Figure 4. LMX1B colocalizes with LC3B... | Rockefeller University Press

Figure 4.

$LMX1B colocalizes with LC3B and is degraded during nutrient starvation. (A) LMX1A-FLAG turnover in stable HEK293T cells treated with CHX (50 µg/ml) for 16 h in the absence or presence of BafA1 (20 nM) or MG132 (10 µM; n = 3). Molecular weight markers are shown in kD. Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) LMX1B-FLAG turnover in stable HEK293T cells treated with CHX (50 µg/ml) for 16 h in the absence or presence of BafA1 (20 nM) or MG132 (10 µM) (n = 3). Molecular weight markers are shown in kD. Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: **P < 0.01, ****P < 0.0001. (C) LMX1B-FLAG degradation in stable HEK293T cells during starvation (6 h) in the presence of CHX (50 µg/ml) ± BafA1 (20 nM) or CHX ± MG132 (10 µM; n = 4–6). Molecular weight markers are shown in kD. Mean ± SD; one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, ****P < 0.0001. (D) Immunoblot and densitometry of cytosolic and nuclear fractions from HEK293T cells stably expressing LMX1B-FLAG with or without 2 h nutrient starvation. Molecular weight markers are shown in kD. Lamin B was used to normalize the nuclear fractions and GAPDH for the total and cytosolic fractions (n = 5). Mean ± SD; Student’s t test: ****P < 0.0001. (E) LMX1B relocates to cytosolic puncta that co-label with LC3B during extended nutrient starvation. Representative immunofluorescence images (to the left) and linescans of the indicated areas of LMX1B-FLAG stable HEK293T cells transfected with GFP-LC3B (green), fixed, and stained with anti-FLAG antiserum (magenta) after 2 or 6 h starvation. Bar = 20 µm, full field; 2 µm, zoom inset. (F) FLIP analysis of nucleus to cytoplasmic GFP-LMX1B transport in fed and starved cells, in the absence or presence of human ATG8 proteins (expression induced with DOX: 500 ng/ml; 20 h). Wild-type and ATG8 KO HeLa cells (Nguyen et al., 2016) were maintained in normal growth medium or starved for 2 h before live-cell FLIP analysis for a period of 15 min constant photobleaching of a cytoplasmic region of interest (example image shown top, right). Data are a measure of the loss of GFP-LMX1B fluorescence intensity within the nucleus of the photobleached cell normalized against non-photobleached cells in the same field of view. Bar = 10 µm. Data points represent individual treated cells in multiple fields of view (n = 4–6). Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: **P < 0.01.$

LMX1B colocalizes with LC3B and is degraded during nutrient starvation. (A) LMX1A-FLAG turnover in stable HEK293T cells treated with CHX (50 µg/ml) for 16 h in the absence or presence of BafA1 (20 nM) or MG132 (10 µM; n = 3). Molecular weight markers are shown in kD. Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) LMX1B-FLAG turnover in stable HEK293T cells treated with CHX (50 µg/ml) for 16 h in the absence or presence of BafA1 (20 nM) or MG132 (10 µM) (n = 3). Molecular weight markers are shown in kD. Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: **P < 0.01, ****P < 0.0001. (C) LMX1B-FLAG degradation in stable HEK293T cells during starvation (6 h) in the presence of CHX (50 µg/ml) ± BafA1 (20 nM) or CHX ± MG132 (10 µM; n = 4–6). Molecular weight markers are shown in kD. Mean ± SD; one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05, ****P < 0.0001. (D) Immunoblot and densitometry of cytosolic and nuclear fractions from HEK293T cells stably expressing LMX1B-FLAG with or without 2 h nutrient starvation. Molecular weight markers are shown in kD. Lamin B was used to normalize the nuclear fractions and GAPDH for the total and cytosolic fractions (n = 5). Mean ± SD; Student’s t test: ****P < 0.0001. (E) LMX1B relocates to cytosolic puncta that co-label with LC3B during extended nutrient starvation. Representative immunofluorescence images (to the left) and linescans of the indicated areas of LMX1B-FLAG stable HEK293T cells transfected with GFP-LC3B (green), fixed, and stained with anti-FLAG antiserum (magenta) after 2 or 6 h starvation. Bar = 20 µm, full field; 2 µm, zoom inset. (F) FLIP analysis of nucleus to cytoplasmic GFP-LMX1B transport in fed and starved cells, in the absence or presence of human ATG8 proteins (expression induced with DOX: 500 ng/ml; 20 h). Wild-type and ATG8 KO HeLa cells (Nguyen et al., 2016) were maintained in normal growth medium or starved for 2 h before live-cell FLIP analysis for a period of 15 min constant photobleaching of a cytoplasmic region of interest (example image shown top, right). Data are a measure of the loss of GFP-LMX1B fluorescence intensity within the nucleus of the photobleached cell normalized against non-photobleached cells in the same field of view. Bar = 10 µm. Data points represent individual treated cells in multiple fields of view (n = 4–6). Mean ± SD; one-way ANOVA followed by Tukey’s multiple comparison post-hoc test: **P < 0.01.