Fig 2: Integrin α1 is upregulated in Pkd1nl/nl and human kidneys and is expressed in the interstitial mesenchymeA) Heat map showing expression of all the integrin subunits detected in the Pkd1nl/nl kidney proteomics dataset. B) Box and violin plots showing human kidney scRNA-seq data for ITGA1, ITGA2, ITGA10 and ITGA11. C) Panels showing super-resolution microscopy images of human normal healthy kidney (top panels) and early stage ADPKD human kidney (bottom panels). Left panels are labelled for αSMA, middle panels are labelled for integrin α1 and right panels show the merge. Scale bar indicates 20 μm. D) Box and violin plots showing the extent of αSMA+ immunostaining (by area) in the indicated groupings. Each symbol represents a biological replicate, for each replicate three random snapshots were taken. E) Dot plot of ITGA1 expression in a scRNAseq dataset published by the kidney precision medicine project. For cell cluster abbreviation definitions, see Methods, MyoF, Myofibroblast; Fb, Fibroblast.

Fig 3: Uncovering the senescence‐associated surface proteome. (a) SA‐β‐Gal activity was analyzed in WI‐38 fibroblasts at ~PDL24 (proliferating, P), after reaching replicative exhaustion at ~PDL54 (RS), or after exposure to 10 Gy of ionizing radiation and culture for an additional 10 days (IRIS). (b) Workflow for cell‐surface protein biotinylation and isolation for proteomic analysis; prepared using BioRender. (c) Overlap of the cell surface proteins elevated in RS vs P (green) and IRIS vs P (pink) after processing as in panels (a, b). (d) Top significantly (FDR <0.05) enriched GO Molecular Function terms after ShinyGO analysis performed on the shared cell surface proteins upregulated in senescent cells. (e) Heatmap illustrating the magnitude of induction in a subset of surface proteins, comparing RS to P cells and IRIS to P cells that were prepared as explained in (a). In the heatmap, 5NTD, ICAM1, DPP4, NECT2, and ITA1 are CD73, CD54, CD26, CD112, and CD49A, respectively. (f) WI‐38 cells were processed as in (a), and the levels of CD49A, CD73, CD109, CD54, CD112, CD44, HLA‐ABC, CD26, and loading control β‐Actin (ACTB) were assessed in Input (whole‐cell proteins) and Surface (cell‐surface proteins) by western blot analysis. “Input”, 10 μg of the protein lysate before pulldown; “Surface”, 5% of the eluted protein volume after pulldown. As the lysis buffer did not fully solubilize membrane proteins, only “Surface” proteins are quantified (ImageJ). Data are representative of three independent replicates.

Fig 4: Immunoblots of GO Term-selected proteins show divergent phenotypes in isogenic COX17- and CTR1-null cells.A-C. Immunoblots of whole cell extracts from wild type (gray), two COX17 KO clonal lines (purple), and two CTR1 KO clonal lines (blue) probed for proteins involved in actin filaments or the cytoskeleton (MYLK, TALGN, ITGA1), components of the THO complex (THOC2, THOC5), the metalloreductase STEAP3, and secretory proteins (CTSB, CTSD, CHGB, and DBH; immature cathepsins are indicated with i). HSP90 was used as a loading control. Immunoblots were quantified by normalizing protein abundance to wild-type cells. The left column of images are from the same immunoblot and cropped for clarity. B. Quantification of COX17 KO protein abundance relative to wild type. Italicized numbers represent p values analyzed by unpaired mean difference two-sided permutation t-test. C. Quantification of COX17 and CTR1 KO protein abundance relative to wild-type cells. Italicized numbers represent p values analyzed by unpaired t-test with the following exceptions: MYLK and TGLN were analyzed by Fisher Exact Probability Test and CHGB was analyzed by Mann Whitney test. D. Immunofluorescence staining of WT and COX17 KO cells stained with the Golgi complex marker GM130 (green) and DAPI (blue) and quantification of Golgi area (pixels, each dot represents one cell). p value analyzed by unpaired t-test.