Journal: bioRxiv

Article Title: Stress adaptation pathways and HA–CD44 signaling maintain the survival of pancreatic cancer cells with centrosome amplification

doi: 10.64898/2026.01.24.701523

Figure Lengend Snippet: A) Centrosome amplification in PDAC cell lines Panc1 and Mia Paca-2. Top panel: Confocal microscopy images. Blue: DAPI, nuclei; Red: γ-tubulin, centrosomes. Bottom panel: Induction of PLK4 expression by doxycycline. GAPDH was used as loading control. B) PDAC cells sustain high levels of CA over time. C) Persistent CA reduces cell proliferation rates in PDAC cells. D) PDAC cells with CA exhibit increased sensitivity to GLS1 inhibition by CB-839. Left panel: Panc1 cells, Right panel: Mia Paca-2 cells. E-H) CB-839 treatment significantly decreases the colony-formation ability of PDAC cells with CA. (E) Panc1 cells (F) Mia Paca-2 cells. (G) BxPC-3 cells. (H) Quantification results of colony formation experiments. Statistical significances were measured by two-way ANOVA in B and H, by two-tailed t-test on C, by non-linear curve fitting in D. p values were reported on graphs.

Article Snippet: Human pancreatic ductal adenocarcinoma cell lines Panc1 (CRL-1469), MiaPaCa-2 (CRL-1420), BxPC-3 (CRL-1687), and U2OS osteosarcoma cell line (HTB-96) were obtained from ATCC.

Techniques: Amplification, Confocal Microscopy, Expressing, Control, Inhibition, Two Tailed Test

Journal: bioRxiv

Article Title: Stress adaptation pathways and HA–CD44 signaling maintain the survival of pancreatic cancer cells with centrosome amplification

doi: 10.64898/2026.01.24.701523

Figure Lengend Snippet: A) Schematic representation of L-glutamine metabolism pathways and enzymes targeted by specific inhibitors in the following experiments. B) Diagram of the NRF2 signaling pathway and inhibitors targeting NRF2 and Keap1. C) CA increases intracellular ROS levels in Panc1, Mia Paca-2 and BxPC-3 cells. D) Quantification of ROS measurement results in 2C. Left panel: Changes in histogram median values. Right panel: Percentage of cells with high ROS levels. E) Induction of CA decreases GSH:GSSG ratios in PDAC cell lines. F) Induction of CA increases nuclear localization of NRF2 in Panc1 cells. GAPDH and Histone H3 blots represent cytoplasmic and nuclear fractionation. G) CA increases the Antioxidant Response Element (ARE)-mediated gene expression in Panc1 cells. H) Overview of the competition experiments performed in panels H-K. I-J) Treatment with CB-839, BSO and ML385 significantly reduces the viability of Panc1 cells with CA in in-vitro competition assays. K) CB-839 and ML385 treatments diminish the survival of Mia Paca-2 cells with CA in in vitro competition assays. L) Inhibition of SNAT1-mediated glutamine uptake reduces the viability of Panc1 cells with CA in in vitro competition assays. Statistical significances were measured by two-tailed t-test in D (left panel), and by two-way ANOVA in D (right panel), E, and I-L. Dots represent individual repeats. p values were reported on graphs.

Article Snippet: Human pancreatic ductal adenocarcinoma cell lines Panc1 (CRL-1469), MiaPaCa-2 (CRL-1420), BxPC-3 (CRL-1687), and U2OS osteosarcoma cell line (HTB-96) were obtained from ATCC.

Techniques: Fractionation, Gene Expression, In Vitro, Inhibition, Two Tailed Test

Journal: bioRxiv

Article Title: Stress adaptation pathways and HA–CD44 signaling maintain the survival of pancreatic cancer cells with centrosome amplification

doi: 10.64898/2026.01.24.701523

Figure Lengend Snippet: A) Schematic representation of metabolic enzyme focused CRISPR screen experiment design. B) Top depleted hits in dox+ and dox- cells compared to initial sample. Left panel: Scatterplot of beta scores for dox+ and dox- sample. Pink dots in the scatterplot represent genes with a beta score that increased after CA. Blue dots represent genes with a beta score that decreased after CA. Right panel: Rank plot showing the genes based on differential beta score in which dox- beta score is subtracted from the dox+ beta score. C) Top 50 differentially depleted genes in dox+ samples. Pink dots represent beta score in dox- comparison, blue dots represent beta score in dox+ comparison. D) GSEA analysis of CRISPR screen results. E) Comparison of differential beta score values of CRISPR screen with Panc1 DepMap essentialities. F) MCL clustering results of top differentially depleted metabolic genes in cells with CA. Genes that were not included in a cluster (singletons) and clusters contain less than three proteins were removed. G) Enrichment analysis of protein-protein interaction network. H-J) Pathway-specific differentially depleted genes in cells with CA. (H) Response to superoxide (GO:0000303). (I) UDP- N -acetylglucosamine metabolic process (GO:0006047). (J) Glycosaminoglycan biosynthetic process (GO:0006024). K) UMAP projection of TCGA PDAC data for selected genes. CIN25 and CA20 gene expression scores was shown on the left side plots. PLK4 and NEK2: CA20 genes; PRDX1 and DHFR: ROS elimination; UGDH and DPAGT1: N-glycan synthesis/nucleotide sugar metabolism. Gene expression Z-scores were used in plots.

Article Snippet: Human pancreatic ductal adenocarcinoma cell lines Panc1 (CRL-1469), MiaPaCa-2 (CRL-1420), BxPC-3 (CRL-1687), and U2OS osteosarcoma cell line (HTB-96) were obtained from ATCC.

Techniques: CRISPR, Comparison, Gene Expression, Glycoproteomics

Journal: bioRxiv

Article Title: Stress adaptation pathways and HA–CD44 signaling maintain the survival of pancreatic cancer cells with centrosome amplification

doi: 10.64898/2026.01.24.701523

Figure Lengend Snippet: A) 4-MU, tunicamycin and FR054 treatments increase DNA content of individual cells. B) Quantification of the G1-peak intensities in . C) Tunicamycin treatment significantly reduces proliferation of centrosome-amplified Panc1 and Mia Paca-2 cells compared to control. D) 4-MU treatment significantly reduces proliferation of centrosome-amplified Panc1, Mia Paca-2, and BxPC-3 cells compared to control. E) Long-term 4-MU treatment results in generation of multinucleated cells. Left panel: Inverted confocal images. Purple color shows DNA content of the cells, and orange color shows centrosomes and cell boundaries. Scale bar: 20 µm. Right panel: Quantification of cell area and nucleus area in pixel squares. F) Quantification of multinucleated giant cells in DMSO and 4-MU treated cells with CA. Left panel: Quantification of CA. Right panel: Quantification of multinucleated cells. G) CRISPR/Cas9 targeted disruption of UGDH gene results in generation of multinucleated cells. Purple color shows DNA content of the cells, and orange color shows centrosomes and cell boundaries. Scale bar: 20 µm. H) Quantification of multinucleated cells in sgAAVS1 and sgUGDH expressing centrosome-amplified and control cells. I) Quantification of multinucleated cells in sgAAVS1 and sgUGDH expressing HA or Vehicle treated cells with CA. Significance was determined by two-tailed t-test in C, by two-way ANOVA test in D, F, H, and I, by one-way ANOVA in E. Dots represent individual repeats. p values were reported on graph.

Article Snippet: Human pancreatic ductal adenocarcinoma cell lines Panc1 (CRL-1469), MiaPaCa-2 (CRL-1420), BxPC-3 (CRL-1687), and U2OS osteosarcoma cell line (HTB-96) were obtained from ATCC.

Techniques: Amplification, Control, CRISPR, Disruption, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Stress adaptation pathways and HA–CD44 signaling maintain the survival of pancreatic cancer cells with centrosome amplification

doi: 10.64898/2026.01.24.701523

Figure Lengend Snippet: A) Flow cytometry analysis showing elevated CD44 surface levels in centrosome-amplified PDAC cells. B) CD44-low Panc1 cells are more sensitive to CA. Left panel: FACS-sorted CD44-low and CD44-high populations in Panc1 and BxPC-3 cells. Right panel: Cell proliferation following different durations (3, 5, and 10 days) of CA. C) CD44-KO Panc1 cells are more sensitive to CA. Left panel: Flow cytometry confirming loss of CD44 expression in CD44-KO cells. Right panel: Cell proliferation following CA (5 and 10 days). D) Schematic of the competition assay design used in panel E. E) CD44-KO generates an increased vulnerability for UPR reduction in centrosome-amplified Panc1 cells. F) Representative confocal images of metaphase spindle organizations in Panc1 cells with CA. Top panel: bipolar clustered spindles; Bottom panel: multipolar spindles. G) Quantification showing reduced centrosome clustering in CD44-KO Panc1-PLK4 cells. H) CD44-low Panc1-PLK4 cells have increased multipolar spindle formation in metaphase. I) CD44-low Mia Paca-2-PLK4 cells have increased multipolar spindle formation in metaphase. Significance was determined by two-way ANOVA test in B, by one-way ANOVA in C, G, H, and I. Dots represent individual repeats. p values were reported on graph.

Article Snippet: Human pancreatic ductal adenocarcinoma cell lines Panc1 (CRL-1469), MiaPaCa-2 (CRL-1420), BxPC-3 (CRL-1687), and U2OS osteosarcoma cell line (HTB-96) were obtained from ATCC.

Techniques: Flow Cytometry, Amplification, Expressing, Competitive Binding Assay

Journal: Stem Cell Reviews and Reports

Article Title: Nestin and SOX2 Maintain self-renewal Abilities of Different Pancreatic Cancer Stem Cell Populations

doi: 10.1007/s12015-025-11006-3

Figure Lengend Snippet: Confirmation of siRNA-mediated KD of NES expression in Panc1 cell variants and SOX2 expression in Panc89 cell variants. 5 × 10 4 cells were subjected to siRNA-mediated KD of NES in Panc1 cell variants, SOX2 in Panc89 cell variants and double KD of NES/SOX2 in all cell variants or to CTRLsi transfection for 72 h. Afterward, the KD was confirmed by RT-qPCR (a, c, e, g) and IFS (b, d, f, h). Every analysis was performed with n = 3 independent experiments. Gene expression data of NES and SOX2 (a, c, e, g) are normalized to CTRLsi conditions. All data sets were tested for normal distribution using Shapiro-Wilk test, followed by one-way ANOVA for statistical significance. Significances are indicated by asterisk: p ≤ 0.033 = *, p ≤ 0.002 = **, p ≤ 0.001 = ***. Data are presented by mean with SD. Representative IFS images from n = 3 independent experiments are shown (b, d, f, h). Scale bars IFS = 1000 μm. (KD = knockdown; RT-qPCR = real time quantitative polymerase chain reaction, IFS = immunofluorescence staining, CTRLsi = control siRNA; SD = standard deviation; Holo = Holoclone)

Article Snippet: As a model for mesenchymal-like PDAC cells, the human cell line Panc1 was used (ATCC, Manassas, Virginia, US) originating from a primary tumor of a male PDAC patient.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Gene Expression, Knockdown, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Control, Standard Deviation

Journal: Stem Cell Reviews and Reports

Article Title: Nestin and SOX2 Maintain self-renewal Abilities of Different Pancreatic Cancer Stem Cell Populations

doi: 10.1007/s12015-025-11006-3

Figure Lengend Snippet: Gene expression analysis of EMT markers and plasticity modulators after single and double KD of NES and SOX2 in Panc1 and Panc89 cell variants. 5 × 10 4 cells were subjected to siRNA-mediated KD of NES in Panc1 cell variants (a), SOX2 in Panc89 cell variants (c), double KD of NES/SOX2 in Panc1 cell variants (b) and Panc89 cell variants (d) or to CTRLsi transfection (a-d) for 72 h. Afterward, gene expression of EMT markers (a-d top: CDH1 , L1CAM , VIM ) and plasticity modulators (a-d bottom: ZEB1 , ZEB2 , OVOL2 ) was determined via RT-qPCR. Analysis was performed with n = 3 independent experiments. Gene expression data of all genes of interest were first normalized to the reference gene GAPDH and then normalized to CTRLsi conditions. All data sets were tested for normal distribution using Shapiro-Wilk test. Parametric data were tested by one-way ANOVA and are presented by mean with SD. Non-parametric data were tested by Kruskal-Wallis one-way ANOVA on ranks and are presented as median with interquartile range. Significances are indicated by asterisk: p ≤ 0.033 = *, p ≤ 0.002 = **, p ≤ 0.001 = ***. (CSC = cancer stem cell; EMT = Epithelial-to-Mesenchymal Transition; KD = knockdown; RT-qPCR = real time quantitative polymerase chain reaction; CTRLsi = control siRNA; SD = standard deviation; Holo = Holoclone)

Article Snippet: As a model for mesenchymal-like PDAC cells, the human cell line Panc1 was used (ATCC, Manassas, Virginia, US) originating from a primary tumor of a male PDAC patient.

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Knockdown, Real-time Polymerase Chain Reaction, Control, Standard Deviation

Journal: Stem Cell Reviews and Reports

Article Title: Nestin and SOX2 Maintain self-renewal Abilities of Different Pancreatic Cancer Stem Cell Populations

doi: 10.1007/s12015-025-11006-3

Figure Lengend Snippet: KD of NES in Panc1 cell variants and KD of SOX2 in Panc89 cell variants marginally impact cell growth but significantly decrease self-renewing properties. 5 × 10 4 cells were either left untreated (a) or were subjected to siRNA-mediated KD of NES in Panc1 cell variants (b, d), SOX2 in Panc89 cell variants (c, e), double KD of NES/SOX2 in all cell variants (c-e) or to CTRLsi transfection (b-e) for 72 h. Afterward, the number of viable cells was determined by Trypan blue staining (a-c) and cells were re-seeded with 400 cells/well for Panc1 cell variants or 200 cells/well for Panc89 cell variants for CFA in 12-well plates (d-e). The number of viable cells under KD of NES (b), SOX2 (b) or NES / SOX2 (c) was normalized to CTRLsi conditions. Data were tested for normal distribution by Shapiro-Wilk analysis, followed by one-way ANOVA and Tukey´s multiple comparison test. d-e) CFAs were monitored for 6–10 d, fixed with PFA and stained with crystal violet. CFA data were analyzed by two-way ANOVA and Tukey´s multiple comparison. Every analysis was performed with n = 3 independent experiments plus technical replicates and data are shown as mean with SEM. Significances are indicated by asterisk: p ≤ 0.033 = *, p ≤ 0.002 = **, p ≤ 0.001 = ***. (KD = knockdown; CTRLsi = control siRNA; CFA = colony formation assay; SEM = standard error of means; Holo = Holoclone; PFA = paraformaldehyde; SEM = standard error of means; Holo = Holoclone; No. = Number; w/o KD = without knockdown)

Article Snippet: As a model for mesenchymal-like PDAC cells, the human cell line Panc1 was used (ATCC, Manassas, Virginia, US) originating from a primary tumor of a male PDAC patient.

Techniques: Transfection, Staining, Comparison, Knockdown, Control, Colony Assay

Journal: Stem Cell Reviews and Reports

Article Title: Nestin and SOX2 Maintain self-renewal Abilities of Different Pancreatic Cancer Stem Cell Populations

doi: 10.1007/s12015-025-11006-3

Figure Lengend Snippet: siRNA-mediated KD of NES in Panc1 and SOX2 in Panc89 cell variants marginally impacts migration and invasion properties. 5 × 10 4 cells were subjected to siRNA-mediated KD of NES in Panc1 and SOX2 Panc89 cell variants or to CTRLsi transfection for 72 h. Afterward, transfected cells were detached and seeded in (a) Ibidi ® 2-well culture inserts (4 × 10 4 for Panc1; 3 × 10 4 for Panc89 cell variants) and migration was analyzed after 24 h for Panc1 and Panc89 cell variants, or in (b) 96-well ULA plates for spheroid formation (1 × 10 4 for Panc1 cell variants; 1.5 × 10 4 for Panc89 cell variants) and determination of invasion properties. Cell invasion (number of invasive fronts [#]; invasion distance [µm]) was analyzed after 72 and 120 h for Panc1 and Panc89 cell variants, respectively. Every analysis was performed with n = 3 independent experiments plus technical replicates and NES and SOX2 KD values, respectively, were normalized to CTRLsi conditions. All data sets were tested for normal distribution using Shapiro-Wilk test. Parametric data were tested by one-way ANOVA with Tukey´s multiple comparison and are presented by mean with SEM. Non-parametric data were tested by Kruskal-Wallis one-way ANOVA on ranks and are presented as median with interquartile range. Representative images from n = 3 independent experiments are shown (a, b; top). Scale bar a) = 200 μm, b) left = 400 μm, b) right = 200 μm. (KD = knockdown; CTRLsi = control siRNA; ULA = ultra-low-attachment plate; SEM = standard error of means; Holo = Holoclone)

Article Snippet: As a model for mesenchymal-like PDAC cells, the human cell line Panc1 was used (ATCC, Manassas, Virginia, US) originating from a primary tumor of a male PDAC patient.

Techniques: Migration, Transfection, Comparison, Knockdown, Control

Journal: Stem Cell Reviews and Reports

Article Title: Nestin and SOX2 Maintain self-renewal Abilities of Different Pancreatic Cancer Stem Cell Populations

doi: 10.1007/s12015-025-11006-3

Figure Lengend Snippet: siRNA-mediated KD of NES in Panc1 cell variants and SOX2 in Panc89 cell variants marginally impacts the response to cytostatic drugs. 5 × 10 4 cells were subjected to siRNA-mediated KD of NES in Panc1 and SOX2 Panc89 cell variants or to CTRLsi transfection for 72 h. Afterward, 1 × 10 3 cells/well of either cell variants and conditions were re-seeded in a 96-well plate and after 24 h, the cells were treated with Gemcitabine, 5-FU or Oxaliplatin for 72 h. Then, (a) Panc1 and (b) Panc89 cell variants were stained with Hoechst 33342 (1:5000) and PI (1:50) and imaged to determine the total number of cells (Hoechst-positive, nuclei count) and the number of dead cells (Hoechst/PI-positive, cell death), respectively. Every analysis was performed with n = 3 independent experiments. The nuclei count and cell death values after KD of NES and SOX2 , respectively, were normalized to CTRLsi conditions. All data sets were analyzed by two-way ANOVA and Tukey´s multiple comparison. Every analysis was performed with n = 3 independent experiments plus technical replicates and data are shown as mean with SEM. Significances are indicated by asterisk: p ≤ 0.033 = *, p ≤ 0.002 = **, p ≤ 0.001 = ***. (KD = knockdown; CTRLsi = control siRNA; 5-FU = 5-Fluorouracil; PI = propidium iodide; SEM = standard error of means; Holo = Holoclone)

Article Snippet: As a model for mesenchymal-like PDAC cells, the human cell line Panc1 was used (ATCC, Manassas, Virginia, US) originating from a primary tumor of a male PDAC patient.

Techniques: Transfection, Staining, Comparison, Knockdown, Control