Figure 1.
LMX1B is an autophagy transcription factor in HEK293T cells. (A) ChIP qPCR analysis of LMX1B promoter occupancy in HEK293T cells. This experiment was repeated three times with consistent results. (B) qRT-PCR analysis of selected genes in HEK293T cells transfected with siLMX1B smartpool or non-targeting siControl. mRNA levels were normalized to GAPDH. Mean ± SD (n = 3); Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. siControl. (C) qRT-PCR analysis of selected genes in HEK293T cells transfected with LMX1B shRNA or non-targeting shControl. mRNA levels were normalized to GAPDH. Mean ± SD (n = 3); Student’s t test: **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. shControl. (D) qRT-PCR analysis of rescue of LMX1B suppression in HEK293T cells double co-transfected with LMX1B smartpool siRNA and codon optimized, siRNA-resistant LMX1B plasmid (or empty vector control). Values are mean ± SD of triplicate treatments from a single knockdown/rescue experiment, representative of >3 experiments with consistent results, and were normalized to siRNA control. Student’s t test: *P < 0.05 and **P < 0.01; siRNA + empty vector vs. siRNA + LMX1B. (E) LMX1B siRNA reduces expression of key autophagy proteins. Left, example immunoblots; right, quantitation relative to GAPDH levels. Molecular weight markers are shown in kD. Mean ± SD; Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. siControl. (F) Imaging-based autophagic flux assay in GFP-LC3B HEK293 cells transfected with non-targeting siControl or siLMX1B, stained with anti-WIPI2 antibodies. To the top, selected example inverted single channel images with color overlays below (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 bottom, puncta counts for GFP-LC3B and WIPI2 in siControl and siLMX1B-treated HEK293 cells in full nutrients (fed) or following 2 h starvation in the presence or absence of BafA1 (20 nM, 2 h). Mean ± SD of >35 cells from three independent experiments; one-way ANOVA followed by Fisher’s least significant difference (LSD) test for planned comparisons: *P < 0.05, **P < 0.01, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) qRT-PCR analysis of autophagy and selected gene expression in two LMX1B KO HEK293T clones (KO1 and KO2). Data show means ± SD (n = 4 samples from two early passage cultures); two-way ANOVA followed by Tukey’s multiple comparison post-hoc test against control cells: *P < 0.05, **P < 0.01,****P < 0.0001. (H) Imaging-based autophagic flux assay in LMX1B CRISPR KO HEK293T cells stained with anti-LC3B and anti-WIPI2 antibodies. Puncta counts for LC3B and WIPI2 in Control and LMX1B KO HEK293T cells in full nutrients (fed) or following 2 h starvation in the presence or absence of BafA1 (20 nM). Mean ± SD of 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 < 0.01, ****P < 0.0001. (I) Immunoblot showing LMX1B levels in CRISPR KO cell line KO2 compared to controls, with analysis of p62 levels and LC3B lipidation in fed and starved conditions (2 h), in the absence and presence of BafA1 (20 nM). Molecular weight markers are shown in kD. Densitometry quantitation is shown to the right (n = 4); one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05.

LMX1B is an autophagy transcription factor in HEK293T cells. (A) ChIP qPCR analysis of LMX1B promoter occupancy in HEK293T cells. This experiment was repeated three times with consistent results. (B) qRT-PCR analysis of selected genes in HEK293T cells transfected with siLMX1B smartpool or non-targeting siControl. mRNA levels were normalized to GAPDH. Mean ± SD (n = 3); Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. siControl. (C) qRT-PCR analysis of selected genes in HEK293T cells transfected with LMX1B shRNA or non-targeting shControl. mRNA levels were normalized to GAPDH. Mean ± SD (n = 3); Student’s t test: **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. shControl. (D) qRT-PCR analysis of rescue of LMX1B suppression in HEK293T cells double co-transfected with LMX1B smartpool siRNA and codon optimized, siRNA-resistant LMX1B plasmid (or empty vector control). Values are mean ± SD of triplicate treatments from a single knockdown/rescue experiment, representative of >3 experiments with consistent results, and were normalized to siRNA control. Student’s t test: *P < 0.05 and **P < 0.01; siRNA + empty vector vs. siRNA + LMX1B. (E) LMX1B siRNA reduces expression of key autophagy proteins. Left, example immunoblots; right, quantitation relative to GAPDH levels. Molecular weight markers are shown in kD. Mean ± SD; Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. siControl. (F) Imaging-based autophagic flux assay in GFP-LC3B HEK293 cells transfected with non-targeting siControl or siLMX1B, stained with anti-WIPI2 antibodies. To the top, selected example inverted single channel images with color overlays below (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 bottom, puncta counts for GFP-LC3B and WIPI2 in siControl and siLMX1B-treated HEK293 cells in full nutrients (fed) or following 2 h starvation in the presence or absence of BafA1 (20 nM, 2 h). Mean ± SD of >35 cells from three independent experiments; one-way ANOVA followed by Fisher’s least significant difference (LSD) test for planned comparisons: *P < 0.05, **P < 0.01, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) qRT-PCR analysis of autophagy and selected gene expression in two LMX1B KO HEK293T clones (KO1 and KO2). Data show means ± SD (n = 4 samples from two early passage cultures); two-way ANOVA followed by Tukey’s multiple comparison post-hoc test against control cells: *P < 0.05, **P < 0.01,****P < 0.0001. (H) Imaging-based autophagic flux assay in LMX1B CRISPR KO HEK293T cells stained with anti-LC3B and anti-WIPI2 antibodies. Puncta counts for LC3B and WIPI2 in Control and LMX1B KO HEK293T cells in full nutrients (fed) or following 2 h starvation in the presence or absence of BafA1 (20 nM). Mean ± SD of 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 < 0.01, ****P < 0.0001. (I) Immunoblot showing LMX1B levels in CRISPR KO cell line KO2 compared to controls, with analysis of p62 levels and LC3B lipidation in fed and starved conditions (2 h), in the absence and presence of BafA1 (20 nM). Molecular weight markers are shown in kD. Densitometry quantitation is shown to the right (n = 4); one-way ANOVA followed by Fisher’s LSD test for planned comparisons: *P < 0.05.

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