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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Tuberous sclerosis complex neuropathology requires glutamate-cysteine ligase

Fig. 1

GCLC knockdown reverses the phenotype of model dysmorphic neurons. a TSC2 knockdown in rat cortical neurons as a model of TSC-related dysmorphic neurons. Neurons were transfected with empty pSuper vector, TSC2sh#1, or sh#2 together with GFP. Next, neurons were treated with 20 nM rapamycin or DMSO (control), fixed, and immunostained for P-S6. Representative images, results of analysis of P-S6 intensity, and neuron soma area. Scale bar: 25 μm. Plots represent mean +/−SEM. **p < 0.01, ***p < 0.001 in Kruskal-Wallis with Dunn’s post-hoc test. Sample sizes for experimental groups are provided in Supplementary materials and methods (Additional file 1). For additional results of TSC2sh validation, see Additional file 3: Figure S1. b Results of analysis of neuronal soma area from screening experiments. Each point represents mean +/− SEM obtained from two independent experiments. For more details see Additional file 2: Table S1 and Additional file 3: Table S2. c Western blot analysis of GCLC level in rat cortical neurons 4 days after nucleofection with empty pSuper vector, GCLCsh-mix used in the screening experiments or individual GCLCsh#1, sh#2, and sh#3. α-tubulin is shown as a loading control. d Representative images of rat cortical neurons after TSC2 knockdown with use of 2 different shRNAs and simultaneous GCLC knockdown with use of mixed shRNAs (as in the screen) or 2 individual shRNAs. GFP-encoding plasmid was co-transfected for visualization of modified neurons. Scale bar: 50 μm. e Neuronal soma area of rat cortical neurons after TSC2 and GCLC knockdown. Plot represents mean +/−SEM. ns- not significant, *p < 0.05, **p < 0.01, ***p < 0.001 in Kruskal-Wallis with Dunn’s post-hoc test. Sample sizes for experimental groups are provided in Supplementary materials and methods (Additional file 1)

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