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STEM_1408_sm_SuppFigure1.tif551KFigure S1: FC treatment has no toxic effect on astrocytes in vivo and in vitro. (A): Confocal images of GFAP+ cells are shown. Scale bar: 50 μm. (B): To characterize the toxic effect of FC on astrocytes in vivo, the mice were subjected to a microinjection of FC (0.25 mM/1μl) or vehicle into the left side of the hippocampus, and brain slices were prepared for GFAP (rabbit polyclonal anti-GFAP antibody, 1:500, Millipore) staining 24 hours after the injection. The morphology and density of GFAP+ cells in hippocampus was not altered by FC microinjection [t (1, 6) = 0.551, P = 0.584; n = 4]. (C): GFAP immunofluorescent staining in vitro is shown. Scale bar: 10 μm. (D): The astrocytes isolated from the adult hippocampus were treated with FC or vehicle for 2 days, and the cells were fixed for GFAP staining or harvested for an MTT assay. FC treatment had no effect on the morphology and the viability of astrocytes [t (1, 22) = 0.437, P = 0.665; n = 12 wells].
STEM_1408_sm_SuppFigure2.tif529KFigure S2: Most ANSCs actively proliferate in the ANSC expansion medium. (A): Confocal images of Nestin+ ANSCs are shown. Most ANSCs expressed Nestin (rabbit polyclonal anti-Nestin antibody, 1:500, Millipore). Scale bar: 10 μm. (B) Twenty-four hours after BrdU (at a 2.5 μM final concentration) incorporation, the cells were fixed for BrdU staining. The rate of BrdU incorporation of ANSCs is 91.8 ± 0.4 %, n = 4 wells.
STEM_1408_sm_SuppFigure3.tif396KFigure S3: The effect of medium transfer on cultured astrocytes. Astrocytes were isolated from the adult hippocampus. After astrocyte purification, either DMEM-F12 was continually used as the culture media, or the medium was changed to ANSC expansion medium. Two days later, conditioned media were collected for ATP or ADP measurements, and the cells were fixed for GFAP staining. (A) Confocal images of GFAP+ cells are shown. Scale bar: 10 μm. (B): MTT analysis indicated that the medium transfer had no effect on astrocyte viability [t (1, 22) = 0.188, P = 0.852; n = 12 wells]. (C): Medium transfer had no effect on astrocytes releasing ATP [t(1,22) = 1.093, P = 0.286; n = 12 wells].
STEM_1408_sm_SuppFigure4.tif200KFigure S4: FC treatment has no toxic effect on NSCs. (A): Analysis of the proliferative effect of FC (0.1 – 1 mM) on the NSCs [F(4, 45) = 2.008, P = 0.11; n = 10 wells] is shown. (B): The total cell number identified by counting the DAPI+ cells is shown. F(4, 45) = 0.309, P = 0.905; n = 10 wells.
STEM_1408_sm_SuppFigure5.tif194KFigure S5: The activity of ADH retained < 50 kDa. (A): A schematic diagram of the medium cutoff and cross-remixed procedure is shown. (B) - (C): While the medium containing the NBS < 50 kDa and the ADH > 50 kDa had no effect, the medium containing the NBS > 50 kDa and the ADH < 50 kDa increased both the percent of BrdU+/DAPI+ [F(4, 55) = 72.805, P < 0.001; n = 12 wells] (B) and the total cell numbers [for day 0, F(4, 55) = 0.124, P = 0.973; for day 1, F(4, 55) = 9.396, P < 0.001; n = 12 wells] (C). *** P < 0.001 compared with control.
STEM_1408_sm_SuppFigure6.tif391KFigure S6: Related to Figure 2. (A): The total cell numbers related to Figure 2B. For day 0, F(4,75) = 0.073, P = 0.99; for day 1, F(4,75) = 15.399, P < 0.001; n = 16 wells. (B): The neurogenic effects of ATP, ATPγs and ADP are shown at the concentrations of 0.1 μM [F(4, 75) = 33.057, P < 0.001; n = 16 wells] and 100 μM [F(4, 75) = 11.637, P < 0.001; n = 16 wells]. (C): Related to Figure 2C. ATP F(6, 105) = 27.070, P < 0.001; n = 16 wells; ATPγs F(6, 105) = 10.911, P < 0.001; n = 16 wells; ADP: F (6, 105) = 14.019, P < 0.001; n = 16 wells; Adenosine F (6, 105) = 0.784, P = 0.539; n = 16 wells. (D): Related to Figure 2D. For day 0, F(6, 77) = 0.383, P = 0.888; for day 1, F(6, 77) = 5.2, P < 0.001; n = 12 wells. (E): Related to Figure 2E. For day 0, F(5, 90) = 0.935, P = 0.463; for day 1, F(5, 90) = 22.967, P < 0.001; n = 16 wells. ** P < 0.01; *** P < 0.001 compared with control.
STEM_1408_sm_SuppFigure7.tif206KFigure S7: The proliferative effects of glutamate and D-serine on NSCs. (A): Glutamate (5 μM, 50 μM, 0. 1 mM), D-serine (1 μM, 10 μM, 20 μM, 100 μM, 1 mM) or glutamate plus D-serine (glutamate 1 mM/D-serine 10 μM, glutamate 1 mM/D-serine 50 μM) did not increase the ratio of BrdU+/DAPI+ [F(10, 145) = 5.6, P < 0.001; n = 12 – 16 wells]. (B): Glutamate, D-serine or glutamate plus D-serine did not increase the total cell number [F(10, 145) = 7.284, P < 0.001; n = 12 – 16 wells]. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with control.
STEM_1408_sm_SuppFigure8.tif160KFigure S8: ANSCs expressed P2Y1 receptors. (A): RT-PCR analysis shows both the cultured embryonic stem cells (ESCs) and the ANSCs expressing P2Y1, P2Y2, P2Y4 and P2Y6 receptors. (B): Immunofluorescent staining demonstrated those ANSCs expressed P2Y1 receptors (rabbit polyclonal anti-P2Y1 antibody, 1:800, Alomone Labs, Israel). Scale bar: 5 μm.
STEM_1408_sm_SuppFigure9.tif285KFigure S9: Effects of antagonists for purinergic P2 receptors. (A): The proliferative effects of ATP, ATPγs or ADP were blocked by PPADs or suramin [F(9, 110) = 19.148, P < 0.001; n = 12 wells]. (B) The increased total cell number induced by ATP, ATPγs or ADP were blocked by PPADs or suramin [for day 0, F(9, 110) = 0.148, P = 0.998; for day 1, F(9, 110) = 15.878, P < 0.001; n = 12 wells]. (C) and (D): PPADs, suramin, MRS2179, BA1 or GD3+ alone had no effect on both the ratio of BrdU+/DAPI+ (C) [F(5, 90) = 0.489, P = 0.783; n = 16 wells] and the total cell number (D) [for day 0, F(5, 114) = 0.489, P = 0.783; F(5, 114) = 1.966, P = 0.289; n = 16 wells]. ** P < 0.01, *** P < 0.001 compared with control.
STEM_1408_sm_SuppFigure10.tif151KFigure S10: Total cell number related to Figure 3B. For day 0, F(5, 66) = 0.399, P = 0.848; for day 1, F(5, 66) = 10.825, P < 0.001; n = 12 wells. * P < 0.05, ** P < 0.01 compared with control (NBS).
STEM_1408_sm_SuppFigure11.tif123KFigure S11: Total cell number related to Figure 4B. For day 0, F(4, 75) = 0.42, P = 0.794; for day 1, F(4,75) = 17.465, P < 0.001; n = 16 wells. ** P < 0.01, *** P < 0.001 compared with control.
STEM_1408_sm_SuppFigure12.tif144KFigure S12: Injection of ATP reversed the brain ATP level of dn-SNRE mice to that of control littermates. Acute hippocampal slices isolated from adult dn-SNARE mice and control littermates were incubated in oxygenized ACSF and sampled 12 min later for ATP measurements. For ATP injection (125 mg/kg, i.p.), hippocampal slices were prepared 30 min after the injection. We observed that the amount of ATP was lower in the media incubating hippocampal slices derived from dn-SNARE mice [F(2,15) = 24.694, P < 0.001; n = 6], suggesting that astrocytic ATP release was deficient in adult dn-SNARE mice. Moreover, ATP injection reversed the brain level of ATP of dn-SNARE mice to that of control littermates. *** P < 0.001.
STEM_1408_sm_SuppInfo.pdf92KSupporting Information.

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