Figure S5.
Properties of the LMX1B Y309A/L312A mutant. (A) GFP-trap immunoprecipitates of wild-type and LIR mutant (Y309A/L312A) LMX1B-FLAG in lysates of HEK293T co-expressing GFP-ATG8 family members. 5% of protein lysate from equivalent GFP-expressing cells is shown as a representative of “input.” Immunoblotting for p62 is included as a positive control for binding. Molecular weight markers are shown in kD. (B) Immunofluorescence imaging of Y309A/L312A LMX1B-FLAG in HEK293T cells. Bar = 20 µm. (C and D) LMX1B turnover in HEK293T expressing wild-type or Y309A/L312A LMX1B-FLAG. Cells were treated with CHX (50 µg/ml; 16 h) in the absence or presence BafA1 (20 nM). Representative blot (C) and quantitation relative to GAPDH (D). Molecular weight markers are shown in kD. Mean ± SD (n = 3); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: *P < 0.05; ***P < 0.001, ****P < 0.0001. (E) Y309A/L312A LMX1B stimulates FLAT-luciferase activity to wild-type levels. To the left, luciferase FLAT element sequences (top), and schematic of the proposed role of ATG8 proteins as LMX1B co-factors (bottom). To the right, quantitation of expression driven by wild-type and Y309A/L312A LMX1B in HEK293T cells. Levels are presented normalized to empty vector control (pcDNA 3.1). Mean ± SEM (n = 3–5); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test comparing scrambled sequence against NURR1, pro-insulin, TFEB, ULK1, UVRAG, and PINK1: *P < 0.05, **P < 0.01, ****P < 0.0001. (F) Y309A/L312A LMX1B rescues autophagy gene transcription in HEK293T cells. HEK293T cells were double co-transfected with 50 nM smartpool LMX1B siRNA and codon optimized, siRNA-resistant wild-type, Y309A/L312A LMX1B (or empty vector control). mRNA levels were normalized siControl + empty vector. Mean ± SEM (n = 3); one-way ANOVA followed by Dunnett’s multiple comparison post-hoc test comparing the three different LMX1B constructs to empty vector (*P < 0.05; **P < 0.01) and a Student’s t test to compare Y309A/L312A with wild-type LMX1B (no data sets statistically significant). (Note: the data sets for “empty vector” and “siLMX1B + LMX1B” are identical to those shown in Fig. 7 A.) (G) Y309A/L312A LMX1B rescues autophagy gene transcription in iPSC-derived human mDANs. Mature (day ∼50) mDANs were transduced with hsyn-GFP-U6-shLMX1B and LMX1B levels were rescued with codon optimized, shRNA-resistant wild-type or Y309A/L312A TRE-LMX1B (or empty vector as a control). mRNA levels were normalized shControl + TRE-empty. Mean ± SEM (n = 3 wells from a single neuralization); one-way ANOVA followed by Dunnett’s multiple comparison post-hoc test comparing the two LMX1B constructs to empty vector (*P < 0.05, **P < 0.01, ***P < 0.001) and a Student’s t test to compare Y309A/L312A with wild-type LMX1B (*/#P < 0.05).

Properties of the LMX1B Y309A/L312A mutant. (A) GFP-trap immunoprecipitates of wild-type and LIR mutant (Y309A/L312A) LMX1B-FLAG in lysates of HEK293T co-expressing GFP-ATG8 family members. 5% of protein lysate from equivalent GFP-expressing cells is shown as a representative of “input.” Immunoblotting for p62 is included as a positive control for binding. Molecular weight markers are shown in kD. (B) Immunofluorescence imaging of Y309A/L312A LMX1B-FLAG in HEK293T cells. Bar = 20 µm. (C and D) LMX1B turnover in HEK293T expressing wild-type or Y309A/L312A LMX1B-FLAG. Cells were treated with CHX (50 µg/ml; 16 h) in the absence or presence BafA1 (20 nM). Representative blot (C) and quantitation relative to GAPDH (D). Molecular weight markers are shown in kD. Mean ± SD (n = 3); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: *P < 0.05; ***P < 0.001, ****P < 0.0001. (E) Y309A/L312A LMX1B stimulates FLAT-luciferase activity to wild-type levels. To the left, luciferase FLAT element sequences (top), and schematic of the proposed role of ATG8 proteins as LMX1B co-factors (bottom). To the right, quantitation of expression driven by wild-type and Y309A/L312A LMX1B in HEK293T cells. Levels are presented normalized to empty vector control (pcDNA 3.1). Mean ± SEM (n = 3–5); one-way ANOVA followed by Tukey’s multiple comparison post-hoc test comparing scrambled sequence against NURR1, pro-insulin, TFEB, ULK1, UVRAG, and PINK1: *P < 0.05, **P < 0.01, ****P < 0.0001. (F) Y309A/L312A LMX1B rescues autophagy gene transcription in HEK293T cells. HEK293T cells were double co-transfected with 50 nM smartpool LMX1B siRNA and codon optimized, siRNA-resistant wild-type, Y309A/L312A LMX1B (or empty vector control). mRNA levels were normalized siControl + empty vector. Mean ± SEM (n = 3); one-way ANOVA followed by Dunnett’s multiple comparison post-hoc test comparing the three different LMX1B constructs to empty vector (*P < 0.05; **P < 0.01) and a Student’s t test to compare Y309A/L312A with wild-type LMX1B (no data sets statistically significant). (Note: the data sets for “empty vector” and “siLMX1B + LMX1B” are identical to those shown in Fig. 7 A.) (G) Y309A/L312A LMX1B rescues autophagy gene transcription in iPSC-derived human mDANs. Mature (day ∼50) mDANs were transduced with hsyn-GFP-U6-shLMX1B and LMX1B levels were rescued with codon optimized, shRNA-resistant wild-type or Y309A/L312A TRE-LMX1B (or empty vector as a control). mRNA levels were normalized shControl + TRE-empty. Mean ± SEM (n = 3 wells from a single neuralization); one-way ANOVA followed by Dunnett’s multiple comparison post-hoc test comparing the two LMX1B constructs to empty vector (*P < 0.05, **P < 0.01, ***P < 0.001) and a Student’s t test to compare Y309A/L312A with wild-type LMX1B (*/#P < 0.05).

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