yap plasmid  (Addgene inc)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: Soft matrix confines pancreatic cancer cells in cellular dormancy in association with autophagic flux, lysosomal biogenesis, and YAP1 degradation. ( A ) Schematic illustrating cell culture on 2D stifness or in 3D softness. ( B ) RNA-seq analysis of genes involved in YAP pathway in KPC cells cultured on 2D monolayers or embedded in 3D collagen ( N = 2). ( C ) Western blot analysis of KPC cells cultured on 2D monolayers or embedded in 3D collagen. ( D ) Western blot analysis of KPC cells embedded in 3D collagen at the indicated time points. ( E - G ) RNA-seq analysis of genes involved in lysosome biogenesis and autophagy pathways in KPC cells cultured on 2D monolayers or embedded in 3D collagen ( N = 2). ( H ) Lysosome biogenesis was visualized through confocal microscopy analysis of Lysotracker/Hoechst staining in BxPC3 cells cultured on 2D monolayers or embedded in 3D collagen. ( I ) Confocal microscopy analysis of autophagic flux of murine or human pancreatic cancer cells expressing LC3-tandemRFP-GFP in cells cultured on 2D monolayers or embedded in 3D collagen. The experiments were repeated 2–3 times, and typical results were presented. (J-K) Western blot analysis of KPC and KC cells cultured on 2D monolayers or embedded in 3D collagen. Scale bar = 25 μm

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Cell Culture, RNA Sequencing, Western Blot, Confocal Microscopy, Staining, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: Stromal softness induces YAP1 degradation in cancer cells through lysosomal flux ( A ) Western blot analysis of KPC cells, seeded on 2D monolayers and treated with the proteasome inhibitor MG132 at 0, 10, 20 and 50 µM for 6 h. ( B ) Western blot analysis of KPC cells cultured on 2D monolayers and treated with the lysosome inhibitor chloroquine at 0, 20 and 50 µM for 6 h. ( C ) Western blot analysis of cells embedded in 3D collagen, treated with DMSO, MG132, E64 and chloroquine for 12 h. ( D - E ) KPC cells were treated with MG132 (20 µM) or CQ (50 µM) for different periods of time (0, 3, 6–9 h), followed by Western blot analysis. ( F ) The knockdown efficiency of lenti-shRNA on CtsB and CtsL in KPC cells was determined by RT-qPCR analysis. ( G ) Western blot analysis of the cells transfected with the indicated shRNA and cultured on 2D monolayers or embedded in 3D collagen for 24 h. ( H - I ) Western blot analysis of KC cells over-expressing Flag-Cystatin A and Flag-Cystatin B in 3D collagen. ( J ) Western blot analysis of KPC cells embedded in 3D fibrin gel at 3 (0.6 kPa), 6, 9, 12, and 24 (2.6 kPa) mg/mL or seeded on the fibrin coated plastic for 24 h. The experiments were repeated more than twice, and typical results are presented. The values are presented as mean ± SD; *** p < 0.001 vs. sh-ctrl group; ### p < 0.001 vs. sh-ctrl group

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Western Blot, Cell Culture, Knockdown, shRNA, Quantitative RT-PCR, Transfection, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: Lysosome destinating CTSL digests YAP1 from the N-terminus ( A ) Western blot analysis of 293T cells embedded in 3D collagen or seeded on plastic for 24 h for expression of wild-type human YAP1 or the Hippo phosphorylation null variant (5SA) of YAP1. ( B ) Western blot analysis of expression of CTSB, CTSL and YAP1 in 293T cells embedded in 3D collagen. ( C ) Schematic representation of YAP1 and CTSL variant constructs. ( D ) CTSL-mediated degradation of YAP1 was analyzed by Western blot analysis of 293T cells expressing wild type CTSL or catalytic inactive CTSL (C138S) together with FLAG-YAP1 (tagged at the N-terminus). The C-terminal fragments of YAP1 and N-terminus cleaved histone H3 are indicated by arrows. ( E ) Western blot analysis of 293T cells expressing wild-type or the signal-peptide-deletion variants of CTSL together with Flag-YAP1. ( F ) Confocal immunofluorescence microscopy of HA tag in 293T cells expressing HA-CTSL, HA-M1-CTSL and HA-ΔSP-CTSL. Nuclei are marked by DAPI. ( G and H ) Western blot analysis of 293T cells expressing CTSL or CTSB together with YAP1-FLAG. C-terminal fragments of YAP1 are indicated by arrows. ( I ) In vitro degradation assay of the CTSL-mediated digestion of YAP1 was carried out by incubating purified His-tagged-YAP1 with increasing doses of purified human CTSL at pH 4.0 and 37 ℃ for 30 min. The levels of C-terminal fragments of YAP1 were determined by Western blot analysis. ( J and K ) In vitro degradation of YAP1 by CTSL was inhibited by E64 and chloroquine. Scale bar = 10 μm. Representative images of experiments repeated 2–3 times are shown

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Western Blot, Expressing, Phospho-proteomics, Variant Assay, Construct, Immunofluorescence, Microscopy, In Vitro, Degradation Assay, Purification

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: Stromal softness up-regulates PTEN to promote autophagic-lysosomal flux for YAP1 degradation ( A ) RT-qPCR analysis of Pten mRNA levels in KPC cells cultured on 2D monolayers or embedded in 3D collagen. ( B - C ) Western blot analysis of KPC or BxPC-3 cells cultured on 2D monolayers or embedded in 3D collagen. ( D ) Western blot analysis of KPC cells embedded in 3D fibrin gel at different stiffnesses (0.6 kPa to 1.3 kPa). ( E ) Western blot analysis of control (sh-GFP) or PTEN knockdown (PTEN shRNA) KPC cells embedded in 3D collagen. ( F ) Confocal imaging of Lysotracker/Hoechst staining in control (sh-GFP) or PTEN knockdown (PTEN shRNA) KPC cells embedded in 3D collagen. ( G and H ) Western blot analysis of control (sh-GFP), ATG5 knockdown (ATG5 shRNA) or ATG7 knockdown (ATG7 shRNA) BxPC-3 cells embedded in 3D collagen. Representative images of experiments repeated 2–3 times are shown. The values are presented as mean ± SD; *** p < 0.001. Scale bar = 25 μm

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Quantitative RT-PCR, Cell Culture, Western Blot, Control, Knockdown, shRNA, Imaging, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: PDAC tumorigenesis and metastasis are promoted in the fibrotic liver ( A ) Schematic representation of the experimental design used to determine the impact of liver fibrosis/stiffness on tumorigenicity. The mice were treated with or without TAA for 4 weeks, followed by inoculation of UN-KPC961 cells for 2 weeks ( n = 6). ( B ) Gross examination (upper row) and H&E staining (lower row) of control and tumor-bearing livers. Scale bar = 500 μm. ( C ) Quantitation of tumor numbers in normal or fibrotic liver sections. ( D ) H&E staining of normal or fibrotic liver sections, scale bar = 200 μm. ( E ) Immunohistochemical staining of type-I collagen, Ki67 and CK19 in normal or fibrotic liver sections, scale bar = 100 μm. ( F ) Immunohistochemical staining of YAP1 and CtsL in normal or fibrotic liver sections, scale bar = 100 μm. ( G ) Immunofluorescence staining of CtsL and YAP1 in PanIN and adjacent hepatic parenchyma. The PanIN ducts are indicated by brown dashed lines, and malignant cells are indicated by pink arrows, scale bar = 25 μm. The values are presented as mean ± SD; ** p < 0.01

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Staining, Control, Quantitation Assay, Immunohistochemical staining, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: High expression of lysosomal-cathepsins correlates with YAP1 down-regulation, less fibrosis and survival of PDAC patients ( A ) Representative immunohistochemistry images of YAP1 and CTSL staining in pancreatic tissues from PDAC patients ( n = 30 for PDAC, n = 29 for control). ( B ) Semi quantitation of CTSL staining in PDAC cells vs. the control cells was determined by densitometry analysis. ( C ) Fibrotic stiffness in human PDAC tissues was determined as expression of tissue transglutaminase 2 (TGM2) in adjacent to the tumor ducts by immunohistochemistry. ( D ) A typical image of human pancreatic cancer slide stained for CTSL, from the Human Protein Atlas database . Fibrotic stiffness is indicated by dashed green circles. ( E ) Decreased expression of CTSL is associated with poor prognosis in PDAC patients (from the Human Protein Atlas database ). The values are presented as mean ± SD; ** p < 0.01. Scale bar = 50 μm

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Expressing, Immunohistochemistry, Staining, Control, Quantitation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: The primers used for PCR amplification and subcloning

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Amplification, Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

doi: 10.1007/s00018-024-05466-y

Figure Lengend Snippet: Sequences of primers used for RT-qPCR analysis

Article Snippet: Through PCR, the cDNAs of CTSL (#11249), CTSB (#11250), YAP1 (#66853) and YAP5SA (#33096) from Addgene, were amplified, and confirmed by sequencing analysis.

Techniques: Sequencing