Journal: Scientific Reports

Article Title: Analysis of the polycystin complex (PCC) in human urinary exosome–like vesicles (ELVs)

doi: 10.1038/s41598-020-58087-3

Figure Lengend Snippet: Analysis of the polycystins, fibrocystin and CEMIPS_TMEM2. ( a ) Structures of the proteins investigated, polycystin-1 (PC1), polycystin-2 (PC2), fibrocystin (Fibro) and cell surface hyaluronidase (CEMIPS_TMEM2). ( b ) Using peptide data from Elucidator (3.3.0.1.SP3.19) and re-analysis of Hogan et al . data , we focused on peptides that definitely came from the PCC and we selected peptides that were decreased or increased with an uncorrected t –test p–value < 0.01. For proteins PC1, PC2, fibrocystin and CEMIPS_TMEM2, peptide starting positions were plotted on the y axis versus the log 2 of the intensity ratio PKD1 /normal, with point area scaling with intensity (ion current) and color indicating the gel slice. Gel slice A, 270 kDa–500 kDa; B, 140 kDa–27 kDa; C, 90 kDa–140 kDa; D, 70 kDa–90 kDa; E, 55 kDa–70 kDa; F, 40 kDa–55 kDa; G, 32 kDa–40 kDa; H, 24–32 kDa; I, 15 kDa–24 kDa; and J, 10 kDa–15 kDa . Data in Suppl Database .csv.

Article Snippet: PC2 antibodies (H-280): sc-25749 rabbit polyclonal to 689..968 aa (Santa Cruz), YCE2 Polycystin-2 Antibody (YCE2): sc-47734 (IgG2a κ ) .

Techniques:

Journal: Scientific Reports

Article Title: Analysis of the polycystin complex (PCC) in human urinary exosome–like vesicles (ELVs)

doi: 10.1038/s41598-020-58087-3

Figure Lengend Snippet: Western and MS/MS analysis of the PCC in ELVs. ( a ) Conventional 3–8% SDS PAGE western blots of highly purified ELVs, probed with monoclonal antibodies again the N–terminus of PC1, N–terminus of fibrocystin and PC2 plus a polyclonal rabbit anti–CEMIPS_TMEM2. All proteins are sensitive to PNGase F but resistant to Endo H showing that they have Golgi type carbohydrate modifications and are by definition mature. ( b ) Native gels performed with a mild detergent, lauryl maltoside neopentylglycol and a detergent that can interact with cholesterol, taurocholic acid. Amphipol A8–35 was used as a charge conferring molecule. These data showed that PC2 and the C–terminus of PC1 migrate only a short way into the gel about 5 mm (c2 MDa delineated by the black line) and a small amount of N–terminal PC1 and fibrocystin ectodomain migrate at c300 kDa. The same phenomenon is seen in ‘blue native’ electrophoresis (but has high background due to the Coomassie dye interacting with PVDF). ( c ) In the presence of varying amounts of SDS 0.2–0.8% (non reducing incubated on ice 20 min), the bulk of N–terminal PC1 and fibrocystin were freed from the complex and migrate at c300 kDa. At low SDS levels the PC1 has a complex migration pattern implying that it exists in multiple complexes. PC2 runs in the high molecular 2 MDa complex and as a faint dimer and monomer, black asterisks. Most CEMIPS_TMEM2 resolves at c200 kDa. ( d ) MS/MS analysis of 16 gel slices taken across the region containing the N–terminal PC1 and fibrocystin ectodomains shows that they do not resolve in the same slice and likely do not interact under these conditions. The PC1 ectodomain is smaller than fibrocystin’s ectodomain. Original westerns are in Supplemental Data Fig. .zip and MS/MS data in Suppl Database .csv.

Article Snippet: PC2 antibodies (H-280): sc-25749 rabbit polyclonal to 689..968 aa (Santa Cruz), YCE2 Polycystin-2 Antibody (YCE2): sc-47734 (IgG2a κ ) .

Techniques: Western Blot, Tandem Mass Spectroscopy, SDS Page, Purification, Bioprocessing, Electrophoresis, Incubation, Migration

Journal: iScience

Article Title: Organoid platinum-resistance model identifies KRT17 as a biomarker of targeted therapy in ovarian cancer

doi: 10.1016/j.isci.2025.113999

Figure Lengend Snippet: Post-platinum organoid transcriptomic and proteomic characterization (A) PCA plot of mRNA sequencing of control vs. GPC lines, for two donors, showing patient-specific characteristics dominating expression profiles of the organoids. (B) Volcano plot of top 25 regulated candidate genes (Log2 Foldchange, p-adjust <0.05). (C) Heatmap analysis of the top 25 regulated genes (p-adjust <0.05) illustrates that, despite great differences between donors, samples cluster according to the treatment. (D) GSEA for GO Biological processes in untreated vs. ptGPC1 lines identifies enrichment in pathways regulating the cytoskeleton and differentiation. (P-adjust <0,05). (E) PCA plot of mass spectrometry measurements ( n = 4 independent technical replicates for two sets of lines, ctrl vs. GPC, total 16 samples), confirms that patient-specific characteristics are defining components in the global proteome profile. (F) GSEA of top enriched pathways for proteome (NES) identifies enrichment in downregulation for pathways regulating cell adhesion and epithelium development. (G) ES plots for cell adhesion in transcriptome and proteome data. (H) Loss of vimentin (magenta) IF staining visualized by confocal imaging in ptGPC HGSOC_14 organoids in comparison to the control line. Scale bar, 25 μm. (I) Quantification plot, showing differences in mean fluorescence intensity of different fields of view. Error bars, mean ± SEM. (J) qPCR validation of the induction of KRT17 (red) in ptGPC1 organoids ( n = 4 independent biological samples, Error bars, mean ± SEM) and (K), the confocal image representative of stainings from two different WT/ptGPC pairs, confirms an increase in KRT17 expression. Error bars, mean ± SEM. Scalebar, 20 μm.

Article Snippet: Cytokeratin 17 (KRT17) Human Tagged Lenti ORF Clone , OriGene , Cat#RC201619L1V.

Techniques: Sequencing, Control, Expressing, Mass Spectrometry, Staining, Imaging, Comparison, Fluorescence, Biomarker Discovery

Journal: iScience

Article Title: Organoid platinum-resistance model identifies KRT17 as a biomarker of targeted therapy in ovarian cancer

doi: 10.1016/j.isci.2025.113999

Figure Lengend Snippet: Platinum hyper-resistance is induced by KRT17 editing (A) qPCR of individual KRT17 exons, in control vs. crKRT17 edited lines, shows the loss of expression of exon 4 ( n = 3 independent biological replicates, Error bars, mean ± SEM). (B) Western blot for KRT17 confirms the expression of the truncated protein. (C) Comparative phase contrast images after ODR2-test for WT crKRT17 lines illustrating long-term expansion potential after sustaining 25 μM and 100 μM challenge in P0. Scale bars, 500 μm. (D) Bar plot summarizing the shift in GPC levels and a strong increase in the crKRT17 line (μM). (E) KRT17 RNA expression by qPCR of treated and untreated, WT and crKRT17 lines, illustrates strong induction in the expression of KRT17 in crKRT17 organoids ( n = 3 independent biological replicates). Error bars, mean ± SEM. (F) Mass spectrometry data show a significantly lower or absent MS/MS intensity of the DAEDWFFSK peptide, corresponding to the coding region of exon 4, in edited organoid lines compared to total KRT17 protein intensity ( n = 4 independent biological samples), confirming the overexpression of the remaining portion of the protein in ptGPC2 organoids. (G) Confocal imaging validates a strong increase in KRT17 levels (red) and (H), KRT5 levels (magenta). Scalebars, 20 μm. (I) Redistribution of Occludin staining signal (magenta) to lateral membranes in crKRT17 ptGPC2 organoids indicates loss of polarity. Scalebar, 50 μm and 25 μm for zoomed-in images (lower right). (J) Western blot for KRT17 expression in FL-KRT17 and control lines, with Flag and endogenous KRT17 antibody, confirms the expression of the exogenous construct as well as the induction of endogenous protein in response to carboplatin. (K) Quantification of the ODR1-test of FL-KRT17 vs. lenti-ctrl organoids showing the increase in organoid counts and rise in GPC. GPC1 = 25 μM (lenti-ctrl) and GPC1 = 50 μM (FL-KRT17). (L) Analysis of the distribution of organoid sizes reveals a group of significantly larger organoids (dotted circles) in ptGPC FL KRT17 culture, suggestive of increased progenitor potential. Error bars represent mean ± SEM of measurements in independent wells. P-values are calculated by the Student’s t test for the quantification of organoid counts, and the Mann-Whitney U test for comparison of differences in organoid sizes (∗ p < 0.05, ∗∗ p < 0.01; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.001).

Article Snippet: Cytokeratin 17 (KRT17) Human Tagged Lenti ORF Clone , OriGene , Cat#RC201619L1V.

Techniques: Control, Expressing, Western Blot, RNA Expression, Mass Spectrometry, Tandem Mass Spectroscopy, Over Expression, Imaging, Staining, Construct, MANN-WHITNEY, Comparison

Journal: iScience

Article Title: Organoid platinum-resistance model identifies KRT17 as a biomarker of targeted therapy in ovarian cancer

doi: 10.1016/j.isci.2025.113999

Figure Lengend Snippet: Prognostic K-score as a stratification for PIK3/Akt-targeted therapy (A) Images of KRT17 immunohistochemical staining representing TMA cores of 5 exemplary patients displaying different levels of expression. Scale bar 200 µm. (B) Kaplan-Meier analysis in the patient cohort of advanced HGSOC cases ( n = 376). K-score cutoff 5 reveals significantly shorter OS ( p -value (Log Rank) = 0.0043) and (C), PFS ( p -value (Log Rank) = 0.008) for the group of patients with KRT17 high . (D) Multivariate Cox regression analysis for OS and (E), for PFS, confirms the prognostic value of the K-score as an independent risk factor (HR 1.84, p -value = 0.008 for OS, and HR 1.58, p -value = 0.024 for PFS). (F) Log-transformed dose-response curves for alpelisib in untreated and carboplatin-treated (ODR test) WT and crKRT17 lines show a strong increase in sensitivity in the lines expressing elevated KRT17 levels. Upper right panel: IC50 values (μM), obtained from two ( n = 2) independent repetitions of the assay performed with the same organoid lines. (G) Drug response curves and overview of IC50s of the analogous testing setup for afuresertib. (H) Alpelisib treatment of the FL-KRT17 expressing HGSOC_14 line confirms the capacity of KRT17 to directly modify the sensitivity of the organoids to PI3 kinase pathway inhibition, and an overview of IC50s of independent repetitions of the assay (upper right). Error bars represent mean ± SEM of technical triplicates.

Article Snippet: Cytokeratin 17 (KRT17) Human Tagged Lenti ORF Clone , OriGene , Cat#RC201619L1V.

Techniques: Immunohistochemical staining, Staining, Expressing, Transformation Assay, Inhibition