Journal: bioRxiv

Article Title: Developing potent and selective TBK1 molecular glue degraders for cancer immunotherapy

doi: 10.64898/2026.01.30.702304

Figure Lengend Snippet: (A) Western blot analysis of total TBK1 and phosphorylated TBK1 (p-TBK1) in PANC-1 cells treated with the TBK1 molecular glue CCT412020 (1 µM) in the presence or absence of TNF and/or IFNγ. (B) Cell viability assay in PANC-1 cells treated with the TBK1 molecular glue CCT412020 (1 µM) in the presence or absence of TNF (10 ng/ml)/IFNγ (5 ng/ml). Cells were treated for 48h, and viability was quantified following treatment. (C) Cell viability assay in MC38 hCRBN cells treated with TNF and the pan-caspase inhibitor emricasan to induce necroptosis, in the presence of either the TBK1 molecular glue CCT412020 (1 µM). Cell viability was quantified following treatment as indicated.

Article Snippet: MCF-7, HCC38, BT-549, MDA-MB-231, MDA-MB-468, HeLa, and PANC-1 cell lines were obtained from ATCC.

Techniques: Western Blot, Viability Assay

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