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. 2018 Feb 4;2018:4196961. doi: 10.1155/2018/4196961

Figure 3.

Figure 3

Ropinirole increases phosphorylation of p70S6K in human iPSC-derived DA neurons via activation of D3 receptor. (a, d) Time course of phosphorylated p70S6K (p-p70S6K) induced by ropinirole (10 μM) in (a) F3 and (d) NAS2 DA cultures, respectively, at 0, 2, 5, 10, 30, and 60 min and analysed by densitometry of Western blots (n = 6) (top panels); the specific levels of p-p70S6K were normalised to the corresponding p70S6K, TH, and tubulin levels. The densitometric values are represented as the percentage of vehicle values. (lower panels) Representative Western blots. (b, c) Blockade of p-p70S6K induced by ropinirole (10 μM) in F3 DA neurons after pretreatment (20 min) with the D3R antagonists (b) SB277011-A (100 nM) or (c) S33084 (10 nM) assessed after 2 min and analyzed by densitometry of Western blots (n = 3) (top panels). (lower panels) Representative Western blots. (h–m) Representative photomicrographs of F3 DA neurons showing p-p70S6K increase in the soma and dendrites of TH+ neurons 2 min after ropinirole (10 μM) following pretreatment with vehicle (i). (j) Blockade of p-p70S6K by pretreatment (20 min) with either SB277011-A (100 nM) or (k) S33084 (10 nM) followed by ropinirole (2 min). (h) Vehicle; (l) SB277011-A alone; and (m) S33084 alone. TH (green) and p-p70S6K (red). Cell nuclei were stained with DAPI (blue). (e, f) Blockade of p-p70S6K induced by ropinirole (10 μM) in F3 DA neurons after pretreatment (20 min) with (e) the MEK inhibitor PD98059 (10 μM) or (f) the PI3-K inhibitor LY294002 (10 μM) assessed after 2 min and analyzed by densitometry of Western blots (n = 3) (top panels). (lower panels) Representative Western blots. (n–s) Representative photomicrographs of F3 DA neurons showing p-p70S6K increase in the soma and dendrites of TH+ neurons 2 min after ropinirole (10 μM) following pretreatment with vehicle (o). (p) Blockade of p-p70S6K by pretreatment (20 min) with PD98059 (10 μM) or (q) LY294002 (10 μM) followed by ropinirole (2 min). (n) Vehicle; (r) PD98059 alone; and (s) LY294002 alone. TH (green) and p-p70S6K (red). Cell nuclei were stained with DAPI (blue). Scale bar: 20 μm. (g) Semiquantitative image analysis of p-p70S6K fluorescence intensity in TH+ and TH− neurons. All data are expressed as mean values ± SEM (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 versus vehicle; ooo p < 0.001, oo p < 0.01, and o p < 0.05 versus ropinirole; post hoc Bonferroni's test). V, vehicle; R, ropinirole; SB, SB277011-A; S33, S33084; PD, PD98059; LY, LY294002.