Figure 3. Global proteomics analysis of... | Rockefeller University Press

Figure 3.

$Global proteomics analysis of lysates and aggregates from SETX-depleted U2OS and differentiated SH-SY5Y cells shows similarities with neurodegeneration-associated aggregates. (A) Cell lysates from U2OS cells with mock treatment or SETX shRNA-mediated depletion (three biological replicates for each) were analyzed by mass spectrometry, identifying 1,579 proteins present in all samples. The abundance of each protein in control versus SETX-depleted cells is shown, with non-significant differences in grey and significant differences in red. Significance was determined using a two-tailed t test assuming unequal variance as well as Benjamini–Hochberg control of the false discovery rate at 0.05. See also Table S1. (B) Detergent-resistant aggregates were isolated from control and SETX-depleted cells and analyzed by mass spectrometry with lysate normalization. Red/grey indicators as in A. (C and D) Cell lysates and aggregate fractions were prepared from differentiated SH-SY5Y cells with control or SETX shRNA (three biological replicates for each) and analyzed as described in A and B. See also Table S1. (E) Levels of each aggregated protein, normalized by lysate values, were analyzed by hierarchical clustering (Pearson) comparing shControl samples (3) and shSETX samples (3) in U2OS cells. Only proteins with statistically significant differences between the groups are shown, which includes 405 proteins (of 1,580 total identified in all samples) in U2OS cells. Relationships between samples, as determined by the clustering output, are shown. (F) Levels of each aggregated protein, normalized by lysate values, were analyzed by hierarchical clustering (Pearson) comparing shControl samples (3) and shSETX samples (3) in differentiated SH-SY5Y cells as in E. 446 proteins shown (of 1,712 total identified in all samples) for SH-SY5Y cells. (G) Overlap between increased aggregated proteins identified in U2OS and in differentiated SH-SY5Y cells. See Table S2. (H) Gene Ontology terms and fold enrichment of proteins identified from overlap in G, number of genes and FDR values in parentheses. (I and J) Relative percentages of proteins identified from various disease (model) datasets in the non-aggregated protein group (NIA) and increased aggregated fractions caused by SETX deficiency in U2OS (I) or differentiated SH-SY5Y cells (J), sources for aggregated proteins as indicated (Dammer et al., 2012; Zuo et al., 2021; McCormack et al., 2019; Hales et al., 2016; Kepchia et al., 2020; Mahul-Mellier et al., 2020). (K and L) TANGO scores of proteins identified in NIA and aggregate fractions from U2OS (K) and differentiated SH-SY5Y cells (L). In I and J, The chi-squared test was used to evaluate the statistical significance of differences in distribution. In K and L, Welch’s t test was used to compute P values. (I–L) P values shown on each graph.$

Global proteomics analysis of lysates and aggregates from SETX-depleted U2OS and differentiated SH-SY5Y cells shows similarities with neurodegeneration-associated aggregates. (A) Cell lysates from U2OS cells with mock treatment or SETX shRNA-mediated depletion (three biological replicates for each) were analyzed by mass spectrometry, identifying 1,579 proteins present in all samples. The abundance of each protein in control versus SETX-depleted cells is shown, with non-significant differences in grey and significant differences in red. Significance was determined using a two-tailed t test assuming unequal variance as well as Benjamini–Hochberg control of the false discovery rate at 0.05. See also Table S1. (B) Detergent-resistant aggregates were isolated from control and SETX-depleted cells and analyzed by mass spectrometry with lysate normalization. Red/grey indicators as in A. (C and D) Cell lysates and aggregate fractions were prepared from differentiated SH-SY5Y cells with control or SETX shRNA (three biological replicates for each) and analyzed as described in A and B. See also Table S1. (E) Levels of each aggregated protein, normalized by lysate values, were analyzed by hierarchical clustering (Pearson) comparing shControl samples (3) and shSETX samples (3) in U2OS cells. Only proteins with statistically significant differences between the groups are shown, which includes 405 proteins (of 1,580 total identified in all samples) in U2OS cells. Relationships between samples, as determined by the clustering output, are shown. (F) Levels of each aggregated protein, normalized by lysate values, were analyzed by hierarchical clustering (Pearson) comparing shControl samples (3) and shSETX samples (3) in differentiated SH-SY5Y cells as in E. 446 proteins shown (of 1,712 total identified in all samples) for SH-SY5Y cells. (G) Overlap between increased aggregated proteins identified in U2OS and in differentiated SH-SY5Y cells. See Table S2. (H) Gene Ontology terms and fold enrichment of proteins identified from overlap in G, number of genes and FDR values in parentheses. (I and J) Relative percentages of proteins identified from various disease (model) datasets in the non-aggregated protein group (NIA) and increased aggregated fractions caused by SETX deficiency in U2OS (I) or differentiated SH-SY5Y cells (J), sources for aggregated proteins as indicated (Dammer et al., 2012; Zuo et al., 2021; McCormack et al., 2019; Hales et al., 2016; Kepchia et al., 2020; Mahul-Mellier et al., 2020). (K and L) TANGO scores of proteins identified in NIA and aggregate fractions from U2OS (K) and differentiated SH-SY5Y cells (L). In I and J, The chi-squared test was used to evaluate the statistical significance of differences in distribution. In K and L, Welch’s t test was used to compute P values. (I–L) P values shown on each graph.

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