Journal: The Journal of Experimental Medicine

Article Title: Ca 2+ –mitochondria axis drives cell division in hematopoietic stem cells

doi: 10.1084/jem.20180421

Figure Lengend Snippet: Ca 2+ channel blocker suppresses cell division of HSCs through the down-regulation of ΔΨ m . (A) The frequency of divided SLAM KSL HSCs after single cell culture for 24 or 48 h ( n = 4, four independent experiments). (B and C) ΔΨ m (B) or viability of HSCs (C) after the culture for 48 h under conditions as described above. Each value of ΔΨ m was relative to the value of uncultured HSCs ( n = 5, three independent experiments). (D and F) Intracellular (D) or mitochondrial Ca 2+ level (F) of HSCs cultured under indicated conditions. Data are presented as means ± SD relative to the value of cells treated with SCF and TPO at 0.5 ng/ml (undivided conditions; n > 3, two independent experiments). (E) The plot showing the relationship between ΔΨ m and intracellular Ca 2+ level after 18 h culture under indicated conditions. R represents the correlation coefficient. (G and H) Intracellular Ca 2+ level (G) or ΔΨ m (H) of HSCs cultured with SCF and TPO at 50 ng/ml (divided conditions) in the absence (Control) or presence of 60 µM Nifedipine (+Nifedipine). Data are presented as means ± SD relative to the value of cells treated with SCF and TPO at 0.5 ng/ml (undivided conditions; n = 4, two independent experiments). (I and J) The effect of Nifedipine on HSC division. Single cell culture (I) and CFSE-dilution assay (J) were performed under conditions described above. Data are presented as means ± SD ( n = 4, four independent experiments; *, P < 0.01; **, P < 0.05 by t test).

Article Snippet: As described previously ( , ), CD150 + CD48 − KSL HSCs were sorted and cultured for 5 d in S-Clone SF-03 medium (Sanko-Junyaku Co.) supplemented with 0.5% bovine serum albumin (Sigma), 0.05∼50 ng/ml mouse SCF and 0.05∼50 ng/ml mouse TPO (all from R&D Systems).

Techniques: Cell Culture, Dilution Assay

Journal: The Journal of Experimental Medicine

Article Title: Ca 2+ –mitochondria axis drives cell division in hematopoietic stem cells

doi: 10.1084/jem.20180421

Figure Lengend Snippet: Expression of cell cycle related genes is altered by the treatment with Ca 2+ channel blocker in HSCs. (A and B) After the culture of sorted CD150 + CD48 − KSL HSCs for 18 h under undivided conditions (0.5 ng/ml SCF and TPO) or divided conditions (50 ng/ml SCF and TPO) in the absence (Control) or presence of 60 µM Nifedipine (+Nifedipine), expression of Cyclins or cyclin-dependent kinase (CDKs; A) and CDK inhibitors (B) was examined by RT-PCR. Graphs depict mRNA expression level normalized by B2m expression ( n = 6, two independent experiments). (C) After culturing CD150 + CD48 − KSL HSCs for 18 h under divided conditions (50 ng/ml SCF and TPO) in the absence (Control) or presence of 60 µM Nifedipine (+Nifedipine), phosphorylation of CDK4 at Thr172, CDK6 at Thr177 or Rb at Ser807/811 was examined by flow cytometry. Each graph depicts geometric mean fluorescence intensity (GeoMFI) relative to the value of cells stained with isotype control. Data are presented as means ± SD ( n = 3, three independent experiments; *, P < 0.01; **, P < 0.05 by t test).

Article Snippet: As described previously ( , ), CD150 + CD48 − KSL HSCs were sorted and cultured for 5 d in S-Clone SF-03 medium (Sanko-Junyaku Co.) supplemented with 0.5% bovine serum albumin (Sigma), 0.05∼50 ng/ml mouse SCF and 0.05∼50 ng/ml mouse TPO (all from R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Fluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Ca 2+ –mitochondria axis drives cell division in hematopoietic stem cells

doi: 10.1084/jem.20180421

Figure Lengend Snippet: Extracellular adenosine acts as a regulator to regulate Ca 2+ –mitochondria pathway. (A) The amount of SCF or TPO within BM in untreated or 5-FU–treated mice. Data are expressed as the mean ± SD ( n = 4, two independent experiments). (B) The frequency of lineage − c-kit + cells within total BM cells after 5-FU treatment (250 mg/kg, i.v; n = 4, two independent experiments). (C) ΔΨ m of indicated fractions in untreated mice. Numbers in dot plots represent the frequency of gated cells within total BM cells. Data are presented as means ± SD relative to the value of L − ESLAM HSCs. ( n = 4, two independent experiments). (D and E) ΔΨ m (D) or viability (E) of HSCs after the co-culture of MPs (linage − c-Kir + Sca-1 − ) for 48 h in the absence or presence of 10 µM SCH442416 (SCH: Adora2a antagonist) and/or 10 µM PSB1115 (PSB: Adora2b antagonist). Cells cultured without both MPs and antagonists serve as control. n = 4, four independent experiments. (F) Expression of adenosine receptors in HSCs before and after 5-FU administration. Data are expressed as the mean ± SD relative to B2m expression ( n = 4, two independent experiments by t test). (G and H) ΔΨ m (G) or intracellular Ca 2+ level (H) in HSCs after the culture with adenosine for 48 h or immediately after the stimulation (10 µM adenosine), respectively. Cells cultured without adenosine serve as control. Data are presented as means ± SD ( n = 5, three independent experiments, *, P < 0.01 by t test [G]; n = 4, two independent experiments, *, P < 0.01 by t test [H]). N.S., not significant.

Article Snippet: As described previously ( , ), CD150 + CD48 − KSL HSCs were sorted and cultured for 5 d in S-Clone SF-03 medium (Sanko-Junyaku Co.) supplemented with 0.5% bovine serum albumin (Sigma), 0.05∼50 ng/ml mouse SCF and 0.05∼50 ng/ml mouse TPO (all from R&D Systems).

Techniques: Co-Culture Assay, Cell Culture, Expressing