Fig 2: Localization and solubility of UBQLN2ALS mutants in induced pluripotent stem cells (iPSCs).(A) Extracts from UBQLN2WT, UBQLN2P497H, UBQLN22XALS, UBQLN2I498X, or UBQLN24XALS iPSCs (clone 1 [C1] and clone 2 [C2]) were separated into soluble and insoluble fractions in 1% Triton X-100 (TX-100) buffer and immunoblotted with α-UBQLN2, and α-β-tubulin antibodies. (B) Localization patterns of wild-type and UBQLN2ALS proteins in iPSCs. UBQLN2WT, UBQLN2P497H, UBQLN24XALS, and UBQLN2I498X iPSCs were stained with α-UBQLN2 and α-TIAR antibodies and imaged by confocal microscopy. Note the lack of cytosolic aggregates. (C) Cell extracts from iPSCs of the indicated genotypes were incubated at room temperature with increasing amounts of chymotrypsin for 5 min. After separation by SDS-PAGE, the proteins were immunoblotted with α-UBQLN2 antibodies. Positions of full-length and cleaved UBQLN2 are denoted by arrows. *: non-specific band. (D) Autophagy inhibition with BafA1 reduced solubility of endogenous UBQLN2ALS proteins. UBQLN2WT, UBQLN2P497H, UBQLN24XALS, and UBQLN2I498X iPSCs were treated with 100 nM of BafA1 for 16 hr followed by BafA1 washout and incubation in BafA1-free growth media for 8 hr. Detergent extracts were separated into soluble and insoluble fractions and analyzed by SDS-PAGE and immunoblotting using UBQLN2, UBQLN1, LC3A/B, and β-tubulin antibodies. (E) Quantification of UBQLN2 solubility in UBQLN2WT, UBQLN2P497H, and UBQLN24XALS iPSCs from (D). The bars represent mean with SEM of triplicate samples. Unpaired t-test was used for statistical analysis. *p≤0.05, ***p≤0.001. Figure 6—source data 1.Uncropped Western blot images corresponding to Figure 6A, C, D, E.

Fig 3: Validation of the relationship between UBQLN1 and autophagy-related genes. (A and B) CO-IP and Western blot experiments to detect the interaction between Ubiquilin 1 and Beclin 2. IB (Immunoblot) refers to the results of immunoblotting detection by using the Western blot method. Input refers to the total protein sample used before the immunoprecipitation experiment, serving as a control to confirm that the input protein content is consistent across samples. (C) Molecular docking analysis of the interaction between Beclin 2 and Ubiquilin 1 (Beclin 2 is blue, Ubiquilin 1 is green).

Fig 4: shUbqln1 mES cells Differentiation into Neuronal or Epithelial-like cells. Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. (A) Schematic of doxycycline inducible UBQLN1 shRNA differentiation into mouse embryonic stem cell neuronal (mESN) and mouse embryonic stem cell epithelial (mESE) cells. (B) Representative microscopy images of unmanipulated mES cells and mES cells following doxycycline induction. (C) Representative t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of unmanipulated neuronal/differentiated neuronal and unmanipulated epithelial/differentiated epithelial cells. Neuronal cells revealed identical transcriptional profile throughout the cell populations (green vs black spots) while loss of UBQLN1 found to alter differentiation of epithelial cells (blue vs red spots). (D) Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. UBQLNs has N-terminal UBL domain (ubiquitin-like), C-terminal UBA domain (ubiquitin-associated) and STI regions in the middle. Ubiquilin2 has an additional collagen-like domain between the fourth STI and UBA domains.

Fig 5: Correlation between gene modules and traits, along with their evaluation metrics. (A) Comparison between model prediction results and interaction mapping data from mass spectrometry analysis. (B and C) R2 values and average connectivity between genes under different soft-threshold conditions. (D) Heatmap showing the correlation between module eigengenes and traits. (E) Correlation analysis of gene modules across different groups. (F) Correlation analysis of “geneModuleMembership” and “geneTraitCor” within the brown module. (G) KEGG enrichment analysis of genes in the brown module. (H) Upset plot between enrichment results and core genes of brown module. (I) Box plot of expression of 6 brown module core genes involved in the regulation of autophagy. (J) Scatter plot of correlation analysis between UBQLN1 and 6 core genes of brown module involved in the regulation of autophagy. (K) GSEA pathway enrichment results based on the correlation with the UBQLN1.