Figure 4: Dopamine release and uptake in the four lines of induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic (DA) neurons.

(a) Dopamine release in Hank's Balanced Salt Solution (HBSS), or in HBSS plus KCl (56 mM), or Ca²⁺-free HBSS plus KCl (56 mM) from normal and parkin-deficient iPSC-derived midbrain neuronal cultures. *P<0.05 versus C001 or C002 in the same condition, n=3–8, Student's t-test, unpaired, two-tailed. (b) Ca²⁺-dependent DA release was calculated from KCl-induced DA release in the presence or absence of Ca²⁺. (c) Spontaneous DA release in HBSS from P001 or P002 neurons infected with lentivirus expressing green fluorescent protein (GFP), parkin or T240R mutant parkin. *P<0.05 versus P001 or P002 expressing GFP, respectively, n=4, Student's t-test, unpaired, two-tailed. (d,e) The amount of endogenous dopamine (d) and the percentage of TH⁺ neurons in NeuN⁺ neurons (e) in the four lines of iPSC-derived neuronal cultures. At least 1000 NeuN⁺ neurons were counted for each line of the iPSC-derived neurons. (f) Specific dopamine uptake in the four lines of iPSC-derived midbrain neuronal cultures. *P<0.05 versus C001 or C002, n=5–9, Student's t-test, unpaired, two-tailed. (g) Specific DA uptake in P001 or P002 neurons infected with lentivirus expressing GFP, parkin or T240R mutant parkin. *P<0.05 versus P001 or P002 expressing GFP, respectively, n=4, Student's t-test, unpaired, two-tailed. (h) Specific [³H]CFT binding on dopamine transporter (DAT) in the four lines of iPSC-derived midbrain neuronal cultures. *P<0.05 versus C001 or C002, n=6, Student's t-test, unpaired, two-tailed. (i) Specific [³H]CFT binding in P001 or P002 neurons infected with lentivirus expressing GFP, parkin or T240R mutant parkin. *P<0.05 versus P001 or P002 expressing GFP, respectively, n=5, Student's t-test, unpaired, two-tailed. Error bars represent s.e.m.