Fig. 5. S100A6 null HSCs failed to mobilize intracellular calcium flux upon stimulation and S100A6-calcium flux is p-Akt dependent.

Time gating before (first 100,000 cells were recorded) and immediately after addition of mSCF (a) or HuSDF-1 (c). The c-kit-enriched cells were exposed to mSCF (a) or HuSDF-1 (c) and the data show that only the HSC in the WT responded to both stimuli but not the S100A6 null cells as indicated by a change of the ratio between the violet (Ca²⁺ bound) and blue (free) fluorescence of Indo1. b, d Histograms show the changes in [Ca²⁺]_i, cytoplasmic Ca²⁺ evoked by mSCF and HuSDF-1, respectively. Error bars represent SD in S100A6WT and KO c-kit-enriched bone marrow cells stained with Indo1 gated on CD150⁺CD48⁻CD34⁻Flt⁻ (b n = 6; d n = 3; *p < 0.05; Mann–Whitney U test). Experiments were repeated three times. e (above) The corresponding change in [Ca²⁺]_i measured by Fluo-4 AM fluorescence intensity before, during and after the stimulation with mSCF over 50 s gated on CD150⁺CD48⁻CD34⁻Flt⁻. (below) Trace the period of mSCF application and total samples that respond upon mSCF stimulation with or without SC79 (WT, WT + SC79 n = 6; KO, KO + SC79 n = 8); *** or ****p < 0.0001; analyzed by multiple t-test (above); Mann–Whitney U test (below), which are representative of three independent experiments.