Depletion of JAK2 enhances neurite outgrowth. (A-C) J1 ESCs were infected with lentivirus encoding scrambled or JAK2-targeted small hairpin RNA (shRNA) and cultured under differentiation conditions. Knockdown efficiency was confirmed by semi-quantitative polymerase chain reaction (qPCR) (A) and immunoblotting (B) after three days of differentiation culture. (C) Expression levels of JAK2 as determined by densitometry. (D) Immunofluorescent images of ESCs infected with either a scrambled or JAK2-targeted shRNA and cultured under neuronal differentiation conditions. Representative images were acquired on day 3. The neuronal marker TUJ1 was stained green. (E, F) Expression levels of neuron-specific marker proteins analyzed by immunoblot-ting (E) and qPCR (F) in control and JAK2-knockdown J1 ESCs after 3 days of neuronal differentiation culture. (G) Cleaved caspase 3 expression in differentiated J1 ESC-derived neurons as assessed by immunoblotting. (H) Expression levels of neuronal markers in J1 ESCs treated for 24 with the JAK2 inhibitor SD-1029 or vehicle as estimated by western blotting. (I) ESCs infected with GFP or GFP-JAK2 WT vector and grown in differentiation culture. Whole-cell lysates were prepared on day 3 for western blot analysis of neuron-specific markers. All results are expressed as mean ± SD. Treatment group means were compared by Student’s t-test (n=3; *P < 0.05; **P < 0.01; ***P < 0.005).