Figure 2.
LMX1B is an autophagy transcription factor in iPSC-derived human mDANs. (A) Immunofluorescence images and quantitation of LMX1A/LMX1B and dopaminergic marker expression in human iPSC-derived mDANs (imaged at: [i] D40; [ii, iii] D20). Cells were stained for anti-TH (magenta) and either anti-TUJ1 (green, [i]), anti-LMX1A (green, [ii]), or anti-LMX1B (green, [iii]). Bars = 100 µm. Data show counts of cells positive for the marker combinations shown (mean ± SD; n = 4 experiments). (B–E) qRT-PCR analysis of (B) TUJ1, (C) LMX1A, (D) LMX1B, and (E) NURR1 in iPSC-derived mDNAs differentiated at days 5, 10, 15, 20, 40, and 50 (or iPSC control). Levels were normalized to GAPDH. Mean ± SEM of mDAN cultures (n = 3) plated from a single neuralization; one-way ANOVA followed by a Dunnett’s multiple comparisons test. *P < 0.05, ***P < 0.001 vs. iPSC stage. (F and G) ChIP q-PCR analysis of (F) LMX1A and (G) LMX1B promoter occupancy in human mDANs using antibodies to isolate endogenous protein/DNA complexes. (H) shRNA suppression of LMX1A or LMX1B expression in iPSC-derived mDANs. To the top, a schematic representation of the pRRL plasmid construct expressing GFP under hsyn control and shRNA for LMX1A or LMX1B (or a non-target shRNA control) under U6 promoter control. Below are example fields of mDANs stained for anti-LMX1A (top, magenta) or anti-LMX1B (bottom, magenta) and anti-TH (cyan) after transduction with hsyn-GFP-U6-shControl, hsyn-GFP-U6-shLMX1A, or hsyn-GFP-U6-shLMX1B (green). To the right, LMX1A and LMX1B qRT-PCR quantitation after viral transduction at day 30–45 (normalized to GAPDH) of two different shRNAs for LMX1A and LMX1B (n = 3). Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: *P < 0.05; **P < 0.001; ***P < 0.001 vs. shRNA control. Bars = 20 µm. (I) qRT-PCR analysis of candidate gene expression in iPSC-derived mDANs (day 30–45) following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction (normalized to GAPDH). Means ± SD (n = 3); Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. shRNA control.

LMX1B is an autophagy transcription factor in iPSC-derived human mDANs. (A) Immunofluorescence images and quantitation of LMX1A/LMX1B and dopaminergic marker expression in human iPSC-derived mDANs (imaged at: [i] D40; [ii, iii] D20). Cells were stained for anti-TH (magenta) and either anti-TUJ1 (green, [i]), anti-LMX1A (green, [ii]), or anti-LMX1B (green, [iii]). Bars = 100 µm. Data show counts of cells positive for the marker combinations shown (mean ± SD; n = 4 experiments). (B–E) qRT-PCR analysis of (B) TUJ1, (C) LMX1A, (D) LMX1B, and (E) NURR1 in iPSC-derived mDNAs differentiated at days 5, 10, 15, 20, 40, and 50 (or iPSC control). Levels were normalized to GAPDH. Mean ± SEM of mDAN cultures (n = 3) plated from a single neuralization; one-way ANOVA followed by a Dunnett’s multiple comparisons test. *P < 0.05, ***P < 0.001 vs. iPSC stage. (F and G) ChIP q-PCR analysis of (F) LMX1A and (G) LMX1B promoter occupancy in human mDANs using antibodies to isolate endogenous protein/DNA complexes. (H) shRNA suppression of LMX1A or LMX1B expression in iPSC-derived mDANs. To the top, a schematic representation of the pRRL plasmid construct expressing GFP under hsyn control and shRNA for LMX1A or LMX1B (or a non-target shRNA control) under U6 promoter control. Below are example fields of mDANs stained for anti-LMX1A (top, magenta) or anti-LMX1B (bottom, magenta) and anti-TH (cyan) after transduction with hsyn-GFP-U6-shControl, hsyn-GFP-U6-shLMX1A, or hsyn-GFP-U6-shLMX1B (green). To the right, LMX1A and LMX1B qRT-PCR quantitation after viral transduction at day 30–45 (normalized to GAPDH) of two different shRNAs for LMX1A and LMX1B (n = 3). Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: *P < 0.05; **P < 0.001; ***P < 0.001 vs. shRNA control. Bars = 20 µm. (I) qRT-PCR analysis of candidate gene expression in iPSC-derived mDANs (day 30–45) following hsyn-GFP-U6-shControl/shLMX1A/shLMX1B lentiviral transduction (normalized to GAPDH). Means ± SD (n = 3); Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. shRNA control.

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