LMX1A/LMX1B overexpression drives autophagy transcription in human mDANs. (A) Schematic representation of the hsyn promoter modified Tet-On system. (B) Immunoblot showing TRE-LMX1A and/or TRE-LMX1B overexpression via the Tet-On system in iPSC-derived mDANs. Expression was induced with the addition of DOX (500 ng/ml) for 2 d. Molecular weight markers are shown in kD. (C) Wide-field time-lapse imaging of GFP-LMX1B overexpression via the Tet-On system in iPSC-derived mDANs. Expression was induced with the addition of DOX (500 ng/ml). GFP fluorescence is shown overlayed on phase contrast images. Bar = 10 µm. (D) Immunofluorescence labeling on iPSC-derived mDANs transduced with TRE-GFP, TRE-GFP-LMX1A, or TRE-GFP-LMX1B controlled by the Tet-ON system. Expression was induced with DOX (500 ng/ml) for 24 h. Cells were labeled with an anti-TH antibody (magenta). Bar = 20 µm. (E) mRNA levels of the indicated genes were determined by qRT-PCR and normalized by GAPDH levels. mRNA levels normalized to TRE-GFP. Data show means ± SD of triplicate wells from a single representative experiment. Student’s t test: *P < 0.05, **P < 0.01, and ***P < 0.001 vs. TRE-GFP control.