Drp1) (O) . Dots depict individual cells. (P-R) WT and Cx43 Δ/Δ HSC were transduced with RFP-tagged empty or Drp1 dominant negative (Drp1 K38A ) vector. (P) Left: Representative immunofluorescence image of Dendra2 + mitochondria (green) and Lamp2 (red) staining. (i) Imaris surface (for mitochondria) and spot (for Lamp2) building algorithm was used to define Lamp2 inside (big red spot) and outside (small pink spots) mitochondria surface (green). Nuclei were counterstained with DAPI (blue). Right: Quantification of Dendra2 + mitochondria colocalization with lamp2 (Mito-Lamp2). Dots depict individual cells. (Q) Representative immunofluorescence image depicting OGDH (red) staining (left) and quantification (right) in WT and Cx43 Δ/Δ HSC transduced with empty or Drp1 K38A vector. Nuclei were counterstained with DAPI (blue). Dots show individual cells. (R) ATP levels in empty or Drp1 K38A vector transduced WT and Cx43 Δ/Δ BM HSC. Data point depict individual experiments. (S) Experimental design for transplantation of Drp1 K38A vector transduced WT and Cx43 Δ/Δ mito-Dendra2 + HSC into lethally irradiated B6.SJL- Ptprc a Pepc b /BoyJ recipient mice and analysis. (T) Peripheral blood count of mito-Dendra2 + leukocyte and CD11b + cells, and (U) BM frequency of donor derived mito-Dendra2 + LT-HSC, MPP2, and LSK cells in WT and Cx43 Δ/Δ chimeric mice containing empty or Drp1 K38A vector. Dots depict all individual mice, and two independent experiments were performed. Data represented as mean ± SEM. All immunofluorescence image quantification data are pool 2–3 independent experiments. Scale bars, 3 µm, 2 µm, 1 µm, and 0.5 µm. Statistical significance was assessed using unpaired, two-tailed Student t test except in panel D where two-way ANOVA and P, Q where one-way ANOVA with Tukey’s multiple comparisons test was used, respectively. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Dots depict individual cells. (D) Left: Representative immunofluorescence image showing mitochondria (green) and Parkin (red) staining in WT and Cx43 Δ/Δ dividing HSC. (i) Imaris reconstructed mitochondrial surface (green) and Parkin spots was used to define inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) Parkin to mitochondria surface.

Techniques: Time-lapse Microscopy, Fluorescence, Staining, Transduction, Dominant Negative Mutation, Plasmid Preparation, Immunofluorescence, Transplantation Assay, Irradiation, Derivative Assay, Two Tailed Test

Journal: Blood

Article Title: Metabolic Adaptation of Regenerative Hematopoiesis Depends on Docking-Independent Mitochondrial Connexin-43

doi: 10.1182/blood.2024028079

Figure Lengend Snippet: (A) Graphical representation of Cx43 full length and the truncation mutation, cys-less and Δ CT 257 incorporated into EF1α-MCS-IRES-dsRED retroviral vector. (B-I) WT and Cx43 Δ/Δ HSC transduced with constructs coding for different forms of Cx43 (FL, cys-less or ΔCT 257 ) and analyzed. Representative immunofluorescence image (B) and quantification (C) depicting OGDH expression (red). Nuclei were counterstained with DAPI (blue). Dots show individual cells. (D) ATP levels in WT and Cx43 Δ/Δ BM HSC containing different Cx43 constructs (FL, cys-less or ΔCT 257 ). Data point depict individual experiments. (E) Immunofluorescence staining of mito-Dendra2 + (green) and Mfn2 (red) and (F) quantification depicting Mfn2 expression. Nuclei were counterstained with DAPI (blue). Dots depict individual cells. (G) Representative immunofluorescence staining of Dendra2 + mitochondria (green) and pDrp1 (S616) (red), and (H) quantification of pDrp1 (S616). Nuclei were counterstained with DAPI (blue). Dots depict individual cells. (I) Mitochondrial calcium levels (Rhod-2 AM) in WT and Cx43 Δ/Δ HSC containing empty vector or different Cx43 constructs (FL, cys-less or ΔCT 257 ). n= 4 independent experiments. (J) Schema depicting transplantation of WT and Cx43 Δ/Δ HSC containing different Cx43 constructs (FL, cys-less or ΔCT 257 ) into sub-lethally irradiated (2.5 Gy) congenic NOD- scid IL2Rgamma null , NOD- scid IL2Rg null mice and analyzed as indicated. (K) Peripheral blood count of mito-Dendra2 + /dsRED + leukocytes, neutrophils, and CD11b cells in NSG mice at indicated time post-transplant (*p<0.05, **p<0.01, ***p<0.001 vs Cx43 Δ/Δ Empty vector). Three independent experiments with 4 mice each were performed. (L) BM count for LT-HSC, MPP2, and LSK cell in NSG mice at indicated time post-transplant. Each data point depicts individual mice, and three independent experiments were performed. (M) HSC-enriched LSK BM cells from WT and Cx43 Δ/Δ chimeric mice (49 days post transplantation) containing different Cx43 constructs (FL, cys-less or ΔCT 257 ) were FACS sorted and mitochondrial OCR was measured by Seahorse extracellular flux analyzer. (N) Quantification summary of mitochondrial OCR (basal, maximal, ATP production, and Spare Respiratory Capacity; SRC) in WT and Cx43 Δ/Δ HSC/MPP containing different Cx43 constructs. All data points depict a pool of 4 mice HSC/MPP and three experimental replicates were performed. (O) WT and Cx43 Δ/Δ mito-Dendra2 + HSC transduced with constructs coding for different forms of Cx43 (FL, cys-less or Δ CT 257 ) were co-cultured over WT BM stroma for 16h. Flow cytometry histogram overlay (left) and quantification (right) demonstrate transfer of mitochondria from HSC/MPP transduced with Cx43 constructs to BM stromal cells. n= 4 independent experiments. Data represented as mean ± SEM. All immunofluorescence quantification data represent pools of 2–3 independent experiments. Scale bar, 2 µm. Statistical p values were generated by unpaired, two-tailed t test except in panel C, F, G where one-way ANOVA with Tukey’s multiple comparisons test was used. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Dots depict individual cells. (D) Left: Representative immunofluorescence image showing mitochondria (green) and Parkin (red) staining in WT and Cx43 Δ/Δ dividing HSC. (i) Imaris reconstructed mitochondrial surface (green) and Parkin spots was used to define inside (reconstructed big red spot; white arrow) and outside (reconstructed small pink spots) Parkin to mitochondria surface.

Techniques: Activity Assay, Mutagenesis, Retroviral, Plasmid Preparation, Transduction, Construct, Immunofluorescence, Expressing, Staining, Transplantation Assay, Irradiation, Cell Culture, Flow Cytometry, Generated, Two Tailed Test