Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: UNC13D regulates the migration of pancreatic cancer cells via focal adhesion (FA) turnover. A , B Boyden chamber and wound healing assay of cell migration capacity upon UNC13D knockdown in ASPC-1 and PANC-1. Data are presented as the mean ± SD. Two-tailed unpaired Student T test: ** p < 0.01 and *** p < 0.001. The quantification data shown are representative of three independent experiments. C Analysis of cell invasion capacity upon UNC13D knockdown in MIA PaCa-2 and PANC-1 cells. The quantification data are presented as the mean ± SD of three independent experiments. Two-tailed unpaired Student T test: ** p < 0.01. D Gene set enrichment assay (GSEA) of differentially expressed genes (DEGs) between UNC13D-high vs -low groups in the TCGA cohort indicates the enrichment of the integrin pathway and FA pathway. E FA disassembly assay using nocodazole (NDZ) shows UNC13D regulates FA turnover of pancreatic cancer cells. UNC13D knockdown slows FA disassembly. CAPAN-1 shSCR or shUNC13D were starved, left untreated or incubated with 10 μM nocodazole. After 4 h, nocodazole was washed out at different times to allow microtubule regrowth to trigger FA disassembly. The cells were then fixed and immunostained with anti-PXN antibody. Quantification of FAs number was obtained from an average of ten cells of each condition from three independent experiments. Data were shown as mean ± SD of three independent experiments. Two-tailed unpaired Student T test: * p < 0.05. Scale bar, 10 μm. F FAK autophosphorylation and its total form were evaluated by western blot at different times of nocodazole washed-out as in E . FAK autophosphorylation was quantified and normalized to total FAK expression

Article Snippet: To generate stable knockdown cell lines, CAPAN-1 cells were transfected with shSCR or shUNC13D expression constructs pKAR1/PUR (Addgene, Cat No. #23,105) using Lipofectamine 3000 reagent.

Techniques: Migration, Wound Healing Assay, Knockdown, Two Tailed Test, Incubation, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: RAB11-UNC13D-FAK axis in recycling endosomes regulates the phosphorylation of FAK. A UNC13D knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by p-FAK, p-PXN, FAK, and PXN. Data are presented as the mean ± SD. The experiments were independently performed at least three times. Statistical analysis was performed using Student’s unpaired T test. ** p < 0.01, *** p < 0.001. B Co-immunoprecipitation analysis of the binding between exogenous Flag-UNC13D and HA-FAK in co-transfected HEK293 cells indicates the direct interaction of them. IgG was incubated with cell lysates as negative controls. C Immunoprecipitation of endogenous FAK from CAPAN-1 and PANC-1 cells confirms the binding between endogenous UNC13D and FAK. D Immunoprecipitation of endogenous RAB11 from PANC-1 overexpressing Flag-UNC13D cells indicates the binding between UNC13D and endogenous RAB11. E Co-immunoprecipitation analysis of the binding among RAB11, UNC13D, and FAK indicates the formation of RAB11-UNC13D-FAK complex. F UNC13D knockdown decreased the binding of RAB11 with FAK. RAB11 was immunoprecipitated from PANC-1 expressing shUNC13D or shSCR to examine the dependency of the interaction between RAB11 and FAK on UNC13D. G Co-localization of FAK, UNC13D, ITGB1, and RAB11 in PANC-1 cells. GFP-FAK, mOrange-UNC13D, and mCerulean-Rab11A cells were transduced. For the active integrin staining, 12G10 anti-ITGB1 was used. The data shown are representative of three independent experiments. Scale bar: 10 μm. H RAB11A knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by pFAK, pPaxillin (p-PXN), FAK, and Paxillin (PXN). The quantification of western blotting in ( G ) is presented as the mean ± SD of three independent experiments. Student’s unpaired T test, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: To generate stable knockdown cell lines, CAPAN-1 cells were transfected with shSCR or shUNC13D expression constructs pKAR1/PUR (Addgene, Cat No. #23,105) using Lipofectamine 3000 reagent.

Techniques: Knockdown, Immunoprecipitation, Binding Assay, Transfection, Incubation, Expressing, Staining, Western Blot

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: UNC13D regulates the migration of pancreatic cancer cells via focal adhesion (FA) turnover. A , B Boyden chamber and wound healing assay of cell migration capacity upon UNC13D knockdown in ASPC-1 and PANC-1. Data are presented as the mean ± SD. Two-tailed unpaired Student T test: ** p < 0.01 and *** p < 0.001. The quantification data shown are representative of three independent experiments. C Analysis of cell invasion capacity upon UNC13D knockdown in MIA PaCa-2 and PANC-1 cells. The quantification data are presented as the mean ± SD of three independent experiments. Two-tailed unpaired Student T test: ** p < 0.01. D Gene set enrichment assay (GSEA) of differentially expressed genes (DEGs) between UNC13D-high vs -low groups in the TCGA cohort indicates the enrichment of the integrin pathway and FA pathway. E FA disassembly assay using nocodazole (NDZ) shows UNC13D regulates FA turnover of pancreatic cancer cells. UNC13D knockdown slows FA disassembly. CAPAN-1 shSCR or shUNC13D were starved, left untreated or incubated with 10 μM nocodazole. After 4 h, nocodazole was washed out at different times to allow microtubule regrowth to trigger FA disassembly. The cells were then fixed and immunostained with anti-PXN antibody. Quantification of FAs number was obtained from an average of ten cells of each condition from three independent experiments. Data were shown as mean ± SD of three independent experiments. Two-tailed unpaired Student T test: * p < 0.05. Scale bar, 10 μm. F FAK autophosphorylation and its total form were evaluated by western blot at different times of nocodazole washed-out as in E . FAK autophosphorylation was quantified and normalized to total FAK expression

Article Snippet: AsPC-1, MIA PaCa-2, CAPAN-1, and PANC-1cells were transfected with siRNA or cDNA constructs using DharmaFECT1 (Thermo Scientific, Lafayette, CO, USA) or Lipofectamine 3000 (Cat #L3000075, Thermo Fisher Scientific, Lafayette, CO, USA).

Techniques: Migration, Wound Healing Assay, Knockdown, Two Tailed Test, Incubation, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Recycling machinery of integrin coupled with focal adhesion turnover via RAB11-UNC13D-FAK axis for migration of pancreatic cancer cells

doi: 10.1186/s12967-024-05630-9

Figure Lengend Snippet: RAB11-UNC13D-FAK axis in recycling endosomes regulates the phosphorylation of FAK. A UNC13D knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by p-FAK, p-PXN, FAK, and PXN. Data are presented as the mean ± SD. The experiments were independently performed at least three times. Statistical analysis was performed using Student’s unpaired T test. ** p < 0.01, *** p < 0.001. B Co-immunoprecipitation analysis of the binding between exogenous Flag-UNC13D and HA-FAK in co-transfected HEK293 cells indicates the direct interaction of them. IgG was incubated with cell lysates as negative controls. C Immunoprecipitation of endogenous FAK from CAPAN-1 and PANC-1 cells confirms the binding between endogenous UNC13D and FAK. D Immunoprecipitation of endogenous RAB11 from PANC-1 overexpressing Flag-UNC13D cells indicates the binding between UNC13D and endogenous RAB11. E Co-immunoprecipitation analysis of the binding among RAB11, UNC13D, and FAK indicates the formation of RAB11-UNC13D-FAK complex. F UNC13D knockdown decreased the binding of RAB11 with FAK. RAB11 was immunoprecipitated from PANC-1 expressing shUNC13D or shSCR to examine the dependency of the interaction between RAB11 and FAK on UNC13D. G Co-localization of FAK, UNC13D, ITGB1, and RAB11 in PANC-1 cells. GFP-FAK, mOrange-UNC13D, and mCerulean-Rab11A cells were transduced. For the active integrin staining, 12G10 anti-ITGB1 was used. The data shown are representative of three independent experiments. Scale bar: 10 μm. H RAB11A knockdown in CAPAN-1 and PANC-1 cells decreased FAK/PXN signaling, which was measured by pFAK, pPaxillin (p-PXN), FAK, and Paxillin (PXN). The quantification of western blotting in ( G ) is presented as the mean ± SD of three independent experiments. Student’s unpaired T test, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: AsPC-1, MIA PaCa-2, CAPAN-1, and PANC-1cells were transfected with siRNA or cDNA constructs using DharmaFECT1 (Thermo Scientific, Lafayette, CO, USA) or Lipofectamine 3000 (Cat #L3000075, Thermo Fisher Scientific, Lafayette, CO, USA).

Techniques: Knockdown, Immunoprecipitation, Binding Assay, Transfection, Incubation, Expressing, Staining, Western Blot

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a Volcano plot showing the suppression of NLRP4 led to a greater susceptibility to olaparib in pancreatic cancer cell lines. b Immunoblot of NLRP4 in control and NLRP4-knockdown BxPC-3 and Capan-1 cells. BxPC-3 and Capan-1 cells were stably transfected with shNC, shNLRP4-1 and shNLRP4-2, and then MTS assays ( c , d ) and colony formation assays ( e , f ) were performed. *** p < 0.001. ** p < 0.01. The analysis of significant differences was performed with Student’s t test. g – j Control and NLRP4-knockdown BxPC-3 and Capan-1 cells were injected into the left flank of nude mice. Tumor volumes were measured every 3 days. Tumors were harvested on day 21, photographed and weighed. Tumor weights are shown in ( h ), BxPC-3 tumor volumes are shown in ( i ), and Capan-1 tumor volumes are shown in ( j ). Data are shown as the mean ± SD ( n = 5). *** p < 0.001. The analysis of significant differences was performed with Student’s t test. *** p < 0.001. ** p < 0.01. k , l Flow cytometry analysis of annexin V/7-ADD staining in the indicated cell lines. Data are presented as the mean ± SD from three independent experiments. The analysis of significant differences was performed with Student’s t test.

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Western Blot, Control, Knockdown, Stable Transfection, Transfection, Injection, Flow Cytometry, Staining

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a , b MTS assays were performed to calculate the IC50 of olaparib in the indicated cells. c , d The indicated cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). MTS assays were performed to investigate the effect of NLRP4 on olaparib resistance. The analysis of significant differences was performed with one-way ANOVA. *** p < 0.001. e The indicated cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). Colony formation assays were performed to investigate the effect of NLRP4 on olaparib resistance. The analysis of significant differences was performed with Student’s t test. *** p < 0.001. ** p < 0.01. f , g Flow cytometry results for annexin V/7-ADD staining in the indicated cell lines after exposure to DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). The analysis of significant differences was performed with Student’s t test. *** p < 0.001. h Control or NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib (500 nM) for RNA sequencing and were subjected to GO pathway enrichment analysis. i Co-IP experiments were conducted in NLRP4 OE cells, followed by LC‒MS/MS analysis. j Control or NLRP4-knockdown BxPC-3 and Capan-1 cells were treated with the indicated dose of olaparib for 48 h. Western blotting was performed with the indicated antibodies.

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Flow Cytometry, Staining, Control, Knockdown, RNA Sequencing Assay, Co-Immunoprecipitation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a – c Representative immunofluorescence micrographs and quantification of γ-H2AX foci in the indicated cells with or without olaparib treatment (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). The analysis of significant differences was performed with Student’s t test. ** p < 0.01, * p < 0.05. Scale bar, 5 μm. d Control or NLRP4-knockdown BxPC-3 and Capan-1 cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h. Western blotting was performed with the indicated antibodies. e – g Representative comet assay micrographs and quantification of tail moments in the indicated cells with or without olaparib treatment (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). The analysis of significant differences was performed with Student’s t test. ** p < 0.01, * p < 0.05. h Representative immunofluorescence micrographs showing BRCA1 and Rad51 IRIF in the indicated cell lines at 6 h after olaparib treatment (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). Scale bar, 5 μm. i Changes in DNA repair genes in control and NLRP4-knockdown Capan-1 cells after olaparib treatment (500 nM olaparib for Capan-1 cells).

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Immunofluorescence, Control, Knockdown, Western Blot, Single Cell Gel Electrophoresis

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a – c Control or NLRP4-knockdown BxPC-3 and Capan-1 cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h. Western blotting analysis and quantification were performed. The analysis of significant differences was performed with Student's t test.** p < 0.01. d – f Effect of NLRP4 on the levels of autophagic flux. The mRFP-GFP-LC3 plasmid was transfected into the indicated cells for 24 h, and then the cells were treated with olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h. Representative images were obtained by laser scanning confocal microscopy. The average fluorescence intensity of autophagic lysosomes (yellow dots in the merged images) and autophagic lysosomes (red in the merged images) in individual cells was quantified. Scale bar, 5 μm. The analysis of significant differences was performed with Student's t test. *** p < 0.001. g – i TEM-based ultrastructure analysis (autophagosomes) in the indicated cells. The analysis of significant differences was performed with Student’s t test. *** p < 0.001.

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Control, Knockdown, Western Blot, Plasmid Preparation, Transfection, Confocal Microscopy, Fluorescence

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a Cells treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h were incubated with an ROS indicator. Representative images were obtained. b , c Cells treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h were incubated with an ROS indicator, and fluorescence intensity was assessed with flow cytometry. The analysis of significant differences was performed with Student’s t test. *** p < 0.001. d , e Cells treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h were incubated with JC-1 working solution, and fluorescence intensity was assessed with flow cytometry. The analysis of significant differences was performed with Student’s t test. *** p < 0.001. f – h Cells treated with olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) plus DMSO or MitoQ for 48 h were incubated with MitoSOX. Representative images were obtained, and the fluorescence intensity was assessed with flow cytometry. The analysis of significant differences was performed with Student’s t test. *** p < 0.001. i Changes in ROS-related genes in control and NLRP4-knockdown Capan-1 cells after olaparib treatment (500 nM olaparib for Capan-1 cells). Scale bar, 5 μm. j Western blotting analysis was performed for the indicated antibodies.

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Incubation, Fluorescence, Flow Cytometry, Control, Knockdown, Western Blot

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a Silver staining of NLRP4-interacting proteins. b Molecular dynamics simulation-derived model structure of NLRP4 bound to Sirt7. c Cell lysates were immunoprecipitated with the indicated antibodies. d , e Western blot analysis was performed using the indicated antibodies. f – l ChIP-qPCR assay to assess H3K18ac status at the NOXO1 genomic region. The analysis of significant differences was performed with Student's t test. ** p < 0.01, *** p < 0.001. m qPCR analysis of NOXO1 expression. n Proposed model depicting the role of NLRP4-induced ROS.

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Silver Staining, Derivative Assay, Immunoprecipitation, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a , b Cells treated with olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) plus DMSO or MitoQ for 48 h were incubated with an ROS indicator, and fluorescence intensity was assessed with flow cytometry. The analysis of significant differences was performed with one-way ANOVA. *** p < 0.001. c – e The mRFP-GFP-LC3 plasmid was transfected into the indicated cells for 24 h, and then the cells were treated with olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) plus DMSO or MitoQ for 48 h. Representative images were obtained by laser scanning confocal microscopy. The average fluorescence intensity of autophagic lysosomes (yellow dots in the merged images) and autophagic lysosomes (red in the merged images) in individual cells was quantified. The analysis of significant differences was performed with Student’s t test. *** p < 0.001. Scale bar, 5 μm. f – h Representative immunofluorescence micrographs and quantification of γ-H2AX foci in the indicated cells treated with olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) plus MitoQ or DMSO for 48 h. The analysis of significant differences was performed by Student’s t test. Scale bar 5 μm. i , j The indicated cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) with or without MitoQ. MTS assays were performed. The analysis of significant differences was performed with one-way ANOVA. *** p < 0.001. k , l The indicated cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) with or without CQ. MTS assays were performed. The analysis of significant differences was performed with one-way ANOVA. *** p < 0.001.

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Incubation, Fluorescence, Flow Cytometry, Plasmid Preparation, Transfection, Confocal Microscopy, Immunofluorescence

Journal: Cell Death & Disease

Article Title: NLRP4 renders pancreatic cancer resistant to olaparib through promotion of the DNA damage response and ROS-induced autophagy

doi: 10.1038/s41419-024-06984-0

Figure Lengend Snippet: a Representative immunofluorescence micrographs of γ-H2AX foci in the indicated cells with or without olaparib treatment (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). b TEM-based ultrastructure analysis (autophagosomes) in the indicated cells. c Cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h. Western blotting analysis was performed for the indicated antibodies. d , e Cells treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells) for 48 h were incubated with an ROS indicator, and fluorescence intensity was assessed with flow cytometry. The analysis of significant differences was performed with one-way ANOVA. *** p < 0.001. f , g The indicated cells were treated with DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). MTS assays were performed to investigate the effect of NLRP4 on olaparib resistance. The analysis of significant differences was performed with one-way ANOVA. *** p < 0.001. h Flow cytometry results for annexin V/7-ADD staining in the indicated cell lines after exposure to DMSO or olaparib (5 μM olaparib for BxPC-3 cells and 500 nM olaparib for Capan-1 cells). The analysis of significant differences was performed with Student’s t test. *** p < 0.001. i Schematic model showing that NLRP4 renders pancreatic cancer resistant to PARPi through the promotion of the DNA damage response and ROS-induced autophagy.

Article Snippet: Briefly, control and NLRP4-knockdown Capan-1 cells were treated with DMSO or olaparib for 48 h. The total RNA was isolated and extracted from the cells with an RNeasy Plus Mini Kit (QIAGEN).

Techniques: Immunofluorescence, Western Blot, Incubation, Fluorescence, Flow Cytometry, Staining