Journal: Nature Communications

Article Title: Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62

doi: 10.1038/s41467-025-60857-4

Figure Lengend Snippet: a Schematic depiction of the cell lines used. b Representative immunoblot of p62 without and with Bafilomycin A1 (BafA1) treatment. ß-actin serves as a loading control. c Quantification of p62 expression levels without and with BafA1 treatment. Data is shown as mean ± s.d. fold change to WT from n = 3 independent experiments, and individual replicates are plotted as dots. P values represent two-tailed unpaired Student’s t -test. d Representative confocal images of p62 foci immunofluorescence (green). DAPI (blue) stains DNA (nuclei). Images are collapsed from Z-stacks. Scale bar is 10 µm. Quantification of ( e ) p62 foci number per cell surface area in µm 2 , and f p62 foci size per cell surface area in µm 2 . Individual data for each cell from n = 3 independent experiments and mean ± s.d. are shown. P values represent non-parametric ANOVA (Kruskal–Wallis statistic for ( e ) 99.54, p < 0.0001; for ( f ) 30.37, p < 0.0001) followed by Dunn’s multiple comparisons test. g Correlations of the mean ± s.d. p62 foci number and size with the number of surplus protein-coding genes in all the cell lines. Two-tailed P values are indicated. Correlations of ( h ) relative transcript abundance, i relative protein abundance, and j gene dependency probability, with aneuploidy score of 127 cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE). R represents Spearman’s rank correlation coefficient. P values are two-sided.

Article Snippet: SQSTM1/p62 antibody (Santa Cruz, sc-28359) and normal mouse IgG (Santa Cruz, sc-2025) were added separately to 1 mg of total protein at 2 μg and 0.25 μg per reaction, respectively, and incubated at 4 o C on rotation overnight.

Techniques: Western Blot, Control, Expressing, Two Tailed Test, Immunofluorescence, Quantitative Proteomics

Journal: Nature Communications

Article Title: Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62

doi: 10.1038/s41467-025-60857-4

Figure Lengend Snippet: a Schematic depiction of the experimental procedure for mass spectrometry-based identification of p62-proximal proteins through APEX2-mediated proximity biotinylation. Image partly created in BioRender. b Scatter plot of the fold changes in global protein abundance in the 13/3, 3/3 and 5/4 polysomic cell lines relative to the parental cell line (WT) and the corresponding changes in abundance of cytosolic p62-proximal proteins relative to WT. Colored dots represent proteins with significantly higher abundance changes of p62-proximal proteins in polysomic cells (FDR < 0.05). Data is generated from mass spectrometry analysis of n = 4 biological replicates (see Supplementary Fig. , and Supplementary Data ). c Gene ontology (GO) over-representation analysis of the enriched p62-proximal proteins in ( b ). Blue bars represent negative log-transformed p values (one-sided hypergeometric test) for GO terms with FDR < 0.05. Representatives from clusters of related GO terms are shown (see “Methods”). d Percentage of mitochondrial proteins in the measured global proteome (grey) and the corresponding percentage within the p62 proximal proteome (magenta) of 13/3, 3/3 and 5/4 cells relative to WT. P values represent results of one-sided hypergeometric test, evaluating the differences in representation of mitochondrial proteins relative to the measured proteome.

Article Snippet: SQSTM1/p62 antibody (Santa Cruz, sc-28359) and normal mouse IgG (Santa Cruz, sc-2025) were added separately to 1 mg of total protein at 2 μg and 0.25 μg per reaction, respectively, and incubated at 4 o C on rotation overnight.

Techniques: Mass Spectrometry, Quantitative Proteomics, Generated, Transformation Assay

Journal: Nature Communications

Article Title: Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62

doi: 10.1038/s41467-025-60857-4

Figure Lengend Snippet: a Schematic depiction of experimental procedure for autophagosome content profiling to identify p62 cargo candidates via APEX2-mediated proximity biotinylation. Image partly created in BioRender. b Volcano plots showing log -transformed fold change in BafA1 versus DMSO (vehicle) treated conditions across the cell lines used. Selected known p62 interactors are annotated. Data is generated from mass spectrometry analysis of n = 3–4 biological replicates (see Supplementary Fig. , and Supplementary Data ). c Gene ontology (GO) over-representation analysis of the p62 cargo candidates common to all karyotypes, shared among all polysomic cell lines, and exclusive to each karyotype (Supplementary Fig. ). Blue bars represent negative log-transformed p values (one-sided hypergeometric test) for GO terms with FDR < 0.05. d Boxplots indicating fold changes in abundance of catalogued p62-specific autophagosome lumen cargo ( , black) and mitochondrial proteins as defined by the MitoCarta3.0 inventory (magenta) compared to all other proteins (grey) enriched among the p62 cargo candidates from ( b ). Boxplots represent median with 25 th and 75 th percentile. Whiskers extend from upper and lower bound of the box to the largest and smallest values, respectively, no further than 1.5x inter-quartile range from the respective bound (Tukey method). P values are derived from two-sided Wilcoxon’s ranks sum tests.

Article Snippet: SQSTM1/p62 antibody (Santa Cruz, sc-28359) and normal mouse IgG (Santa Cruz, sc-2025) were added separately to 1 mg of total protein at 2 μg and 0.25 μg per reaction, respectively, and incubated at 4 o C on rotation overnight.

Techniques: Transformation Assay, Generated, Mass Spectrometry, Derivative Assay

Journal: Nature Communications

Article Title: Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62

doi: 10.1038/s41467-025-60857-4

Figure Lengend Snippet: a Schematic depiction of experimental procedure for p62 pull-down followed by mass spectrometry. Image partly created in BioRender. b Volcano plots showing log 2 -transformed fold change of enriched p62 interactors in WT, 13/3 and 5/4 cells treated with BafA1, highlighting mitochondrial (magenta), karyotype-independent (cyan) and polysomic-specific (orange) interactors. Data is generated from mass spectrometry analysis of n = 4 biological replicates (see Supplementary Fig. , and Supplementary Data ). c Representative immunoblot and d Quantification of p62 expression levels in the diploid parental HCT116 cells (WT-Ctrl.), parental cells transiently overexpressing EGFP (WT-EGFP OE) or p62-EGFP (WT-p62-EGFP OE), and the polysomic 5/4 cells. Data is shown as mean ± s.d. fold change to WT-Ctrl from n = 4 independent experiments, and individual replicates are plotted as dots. P values represent two-tailed unpaired Student’s t -test. e Volcano plots showing log 2 -transformed fold change of enriched p62 interactors in WT-EGFP OE and WT-p62-EGFP OE cells, treated with DMSO or BafA1, highlighting mitochondrial (magenta), karyotype-independent (cyan) and polysomic-specific (orange) interactors. Data is generated from mass spectrometry analysis of n = 4 biological replicates (see Supplementary Fig. , and Supplementary Data ). f Percentage of mitochondrial proteins in the measured proteome (grey) and the corresponding percentage within the p62 interactome (magenta) of WT, WT-p62-EGFP OE, 13/3 and 5/4 cells. P values represent results of one-sided hypergeometric test, evaluating the differences in representation of mitochondrial proteins relative to the measured proteome.

Article Snippet: SQSTM1/p62 antibody (Santa Cruz, sc-28359) and normal mouse IgG (Santa Cruz, sc-2025) were added separately to 1 mg of total protein at 2 μg and 0.25 μg per reaction, respectively, and incubated at 4 o C on rotation overnight.

Techniques: Mass Spectrometry, Transformation Assay, Generated, Western Blot, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62

doi: 10.1038/s41467-025-60857-4

Figure Lengend Snippet: a Representative confocal images of p62 (magenta) and mitochondrial outer membrane translocase TOMM20 (yellow) colocalization in diploid parental (WT) and a polysomic (5/4) cell line. Magnified inserts are shown. White arrow heads indicate p62-TOMM20 colocalization. Images are collapsed from Z-stacks. Scale bar is 10 µm. b Quantification of the Pearson correlation coefficient (PCC) for p62-TOMM20 colocalization in WT, 21/3, 13/3, 5/3, 3/3 and 5/4 cells. Values of individual cells from n = 3 independent experiments and mean ± s.d. are shown. P -values represent one-way ANOVA followed by Sidak’s multiple comparisons test. c Correlation of the mean ± s.d. PCC for p62-TOMM20 colocalization from ( b ) with the number of surplus protein-coding genes in all the cell lines. Two-tailed P -value is indicated. d Representative confocal images of the different mitochondrial architecture (red) observed in the cells. DAPI (blue) is used as a nuclear stain. Images are collapsed from Z-stacks. Scale bar is 10 µm. e Quantification of the cell fractions with each mitochondrial form, classified as healthy (extended network), intermediate (minimally extended network) and stressed (perinuclearly clustered network) as shown in ( d ). Data represent mean ± s.d. percentage of n = 3 independent experiments, and individual replicates are plotted as dots. P values represent two-tailed unpaired Student’s t -test. f Correlation of the mean ± s.d. proportion of cells harboring stressed (perinuclear) mitochondria from ( e ) with the number of surplus protein-coding genes in all the cell lines. Two-tailed P -values are indicated. g Relative mitochondrial DNA (mt-DNA) copy number determined by qPCR of the non-coding mt-DNA D-Loop and respiratory chain complex 1 subunit ND1, normalized to the nuclear ß 2 -microglobulin gene. Data is shown as mean ± s.d. fold change to WT from n = 5 independent experiments, and individual replicates are shown as dots. P values represent two-tailed unpaired Student’s t -test. h Representative immunoblot and i Quantification of expression levels of selected mitochondrial proteins. Ponceau staining is used as loading control. Data is shown as mean ± s.d. fold change to WT from n = 3–4 independent experiments, and individual replicates are plotted as dots. P values represent two-tailed unpaired Student’s t -test.

Article Snippet: SQSTM1/p62 antibody (Santa Cruz, sc-28359) and normal mouse IgG (Santa Cruz, sc-2025) were added separately to 1 mg of total protein at 2 μg and 0.25 μg per reaction, respectively, and incubated at 4 o C on rotation overnight.

Techniques: Membrane, Two Tailed Test, Staining, Western Blot, Expressing, Control

Journal: Nature Communications

Article Title: Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62

doi: 10.1038/s41467-025-60857-4

Figure Lengend Snippet: a Representative immunoblot of GFP showing EGFP and EGFP-p62 overexpression in diploid parental (WT) cells. b Representative confocal images of p62 (magenta) and EGFP (green) foci visualization in WT cells transiently transfected with EGFP and EGFP-p62 plasmids. DAPI (blue) is used as a nuclear stain. Images are collapsed from Z-stacks. Scale bar is 10 µm. Quantifications of ( c ) p62 foci number per cell surface area in µm 2 , d p62 foci size per cell surface area in µm 2 , and e Pearson correlation coefficient for p62-TOMM20 colocalization in respective samples. Violin plots indicate mean and distribution of values, while dots represent individual cells from n = 3 independent experiments. P -values represent two-tailed unpaired t test with Welch’s correction. Heatmaps showing ( f ) p62 foci number per cell surface area in µm 2 , g p62 foci size per cell surface area in µm 2 , and h Pearson correlation coefficient for p62-TOMM20 colocalization in 13/3, 3/3 and 5/4 polysomic cell lines transiently transfected with HSP27, HSP90α-Flag, EGFP-HSF1 and constitutively active HSF1 (see Supplementary Fig. ). Values represent fold changes to mock transfected cells (Ctrl.). P values represent non-parametric ANOVA followed by Dunn’s multiple comparisons test ( f and g ) and one-way ANOVA followed by Sidak’s multiple comparisons test ( h ). * p < 0.05, *** p < 0.001, **** p < 0.0001 (for exact p -values, see Supplementary Fig. ). i – k Quantification of the aggresome dye fluorescence intensity of 13/3, 3/3, and 5/4 cells transiently transfected with HSP27, HSP90α-Flag, EGFP-HSF1 and constitutively active HSF1. Data is shown as mean ± s.e.m fold change to mock transfected cells (Ctrl.) from n = 3 independent experiments, and individual replicates are plotted as dots. P values represent two-tailed unpaired Student’s t -test.

Article Snippet: SQSTM1/p62 antibody (Santa Cruz, sc-28359) and normal mouse IgG (Santa Cruz, sc-2025) were added separately to 1 mg of total protein at 2 μg and 0.25 μg per reaction, respectively, and incubated at 4 o C on rotation overnight.

Techniques: Western Blot, Over Expression, Transfection, Staining, Two Tailed Test, Fluorescence

Journal: Nature Communications

Article Title: Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62

doi: 10.1038/s41467-025-60857-4

Figure Lengend Snippet: a Schematic depiction of the process of posttranslational protein import into mitochondria. Most mitochondrial proteins are synthesized as precursors by cytosolic ribosomes and guarded by chaperones in their unfolded state for import into mitochondria, where the pre-sequences are cleaved, and the proteins folded into their native state. b Immunoblot of selected mitochondrial proteins (from Fig. ) showing precursor and mature forms. Ponceau staining is used as a loading control. *Degradation product. c Representative images showing import kinetic of synthesized 35 S-methonine-labeled su9-DHFR into mitochondria isolated from WT, 13/3, 3/3 and 5/4 cells. The su9-DHFR precursor is processed upon reaching the matrix (i.e., mature form). CCCP, which depletes mitochondrial membrane potential thereby preventing import, is used as a negative control. 20% of the synthesized substrate (precursor) is loaded for comparison. All samples are resolved by SDS-PAGE and visualized by autoradiography. d Quantification of the import kinetic from ( c ). Data represents mean ± s.e.m. of n = 4 (for 13/3, 3/3 and 5/4 cell lines) and n = 12 (for WT cell line; four for each polysomic cell line) independent import assays. Each polysomic cell line is run alongside the parental cell line per experiment as shown in ( c ). The data is normalized to WT, t = 10 mins (set to 100%). P -values represent one-tailed paired t -test. e Proposed model of aneuploidy-induced disruptions of cellular proteostasis and their effect on mitochondria. The gain of extra chromosome leads to translation of excess proteins, which places a severe burden on cytosolic proteostasis. Aberrant cytosolic proteostasis due to the expression of surplus proteins limits chaperone-mediated activity, thereby impairing mitochondrial protein import and leading to the accumulation of mitochondrial precursor proteins in the cytosol, where they are sequestered into p62-positive bodies (right). The p62-positive bodies do not only reside in the cytosol, but also associate with the mitochondrial outer membrane and interfere with mitochondrial functions. Overexpression of protein folding factors decreases the formation of p62-positive bodies and their association with mitochondria.

Article Snippet: SQSTM1/p62 antibody (Santa Cruz, sc-28359) and normal mouse IgG (Santa Cruz, sc-2025) were added separately to 1 mg of total protein at 2 μg and 0.25 μg per reaction, respectively, and incubated at 4 o C on rotation overnight.

Techniques: Synthesized, Western Blot, Staining, Control, Labeling, Isolation, Membrane, Negative Control, Comparison, SDS Page, Autoradiography, One-tailed Test, Expressing, Activity Assay, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Synthesis, target analysis, and cerebroprotective effects of novel imide antioxidants via the Nrf2/HO-1 pathway in cerebral ischemia-reperfusion injury

doi: 10.3389/fphar.2025.1552717

Figure Lengend Snippet: Z3-mediated activation of the sestrin 2/p62/Nrf2/HO-1 signaling pathway in PC12 cells. (A) Immunofluorescence of cells pre-incubated with 10 μmol·L −1 Z3 or TBHQ for 6 h and stained with Nrf2 antibody (red) and DAPI (blue), showing the nuclear translocation of Nrf2. (B,C) Western blotting of sestrin 2, p62, and HO-1 in cells treated with Z3 for 24 h (D) MTT viability assays of cells pretreated with ZnPP for 1 h prior to the addition of Z3 and stimulation with H 2 O 2 . Data expressed as means ± SD (n = 3); #### P < 0.0001; ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The anti-p62 (Cat# sc-48402) and anti-Nrf2 (Cat# sc-365949) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, United States), and the anti-mouse IgG (Cat# 7076S) antibody was from Cell Signaling Technology (Danvers, MA, United States).

Techniques: Activation Assay, Immunofluorescence, Incubation, Staining, Translocation Assay, Western Blot