Data from: A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs and hNPCs. The results of our study build on current evidence that CUL9 may have unique functions in different cell types potentially contributing to the difficulty of identifying CUL9 substrates.

Information on data/files:

Please first unzip Figure_4_CUL9-IP-LCMSMS-data.zip and Figure_7_iTRAQ-data.zip, then follow the description below.

CUL9 immunoprecipitation from whole lysates collected for hPSCs were analyzed using LC/MS-MS. IgG was used as a control to determine proteins specifically enriched in the CUL9 IP. This data correlates to Figure 4 of the associated manuscript.

Initial CUL9 immunoprecipitation was performed by VG and analyzed by LC-MS/MS at UNC.

Fig4-LCMSMS_IPCUL9_Replicate1_UNC

Two more immunoprecipitations were performed by VG and analyzed by LC-MS/MS at Vanderbilt University MSRC Proteomics Core. The first run (replicate 1) was used to produce the STRING figure in the associated manuscript, as well as the volcano plot in Supplemental Figure 8.

Two Scaffold files contain raw data and our analysis parameters:

• Fig4-LCMSMS_IPCUL9_Replicate2_Vanderbilt
• Fig4-LCMSMS_LCMSMS_IPCUL9_Replicate3_Vanderbilt

Excel files exported from the above Scaffold files contain the raw data in excel format including spectral counts, peptide counts, protein probability, and detailed raw data as it pertains to each identified protein. See these data sets in the folders below:

• Fig4-LCMSMS_IPCUL9_Run2_Vanderbilt_CompleteDataSet
• Fig4-LCMSMS_IPCUL9_Run3_Vanderbilt_CompleteDataSet

CUL9 KO clones were used to identify proteins increased or decreased compared to parental wild-type cell lines using iTRAQ. This data correlates to Figure 8 of the associated manuscript. Two iTRAQ experiments were performed to identify proteins altered in CUL9 KO iPSCs, neural stem cells (NSCs), and NPCs. Each set of experiments was duplicated – each in a different isogenic CUL9 KO clone (Clone 20 or Clone B)

iTRAQ labeling of experiment one is as follows:

1. Label 115: Parental WT NSCs
2. Label 117: CUL9 KO Clone 20 or B NSCs
3. Label 114: Parental WT NPCs
4. Label 116: CUL9 KO Clone 20 or B NPCs

Files containing raw and analyzed data of experiment one are labeled as follows:

• Fig7_Experiment1_Clone 002_compared-to-NSC-WT
• Fig7_Experiment1_Clone001-compared-to-NSC-WT
• Fig7_Experiment1_Clone001iPSC_compared-to-iPSCWT
• Fig7_Experiment1_Clone002_compared-to-iPSCWT

iTRAQ labeling of experiment two is as follows:

For Clone 20:

1. Label 115: Parental WT NSCs
2. Label 117: CUL9 KO Clone 20 or B NSCs
3. Label 114: Parental WT iPSCs
4. Label 116: CUL9 KO Clone 20 or B iPSCs

Files containing raw and analyzed data of experiment two are labeled as follows:

• Fig7_Experiment2_Clone001NPC_Compared-to-NPC-WT
• Fig7_Experiment2_Clone001NPC_Compared-to-NSC-WT
• Fig7_Experiment2_Clone002NPC_Compared-to-NPC-WT
• Fig7_Experiment2_Clone002NPC_Compared-to-NSC-WT

Please note that the first tab of each excel document contains the full raw data set before analysis. All other tabs contain analyzed data comparing the proteins identified in two cell lines. Analyzed tabs are labeled to indicate which cell lines are being compared. Please note that the CUL9 isogenic clones Clone 20=Clone#1 and Clone B=Clone#2 as referenced in the associated manuscript.

Detailed information about the protocols used for collection and analysis of this data can be found in the supplemental information within the associated manuscript.