Figure 7.
LIR-dependent LMX1B binding to ATG8 proteins provides cellular stress protection. (A) LIR mutant Δ308-317 LMX1B fails to rescue autophagy gene transcription in HEK293T cells. HEK293T cells were co-transfected twice with 50 nM smartpool LMX1B siRNA and codon optimized, siRNA-resistant wild-type, or Δ308-317 LMX1B (or empty vector control). mRNA levels were normalized to siControl. Mean ± SEM (n = 3); one-way ANOVA followed by Dunnett’s multiple comparison post-hoc test comparing the two LMX1B constructs to empty vector (*P < 0.05) and a Student’s t test to compare Δ308-317 and wild-type LMX1B (#P < 0.05). (B) LIR mutant Δ308-317 LMX1B fails to rescue 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 Δ308-317 TRE- LMX1B (or empty vector as a control). mRNA levels were normalized to shControl. 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) and a Student’s t test to compare Δ308-317 with wild-type LMX1B (#P < 0.05). (C) LIR mutant Δ308-317 LMX1B fails to rescue the full autophagy response in LMX1B CRISPR KO2 HEK293T cells expressing wild-type or Δ308-317 LMX1B. To the left, example inverted single channel images and color overlays (LC3B is depicted in grayscale; WIPI2 is depicted in magenta; DAPI is depicted in blue). Cells were counterstained with DAPI (blue). Bar = 20 µm. To the right, puncta counts for GFP-LC3B and WIPI2 in nutrient replete conditions or following 2 h starvation in the presence or absence of BafA1 (20 nM, 2 h). Mean ± SD of 20–30 fields of cells from three independent experiments, counting ∼50 cells per field; one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, ****P < 0.0001. (D) Rotenone-induced cell death (10 µM, 20 µM; 48 h) in control and LMX1B CRISPR KO HEK293T cells. Data show means ± SD of three independent experiments; two-way ANOVA followed by Tukey’s multiple comparison post hoc test: **P < 0.01, ****P < 0.0001 vs. control cells. (E) Wild-type LMX1B, but not LIR mutant Δ308-317 LMX1B, protects against rotenone toxicity in LMX1B CRISPR KO HEK293T cells. KO1 and KO2 cells were treated with lentiviruses expressing empty vector control, wild-type LMX1B, or LIR mutant Δ308-317 LMX1B, then exposed to rotenone (10 µM) for 48 h. Data show means ± SD of three independent experiments; one-way ANOVA followed by Bonferroni multiple comparison post-hoc test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Rotenone-induced cell death (15 µM; 24 h) assay in iPSC-derived mDAN cultures (day 30–50) transduced with TRE-empty (control), TRE-LMX1B, Y309A/L312A TRE-LMX1B, or Δ308-317 TRE-LMX1B (expression induced with DOX: 500 ng/ml; 3 d). Active caspase fluorescence relative to total protein levels was normalized to TRE-empty control (left graph) or TRE-LMX1B control (right graph) values. Mean ± SD (n = 4–9); one-way ANOVA followed by Bonferroni multiple comparison post-hoc test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

LIR-dependent LMX1B binding to ATG8 proteins provides cellular stress protection. (A) LIR mutant Δ308-317 LMX1B fails to rescue autophagy gene transcription in HEK293T cells. HEK293T cells were co-transfected twice with 50 nM smartpool LMX1B siRNA and codon optimized, siRNA-resistant wild-type, or Δ308-317 LMX1B (or empty vector control). mRNA levels were normalized to siControl. Mean ± SEM (n = 3); one-way ANOVA followed by Dunnett’s multiple comparison post-hoc test comparing the two LMX1B constructs to empty vector (*P < 0.05) and a Student’s t test to compare Δ308-317 and wild-type LMX1B (#P < 0.05). (B) LIR mutant Δ308-317 LMX1B fails to rescue 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 Δ308-317 TRE- LMX1B (or empty vector as a control). mRNA levels were normalized to shControl. 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) and a Student’s t test to compare Δ308-317 with wild-type LMX1B (#P < 0.05). (C) LIR mutant Δ308-317 LMX1B fails to rescue the full autophagy response in LMX1B CRISPR KO2 HEK293T cells expressing wild-type or Δ308-317 LMX1B. To the left, example inverted single channel images and color overlays (LC3B is depicted in grayscale; WIPI2 is depicted in magenta; DAPI is depicted in blue). Cells were counterstained with DAPI (blue). Bar = 20 µm. To the right, puncta counts for GFP-LC3B and WIPI2 in nutrient replete conditions or following 2 h starvation in the presence or absence of BafA1 (20 nM, 2 h). Mean ± SD of 20–30 fields of cells from three independent experiments, counting ∼50 cells per field; one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, ****P < 0.0001. (D) Rotenone-induced cell death (10 µM, 20 µM; 48 h) in control and LMX1B CRISPR KO HEK293T cells. Data show means ± SD of three independent experiments; two-way ANOVA followed by Tukey’s multiple comparison post hoc test: **P < 0.01, ****P < 0.0001 vs. control cells. (E) Wild-type LMX1B, but not LIR mutant Δ308-317 LMX1B, protects against rotenone toxicity in LMX1B CRISPR KO HEK293T cells. KO1 and KO2 cells were treated with lentiviruses expressing empty vector control, wild-type LMX1B, or LIR mutant Δ308-317 LMX1B, then exposed to rotenone (10 µM) for 48 h. Data show means ± SD of three independent experiments; one-way ANOVA followed by Bonferroni multiple comparison post-hoc test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Rotenone-induced cell death (15 µM; 24 h) assay in iPSC-derived mDAN cultures (day 30–50) transduced with TRE-empty (control), TRE-LMX1B, Y309A/L312A TRE-LMX1B, or Δ308-317 TRE-LMX1B (expression induced with DOX: 500 ng/ml; 3 d). Active caspase fluorescence relative to total protein levels was normalized to TRE-empty control (left graph) or TRE-LMX1B control (right graph) values. Mean ± SD (n = 4–9); one-way ANOVA followed by Bonferroni multiple comparison post-hoc test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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