Additional file 1 of Translatome analysis of tuberous sclerosis complex 1 patient-derived neural progenitor cells reveals rapamycin-dependent and independent alterations
Creators: name: Aksoylu, Inci S.
name: Martin, Pauline
name: Robert, Francis
name: Szkop, Krzysztof J.
name: Redmond, Nicholas E.
name: Bhattacharyya, Srirupa
name: Wang, Jennifer
name: Chen, Shan
name: Beauchamp, Roberta L.
name: Nobeli, Irene
name: Pelletier, Jerry
name: Larsson, Ola
name: Ramesh, Vijaya
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1. Figure S1. Quality control of RNA sequencing data of cytosolic and polysome-associated mRNA isolated from NPCs of different conditions. A Barplot showing overall number of reads that are aligned to adapter sequences, rRNA sequences, assigned to genes as well as unmapped/unassigned reads. B Boxplot showing TMM-log2 normalized counts of TSC1 transcript in TSC1−/− (red) and TSC1+/+ (blue) NPCs. C Scree-plot showing the percentage of variance explained by PC1-PC10. D Correlation of principal components (PC1-PC9) to experimental factors. E Projection of samples in principal components 1 and 2, with samples shaped according to library type (circle: polysome-associated mRNA; triangle: cytosolic mRNA) and colored according to genotype of samples (TSC1−/−, red; TSC1+/+, blue). Figure S2. Quality control of RNA sequencing data of postmortem samples of BA19 ASD and control. A Barplot showing overall number of reads that are aligned to adapter sequences, assigned to genes as well as unmapped/unassigned reads. B Scree-plot showing the percentage of variance explained by PC1-PC10. C Correlation of principal components (PC1-PC9) to experimental factors. D Projection of samples in principal components 1 and 2, with samples shaped according to library type (circle: polysome-associated mRNA; triangle:cytosolic mRNA) and colored according to condition (ASD: red; Control: blue). Figure S3. Immunoblotting in NPCs. A Immunoblotting for TSC1 in TSC1−/− compared with CRISPR-corrected TSC1+/+ NPCs. Ribosomal S6 protein serves as a loading control. B Immunoblot of NPCs treated with rapamycin (50 nM) and RMC-6272 (10 nM) for indicated proteins. β-tubulin served as a loading control. Images have been cropped for clarity and conciseness, and entire blots are shown in Additional file 9. Figure S4. Gene ontology analysis comparison for ASD and TSC1−/− NPCs. A and B Gene ontology analysis (similar to Fig. 1F) for genes categorized as “translation up” in TSC1−/− versus TSC1+/+ and ASD versus Ctrl BA19 comparisons (A); and genes categorized as “translation down” in TSC1−/− and TSC1+/+ and ASD versus Ctrl BA19 comparisons (B). The analysis was performed using ClueGO in “cluster mode.” Figure S5. Comparison of rapamycin-treated TSC1−/− to non-treated TSC1+/+ NPCs. A and B anota2seq analysis (A) and kernel densities for p value or FDR from anota2seq analysis (B) are shown comparing rapamycin-treated TSC1−/− to non-treated TSC1+/+ NPCs. (similar to Fig. 1B-C). Figure S6. Additional data related to changes in cells size and proliferation. A Bright field images (left panel) and cell size quantitation (right panel) of TSC1+/+ and TSC1−/− NPCs treated with 50 nM rapamycin or 10 nM RMC-6272 (n = 3 ± SD) are shown. Scale bar = 100 µm. B TSC1+/+ and TSC1−/− NPCs were treated with 10 nM RMC-6272 or DMSO as a control along with 1:500 dilution of the fluorescent nuclear marker NucSpot650 to visualize cell nuclei in the near infrared (NIR) spectrum. Using the Incucyte SX5 system, images were taken every 2 h for a total of 48 h. Graphs of NIR mean intensity (NIRCU) were generated using GraphPad Prism9 showing the average nuclei number/image field (36 non-overlapping image fields/well). Data represent three biological replicates per treatment group (± SEM). *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 calculated by Student’s t test. Figure S7. Additional data related to changes in neurite outgrowth. A and B Quantitation of neurite number (left), length (middle) and extremities (right) from trace images are shown for immunofluorescence staining of TSC1± (A) and TSC1+/+ (B) NPCs treated with DMSO, 50 nM rapamycin or 10 nM RMC-6272 using the neuronal marker MAP2 and HCA-Vision image quantitation software. Data represent eight non-overlapping field images/treatment generated using GraphPad Prism9 with relative fold change normalized to DMSO-treated NPCs (mean, ± SD). ** p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001, ns = not significant calculated by Student’s t test (A-B).