Fig. 9: HypnoS revealed ENT1/2 mediate Ado transportation in cultures. | Nature Communications

Fig. 9: HypnoS revealed ENT1/2 mediate Ado transportation in cultures.

From: A high-performance fluorescent sensor spatiotemporally reveals cell-type specific regulation of intracellular adenosine in vivo

Fig. 9

a Schematic illustration depicting the experimental design for c–e. b Schematic drawing depicting the transmembrane transport of Ado via ENT1/2. Representative pseudocolor images (c, left), traces (c, middle) and group summary (c, right) of HypnoS ΔF/F0 in response to 100 μM Ado in cultured hippocampal neurons from wild-type (WT) mice, ENT1 knockout (ENT1 KO) mice, ENT2 knockout (ENT2 KO) mice and ENT1/2 double knockout (ENT2 KO & sgENT1 KO, dKO) mice. ΔF/F0 denotes the change in fluorescence ratio excited at 488 nm and 405 nm. n = 150, 130, 148, and 150 cells from 3 cultures for WT, ENT1 KO, ENT2 KO, and ENT1/2 KO, respectively. Two-tailed Student’s t-test: P = 0.0000026 between ENT1 KO and ENT2 KO; P = 2.2 × 10−140 between WT and ENT1/2 dKO. Scale bar, 100 μm. Representative pseudocolor images (d, left), traces (d, middle) and group summary (d, right) of HypnoS ΔF/F0 in response to 10 mM 2-DG in cultured hippocampal neurons from WT mice, ENT1 KO mice, ENT2 KO mice and ENT1/2 dKO mice. ΔF/F0 denotes the change in fluorescence response excited at 405 nm. n = 150, 110, 148, and 150 cells from 3 cultures for WT, ENT1 KO, ENT2 KO, and ENT1/2 dKO, respectively. Two-tailed Student’s t-test: P = 2.1 × 10−61 between ENT1 KO and ENT2 KO; P = 2.8 × 10−103 between WT and ENT1/2 dKO. Representative pseudocolor images (e, left), traces (e, middle) and group summary (e, right, 600 pulses) of HypnoS ΔF/F0 in response to 10 Hz, indicated number of pulses in cultured hippocampal neurons from WT mice, ENT1 KO mice, ENT2 KO mice and ENT1/2 dKO mice. ΔF/F0 denotes the change in fluorescence response excited at 405 nm. n = 150, 117, 148, and 144 cells from 3 cultures for WT, ENT1 KO, ENT2 KO, and ENT1/2 KO, respectively. Two-tailed Student’s t-test: P = 4.0 × 10−11 between ENT1 KO and ENT2 KO; P = 3.3 × 10−41 between WT and ENT1/2 dKO. All scale bars, 100 µm. Data are shown as mean ± s.e.m. in c–e with the error bars or shaded regions indicating the s.e.m. ***P < 0.001. Source data are provided as a Source Data file.

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