Data for: Dysregulation of mTOR signaling mediates common neurite and migration defects in both idiopathic and 16p11.2 deletion autism neural precursor cells
Data files
Apr 01, 2024 version files 6.11 MB
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Figure1-Figuresupplement1_SourceData1.csv
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Figure1-FigureSupplement1C_SourceData2.csv
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Figure1B_SourceData.xlsx
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Phosphoproteoeme_16pDelASDvsSib.xlsx
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Phosphoproteome_IASDvsSib.xlsx
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README.md
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TotalProteome_16pDelASDvsSib.xlsx
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Abstract
Autism spectrum disorder (ASD) is defined by common behavioral characteristics, raising the possibility of shared pathogenic mechanisms. Yet, vast clinical and etiological heterogeneity suggests personalized phenotypes. Surprisingly, our iPSC studies find that six individuals from two distinct ASD subtypes, idiopathic and 16p11.2 deletion, have common reductions in neural precursor cell (NPC) neurite outgrowth and migration even though whole genome sequencing demonstrates no genetic overlap between the datasets. To identify signaling differences that may contribute to these developmental defects, an unbiased phospho-(p)-proteome screen was performed. Surprisingly, despite the genetic heterogeneity, hundreds of shared p-peptides were identified between autism subtypes including the mTOR pathway. mTOR signaling alterations were confirmed in all NPCs across both ASD subtypes and mTOR modulation rescued ASD phenotypes and reproduced autism NPC-associated phenotypes in control NPCs. Thus, our studies demonstrate that genetically distinct ASD subtypes have common defects in neurite outgrowth and migration which are driven by the shared pathogenic mechanism of mTOR signaling dysregulation.
README: Data for: Dysregulation of mTOR signaling mediates common neurite and migration defects in both idiopathic and 16p11.2 deletion autism neural precursor cells
https://doi.org/10.5061/dryad.6wwpzgn5v
As described in the methods section, the entire dataset represents source data for the Figures presented in our manuscript published in the journal Elife " Dysregulation of mTOR signaling mediates common neurite and migration defects in both idiopathic and 16p11.2 deletion autism neural precursor cells”. The source data is a requirement for publication in Elife and will aid others who would like to replicate the published figures and potentially utilize the data in their future assessments. With this in mind, much of the methods utilized in collecting our data and the nomenclature of the files in this data bank can largely be contextualized by referencing our manuscript. For example, Figure1B_SourceData is the data utilized to create Figure 1B in our Elife Manuscript. The data includes cell biological measures of neurite outgrowth, neurosphere migration, molecular measures such as Western Blot densitometry ratios, and then proteomic, genomic, and phosphoproteomic data. Please see methods for more details on these measures.
Description of the data and file structure
Figure 1
Figure 1: This data focuses on neurite outgrowth and neurosphere migration data from both the I-ASD and 16pDel cohorts in baseline medium conditions. For I-ASD cohort data (Figure 1B, 1D), data is separated by individual. This cohort consists of 3 individuals with autism (I-ASD-1, I-ASD-2, I-ASD-3) and their three brothers (Sib-1, Sib-2, Sib-3). In the I-ASD cohort, most of our comparisons were made between sibling pairs (I-ASD-1 compared to Sib-1 for example). For the 16pDel cohort data (1E, 1F) we made comparisons across all groups and delineated individual data as well (Individuals 1-3). The groups include Sibs, I-ASD, NIH, and 16pDel (see methods for further description). All the neurite and neurosphere migration data presented here was collected over multiple years (between 2014-2018). Each Excel cell for neurites represents the average % of neurites per dish. Each Excel cell for neurospheres represents the migration of a single neurosphere. For neurite data, one experiment often had 2-3 dishes. For neurosphere data, one experiment usually measured 10-20 spheres. Thus, these sheets are a composite of multiple experiments. For the I-ASD cohort, we very clearly delineated which data was obtained from which iPSC clone using the highlighting formatting and an accompanying key. This is important, as we mentioned in the methods, clones can vary from one another. Furthermore, for those looking to replicate our graphs, clone delineation is required for appropriate replication. Every I-ASD cohort individual has at least 2 clones. Some individuals have up to 5 clones. If a clone was not available for an individual the notation “N/A” was utilized. If an experimental replicate was not conducted for an individual (therefore no data was collected) “null” was used. For formatting-related spaces, we utilized the term “blank”.
Figure 1B: Neurite outgrowth in our idiopathic ASD cohort. Highlight represents the corresponding clone, please see the key. N/A means no clone. Null means this experiment was not conducted even if a clone was available. Each cell is the average % of neurites in a dish.
Figure 1D: Neurosphere migration in our I-ASD cohort. N/A means no clone. Null means this experiment was not conducted even if a clone was available. Each cell represents the migration of a single sphere.
Figure 1E: Neurite outgrowth in all groups: Individuals delineated by color coding key. Clones not delineated. “Null” means the experiment was not replicated.
Figure 1F: Neurosphere migration in all groups: Individuals delineated by color coding key. Clones not delineated. “Null” means the experiment was not replicated.
Figure 1 - Figure Supplement 1A: Migration and initial sphere size (ISS), multiple experiments across multiple groups (Sib, NIH, 16pDel, I-ASD)
Figure 1 - Figure supplement 1B: Actual and projected neurosphere migration data. Projected data was obtained by subdividing the data from Figure 1-Figure Supplement 1A by the group running the linear model Migration~ISS and taking the fitted values from this model to show the change in Migration when ISS is accounted for.
Figure 2
Figure 2: This data once again focuses on neurite outgrowth and neurosphere migration data. However, in this figure, the focus is on comparisons between NPCs in control media conditions (baseline media) or in medium conditions with an extracellular factor added (PACAP, NGF, and 5-HT).
Figure 2A: Neurites under control and PACAP (3nM) conditions in our I-ASD cohort. “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 2B: Neurites under control and NGF (30ng/mL) conditions in our I-ASD cohort. “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 2C: Neurites under control and PACAP (3nM) conditions in all Sibs, NIH, I-ASD, and each 16pDel individual (16p M-1, 16p M-2, 16pF). “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 2D: Neurites under control and NGF (30ng/mL) conditions in all Sibs, NIH, I-ASD, and each 16pDel individual (16p M-1, 16p M-2, 16pF). “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 2E: Neurosphere migration under control and PACAP (3nM) conditions in our I-ASD cohort. “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 2F: Neurosphere migration under control and PACAP (3nM) conditions in all Sibs, NIH, I-ASD, and each 16pDel individual (16p M-1, 16p M-2, 16pF). “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 2 Supplement 1: Neurites under control and 5-HT (100 ug/mL) conditions in our I-ASD cohort. “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 2 Supplement 2: Neurites under control and several PACAP (1nM, 3nM, 10nM, 30nM, 100nM), or control and several NGF (3ng/mL, 10ng/mL, 30ng/mL, 100ng/mL) conditions in our I-ASD cohort.
Figure 2 Supplement 3: Neurites under control and 5-HT (100 ug/mL) conditions in all Sibs, NIH, I-ASD, and each 16pDel individual (16p M-1, 16p M-2, 16pF). “Null” denotes no experimental replicate was conducted, aka there is no data.
Figure 3
Figure 3: Focuses largely on the phosphoproteome, proteome, and whole genome sequencing data. For the P-proteome, proteome for I ASD, this represents the analysis of combined and pooled protein from all three I-ASD individuals when compared to pooled protein from all three Sibs. One tab includes the top 10 most enriched pathways by logPvalue, and the other “IPA readout” shows the results of the canonical pathway analysis in IPA (Ingenuity Pathway Analysis software). The 16pDel data is similarly pooled with all 3 16pDel individuals and compared to pooled Sibs with similar tabs of data. The whole genome sequencing includes IPA pathway readout as well as top pathways as well.
Figure 4
Figure 4: The source data in this figure includes an example of the actual X-ray films (as both PDFs and JPEGs) that were utilized in the generation of the densitometry measurements in the accompanying CSV files. All CSV files show the densitometry ratio of (PS6/GAPDH)/(S6/GAPDH) abbreviated as PS6/S6 or (PAKT/GAPDH)/(AKT/GAPDH) abbreviated as PAKT/AKT. Note the X-ray film files usually only show a small subset of the data in the CSV files-other films were also compiled in the CSV files.
Figures 4A and 4B: Figure 4A image and PDF files show X-ray film examples of P-S6, GAPDH, and S6 levels in Sib-1 and I-ASD-1. Figure 4B is the densitometry ratios obtained from Figure 4A as well as other films that included I-ASD-1 and Sib-1. “Null” again means these westerns were not conducted.
Figures 4C and 4D: Figure 4C image and PDF files show X-ray film examples of P-S6, GAPDH, and S6 levels in Sib-3 and I-ASD-3. Figure 4D is the densitometry ratio obtained from Figure 4C as well as other films that included I-ASD-3 and Sib-3. “Null” again means these westerns were not conducted.
Figures 4E and 4F: Figure 4E image and PDF files show X-ray film examples of P-S6, GAPDH, and S6 levels in Sib-2 and I-ASD-2. Figure 4F is the densitometry ratios obtained from Figure 4E as well as other films that included I-ASD-2 and Sib-2. “Null” again means these westerns were not conducted.
Figures 4G and 4H: Figure 4G image and PDF files show X-ray film examples of P-S6, GAPDH, and S6 levels in Sibs, NIHs, and each 16pDel individual. Figure 4H is the densitometry ratios obtained from Figure 4G as well as other films that included a direct comparison of Sib, NIH, and 16pDel.
Figure 4 - Figure Supplement 1A and 1B: Image and PDF files show X-ray film examples of P-AKT, GAPDH, and AKT levels in Sib-1 and I-ASD-1. 1B is the densitometry ratios obtained from Figure 1A as well as other films that included I-ASD-1 and Sib-1. “Null” again means these westerns were not conducted.
Figure 4 - Figure Supplements 1C and 1D: Image and PDF files show X-ray film examples of P-AKT, GAPDH, and AKT levels in Sib-3 and I-ASD-3. 1D is the densitometry ratio obtained from 1C as well as other films that included I-ASD-3 and Sib-3. “Null” again means these westerns were not conducted.
Figure 4 - Figure Supplements 1E and 1F: Image and PDF files show X-ray film examples of P-AKT, GAPDH, and AKT levels in Sib-2 and I-ASD-2. 1F is the densitometry ratios obtained from 1E as well as other films that included I-ASD-2 and Sib-2. “Null” again means these westerns were not conducted.
Figure 4 - Figure Supplements 1G and 1H: Image and PDF files show X-ray film examples of P-AKT, GAPDH, and AKT levels in Sibs, NIH, and each 16pDel individual. 1H is the densitometry ratio obtained from 1G as well as other films that included a direct comparison of Sib, NIH, and 16pDel.
Figure 4- Figure Supplement 2: Quantified densitometry ratios of cellular P-S6/ S6 levels in all Sibs and I-ASDs.
Figure 5
Figure 5: This figure contains multiple types of data including Western blot X-ray Images/PDFs (labeled and unlabeled), Western blot densitometry data, neurite outgrowth data, and neurosphere migration data. Much of this figure focuses on utilizing small-molecule drugs that affect the mTOR pathway. Specifically, the small molecules include SC-79 (mTOR activator) and MK-2206 (mTOR inhibitor). Much of the figure/data focuses on the application of this drug to the I-ASD-1 and Sib-1 pair, but some data also comes from I-ASD-3 and Sib-3.
Figures 5A and 5B: Image and PDF files of the X-ray film showing an example of Sib-1 and I-ASD-1 NPC proteins when treated with SC-79. 5B is the densitometry measurements of film 5A and other similar films that depict P-S6 levels in I-ASD-1 in control and SC-79 conditions.
Figure 5C: Neurite outgrowth in I-ASD-1 and Sib-1 under control and SC-79 conditions. Null means replicate experiments were not conducted.
Figure 5D: Neurosphere migration in I-ASD-1 and Sib-1 under control and SC-79 conditions. Null means replicate experiments were not conducted.
Figure 5E and 5F: Image and PDF files of the X-ray film showing an example of Sib-1 and I-ASD-1 NPC proteins when treated with MK-2206. 5F is the densitometry measurements of film 5E and other similar films that depict P-S6 levels in I-ASD-1 in control and MK-2206 conditions.
Figure 5G: Neurite outgrowth in I-ASD-1 and Sib-1 under control and MK-2206 conditions. Null means replicate experiments were not conducted.
Figure 5H: Neurosphere migration in I-ASD-1 and Sib-1 under control and MK-2206 conditions. Null means replicate experiments were not conducted.
Figure 5I: Image and PDF files of the X-ray film showing an example of Sib-3 and I-ASD-3 NPC proteins when treated with SC-79.
Figure 5J: Neurite outgrowth in I-ASD-3 and Sib-3 under control and SC-79 conditions. Null means replicate experiments were not conducted.
Figure 5K: Image and PDF files of the X-ray film showing an example of Sib-3 and I-ASD-3 NPC proteins when treated with MK-2206.
Figure 5L: Neurite outgrowth in I-ASD-3 and Sib-3 under control and MK-2206 conditions. Null means replicate experiments were not conducted.
Figure 6
Figure 6: This figure contains multiple types of data including Western blot X-ray Images/PDF, Western blot densitometry data, neurite outgrowth data, and neurosphere migration data. Much of this figure focuses on utilizing small-molecule drugs that affect the mTOR pathway. Specifically, the small molecules include SC-79 (mTOR activator) and MK-2206 (mTOR inhibitor). Much of the figure/data focuses on the application of this drug to the I-ASD-2 and Sib-2 pair, but some data also comes from NIH and 16pDel.
Figures 6A and 6B: Image and PDF files of the X-ray film showing an example of Sib-2 and I-ASD-2 NPC proteins when treated with MK-2206. 6B is the densitometry measurements of film 6A and other similar films that depict P-S6 levels in I-ASD-2 in control and MK-2206 conditions.
Figure 6C: Neurite outgrowth in I-ASD-2 and Sib-2 under control and MK-2206 conditions. Null means replicate experiments were not conducted.
Figure 6D: Neurosphere migration in I-ASD-2 and Sib-2 under control and MK-2206 conditions. Null means replicate experiments were not conducted.
Figures 6E and 6F: Image and PDF files of the X-ray film showing an example of Sib-2 and I-ASD-2 NPC proteins when treated with SC-79. 6F is the densitometry measurements of film 6E and other similar films that depict P-S6 levels in I-ASD-2 in control and SC-79 conditions.
Figure 6G: Neurite outgrowth in I-ASD-2 and Sib-2 under control and SC-79 conditions. Null means replicate experiments were not conducted.
Figure 6H: Neurosphere migration in I-ASD-2 and Sib-2 under control and SC-79 conditions. Null means replicate experiments were not conducted.
Figure 6I: Image and PDF files of the X-ray film showing an example of NIH and 16pDel NPC proteins when treated with MK-2206.
Figure 6J: Neurite outgrowth in NIH and 16pDel under control and MK-2206 conditions. Null means replicate experiments were not conducted.
Figure 6K: Image and PDF files of the X-ray film showing an example of NIH and 16pDel NPC proteins when treated with SC-79
Figure 6L: Neurite outgrowth in NIH and 16pDel under control and SC-79 conditions. Null means replicate experiments were not conducted.
Figure 7
Figure 7: In these experiments, I-ASD-1, Sib-1, and I-ASD-2 neurites were measured in a variety of media conditions per CSV sheet. The conditions included a low dose/concentration of either MK-2206 or SC-79, each extracellular factor previously used (PACAP, NGF, 5-HT), and a “+” condition where the low dose MK-2206 OR SC-79 was combined with the extracellular factor. The clone is indicated on the right side. All cells represent the average % of neurites in a dish.
Figure 7A: Neurite outgrowth in I-ASD-1 NPCs under Con, SC-79 (0.1 ug/mL), PACAP, NGF, 5-HT, and SC79 +Extraceullar factor conditions.
Figure 7B: Neurite outgrowth in Sib-1 NPCs under Con, SC-79 (0.1 ug/mL), PACAP, NGF, 5-HT, and SC79 +Extraceullar factor conditions.
Figure 7C: Neurite outgrowth in Sib-1 NPCs under Con, MK-2206 (1nM), PACAP, NGF, 5-HT, and MK-2206 +Extraceullar factor conditions.
Figure 7D: Neurite outgrowth in I-ASD2 NPCs under Con, MK-2206 (1nM), PACAP, NGF, 5-HT, and MK-2206 +Extraceullar factor conditions.
Output files
Phosphoproteome_IASDvsSib: Output from phosphoproteomic analysis of all I-ASD and all Sib data pooled and compared. The proteins displayed represent the phosphoproteins that were enriched in the I-ASD pooled samples. Other columns indicate various statistical measures of how significantly different these proteins are in I-ASD pooled samples compared to the Sib pooled sample. The yellow highlighting means these were the data that met our significance threshold of LogpValue>5 and were used in the analysis of our manuscript.
TotalProteome_IASDvsSib: Output from proteomic analysis of all I-ASD and all Sib data pooled and compared. Proteins displayed represent the proteins that were enriched in the I-ASD pooled samples. Other columns indicate various statistical measures of how significantly different these proteins are in I-ASD pooled samples compared to the Sib pooled sample. The yellow highlighting means these were the data that met our significance threshold of LogpValue>5 and were used in the analysis of our manuscript.
Phosphoproteome_16pDelvsSib: Output from phosphoproteomic analysis of the 2 male 16pDel and all Sib data pooled and compared. The proteins displayed represent the phosphoproteins that were enriched in the 16pDel pooled samples. Other columns indicate various statistical measures of how significantly different these proteins are in16pDel pooled samples compared to the Sib pooled sample. The yellow highlighting means these were the data that met our significance threshold of LogpValue>5 and were used in the analysis of our manuscript.
Totalproteome_16pDelvsSib: Output from proteomic analysis of the 2 male 16pDel and all Sib data pooled and compared. Proteins displayed represent the proteins that were enriched in the 16pDel pooled samples. Other columns indicate various statistical measures of how significantly different these proteins are in16pDel pooled samples compared to the Sib pooled sample. The yellow highlighting means these were the data that met our significance threshold of LogpValue>5 and were used in the analysis of our manuscript.
WholeGenomeSequencing_IASD: Whole Genome Sequencing results from I-ASD individuals showing the chromosome, variants, and gene of interest, the type of mutation noted, the quality read, the alteration, and then whether or not the variant was present in each Family and which individuals or additional individuals from our I-ASD cohort contain the variant. N/A means no other individual shares the variant.
Sharing/Access information
NA
Code/Software
We have one set of code utilizing R studio (Build 369). This code includes linear models to assess whether initial sphere size (ISS) correlates with the final migration of a neurosphere.
Methods
The entire dataset represents source data for the Figures presented in our manuscript published in the journal Elife " Dysregulation of mTOR signaling mediates common neurite and migration defects in both idiopathic and 16p11.2 deletion autism neural precursor cells”. The source data is a requirement for publication in Elife and will aid others who would like to replicate the Figures and potentially utilize the data in their future assessments. With this in mind, much of the methods utilized in collecting our data and the nomenclature of the files in this data bank can largely be contextualized by referencing our manuscript. For example, Figure1B_SourceData is the data utilized to create Figure 1B in the above manuscript.
The manuscript focused on cell culture data from a bench lab setting. Specifically, this work focused on cells known as neural precursor cells (NPCs), which are early brain cells that ultimately go on to develop into neurons, astrocytes, and oligodendrocytes (the mature cells of the brain). Specifically, these neural precursor cells were derived from individuals with autism and either sibling or unrelated controls (from the NIH) utilizing a technique known as induced pluripotent stem cells (iPSCs). Briefly, white blood cells or fibroblast cells from individuals with autism were reprogrammed into induced pluripotent stem cells using a viral technique and then these induced pluripotent stem cells were grown in a special medium that allows them to form into neural precursor cells. These neural precursor cells were cultured in dishes and then utilized in various ways for our experiments. The goal was to study whether early developmental processes were disrupted in NPCs derived from individuals with Autism
Our source data includes data from NPCs derived from 12 different individuals. 6 of these individuals have autism- 3 with idiopathic autism (underlying genetic cause is unknown) who have been labeled as I-ASD in this data set, and 3 with a genetic form of autism caused by the 16p11.2 deletion notated as 16pDel in this data set. The comparison groups include a sex-matched sibling to the I-ASD cohort and then 3 unrelated sex-matched individuals from the NIH who have no known neuropsychiatric disorders. Our data is presented either as by individual or by group, depending on how the data is displayed in our manuscript figure. The overall groups are then: I-ASD (idiopathic autism), Sib (Sibling to the I-ASD individual who does not have autism or other developmental disorders), 16pDel (Individuals with autism and the 16p11.2 deletion), and then NIH (for the 3 individuals whose stem cells were taken from an NIH repository). Individuals were notated: I-ASD-1, I-ASD-2, I-ASD-3, Sib-1, Sib-2, Sib-3, NIH-1, NIH-2, NIH-3, 16pDelM-1, 16pDelM-2, and 16pDelF, or as “individual 1, individual 2, and individual 3” across the datasets.
In addition, other notations seen include “Clone”. When induced pluripotent stem cells are made from the initial cell derived from an individual, they form clusters of cells known as clones. Due to a variety of different reasons, clones can have different properties from one another, so to increase rigor, often used 2-5 different clones from each of the above individuals were made into NPCs to conduct our experiments. Clones can be considered to be distinct biological replicates and their notation is important for the case of some experiments. We did not do the same number of clones or experiments for every individual. This is because we established this technique on the I-ASD cohort first, specifically in Sib-1 and I-ASD-1 and thus there are more replicates in this group than in any other.
There are several types of NPC-related data and the methods for these are described below with links for further information.
Neurite outgrowth data: As a neural precursor cell grows and develops, one of the important processes it needs to undergo is differentiation so it can form into a cell such as a neuron. Neurons are characterized by the presence of axons and dendrites which are outgrowths from neuronal cells that allow them to communicate with each other and other cells. NPCs are not quiet neurons yet, but as they develop, they begin to grow a neurite which is an early version of an axon or a dendrite. One set of our data includes the study of these neurites, and the methods were as follows: NPCs growing in a dish over time will cover the entire surface area of this dish (this is called confluency). When the NPCs are confluent, they are ready for experimentation. Confluent NPCs were taken out of their growth dish and then replated at the low density of 50,000 cells into alternative dishes. These replated NPCs were allowed to sit in these new dishes for 48 hours. At 48 hours, dishes were imaged on a confocal microscope which allows us to see each NPC. 3 sections of each dish (about 1 cm of the dish) were counted (visually by eye) for the total number of NPCs in that area and then we also kept count of the number of NPCs that had neurites (which were described as extension coming from the cell body that was twice the length of the cell body of the NPC). The percentage of neurites in each area was averaged together to get an estimate of the total neurites (%) in a dish. This is the data found in all the neurite Excel sheets. For more step-by-step information on the neurite assay, please see our methods paper in JOVE. In some cases, when neurites were replated at low density for the experiment, we placed them under different media “conditions” (notated as “conditions”) in the Excel sheets. The altered media conditions included the addition of either a growth factor, neurotransmitter, or signaling molecule (extracellular factor: ex: PACAP, NGF, 5-HT) or a small molecule drug (SC-79, MK-2206) to the media.
Neurosphere migration data: Another important developmental process that NPCs undergo is migration. To assess NPC migration, we first created structures known as neurospheres which are essentially spherical aggregates of NPCs. When we take these spheres and plate them onto a gel coating, over time, some cells will migrate away from the original sphere structure and the ultimate result will look like a carpet of migratory cells spreading out from a dense inner cell mass. To measure migration, we measure the total area of all the cells and subtract from it the area of the inner cell mass size. Thus, migration is represented in um2 as indicated in many of our Excel sheets. Like with the neurite experiments, during the migration phase, different media conditions were used. A detailed step-by-step guide on this assay is also seen in our JOVE visual methods manuscript.
Western Blot data: Western blotting is a semi-quantitative method to assess the presence and amount of a protein. Our proteins of interest are part of a signaling pathway known as the "mTOR pathway" which is an important pathway for regulating development. For our data, the protein was extracted from our NPCs and then this protein was ultimately put through the western blotting technique (which is a quite standard method, and our exact methods are detailed in the manuscript). Once the blot is acquired, immunochemistry and chemiluminescence techniques were utilized to develop an X-ray film that shows an imprint of the protein of interest. We have provided scanned images of these X-ray films as one part of our Western Blot Source data. The lanes (black bars) from these films can be measured as a proxy for protein amount. The size and intensity of the lanes roughly translate to a semi-quantitative measure known as densitometry and give an estimate of how much of the protein of interest is present in our NPCs. ImageJ software was used to measure these bands and obtain a value. The proteins of interest were P-S6, S6, P-AKT, and AKT (all mTOR pathway members). A ratio between the phosphorylated version (P-S6) and the unphosphorylated version (S6) was provided as the data in our Excel sheets. Much like the experiments above, NPCs could be cultured in a base culture medium or culture medium with the addition of a drug or an extracellular factor
Omics Data: Several types of omic data were also collected including genomic, phosphoproteomic, and proteomic data. These data were acquired from other collaborating labs or clusters at Rutgers that have standardized protocols and methods for obtaining these large datasets. Brief non-proprietary methods are included in the original manuscript. Interpretation of this data does not require an understanding of these methods. The protein or gene names and either the gnomad frequency or the logPvalues were fed into software from Qiagen known as Ingenuity to allow for the pathway analysis and network maps shown in our manuscript.
R source code: For one experiment, we needed to show that the initial size of a neurosphere (Initial Sphere Size, ISS) was not an important variable for migration. We utilized ISS, migration, and diagnosis (Sib, I-ASD, NIH, 16pDel) as variables in linear models to show that ISS did not influence final migration. The code and accompanying datasheet are also provided here.