Journal: Journal of Translational Medicine

Article Title: PAFAH1B1 stabilization, through PUM2-mediated competitive binding with miRNAs on its’ 3’-UTR, takes active actions on ferroptosis to overcome the radioresistance to 125 I radioactive particles of pancreatic ductal adenocarcinoma

doi: 10.1186/s12967-026-08948-8

Figure Lengend Snippet: Low PAFAH1B1 level is closely associated with the radioresistance of 125 I particle radiation in PDAC cells. BxPC-3-RR, PANC-1-RR cells and their parental BxPC-3-P and PANC-1-P cells were exposed to 0, 2, 4 and 8 Gy radiation. ( A , C ) Colony formation in PDAC cells was monitored by colony formation assay. ( B , D ) Cell viability of PDAC cells was assessed by CCK-8 assay. BxPC-3-RR, PANC-1-RR cells and their parental BxPC-3-P and PANC-1-P cells were exposed to 4 Gy radiation. ( E ) Cell apoptosis was assessed by Annexin-V-APC/PI staining and flow cytometry analysis. ( F ) The mRNA levels of SMAD4 and PAFAH1B1 in PDAC cells was detected by qRT-PCR. ( G ) The protein levels of SMAD4 and PAFAH1B1 in PDAC cells were detected by western blot. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Immortalized human pancreatic duct epithelial cell line hTERT-HRNE cells and human PDAC cell lines BxPC-3, PANC-1, MIA Paca-2 and CFPAC-1 were from ATCC (Manassas, VA, USA), and cultured in RPMI1640, DMEM or IMDM medium containing 10% FBS (Gibco, Grand Island, NY, USA).

Techniques: Colony Assay, CCK-8 Assay, Staining, Flow Cytometry, Analysis, Quantitative RT-PCR, Western Blot

Journal: Journal of Translational Medicine

Article Title: PAFAH1B1 stabilization, through PUM2-mediated competitive binding with miRNAs on its’ 3’-UTR, takes active actions on ferroptosis to overcome the radioresistance to 125 I radioactive particles of pancreatic ductal adenocarcinoma

doi: 10.1186/s12967-026-08948-8

Figure Lengend Snippet: Reinforced PAFAH1B1 promotes the 125 I particle radiation sensitivity in irradiation-resistant PDAC cells. BxPC-3-RR and PANC-1-RR cells were transfected with vector alone or PAFAH1B1 overexpression construct. BxPC-3-P and PANC-1-P cells were stably transduced with AAV-shNC or AAV-shPAFAH1B1. ( A ) The mRNA level of PAFAH1B1 in PDAC cells was detected by qRT-PCR. ( B ) The protein level of PAFAH1B1 in PDAC cells was detected by western blot. ( C - D ) Cell viability of PDAC cells was assessed by CCK-8 assay. ( E - G ) Colony formation in PDAC cells was monitored by colony formation assay. PDAC cells were subjected to overexpression or knockdown, followed by 4 Gy radiation. ( H ) Cell apoptosis was assessed by Annexin-V-APC/PI staining and flow cytometry analysis. ( I ) DNA damage was assessed by Comet assay. **, P < 0.01; ***, P < 0.001

Article Snippet: Immortalized human pancreatic duct epithelial cell line hTERT-HRNE cells and human PDAC cell lines BxPC-3, PANC-1, MIA Paca-2 and CFPAC-1 were from ATCC (Manassas, VA, USA), and cultured in RPMI1640, DMEM or IMDM medium containing 10% FBS (Gibco, Grand Island, NY, USA).

Techniques: Sensitivity, Irradiation, Transfection, Plasmid Preparation, Over Expression, Construct, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Colony Assay, Knockdown, Staining, Flow Cytometry, Analysis, Single Cell Gel Electrophoresis

Journal: Journal of Translational Medicine

Article Title: PAFAH1B1 stabilization, through PUM2-mediated competitive binding with miRNAs on its’ 3’-UTR, takes active actions on ferroptosis to overcome the radioresistance to 125 I radioactive particles of pancreatic ductal adenocarcinoma

doi: 10.1186/s12967-026-08948-8

Figure Lengend Snippet: Induction of ferroptosis contributes to the radiosensitivity of PDAC cells. BxPC-3-RR and PANC-1-RR cells were exposed to 4 Gy radiation. The intracellular ROS ( A ) and iron levels ( E ) were detected by DCFH-DA and FerroOrange staining, respectively. Green, DCFH-DA; Red, FerroOrange. Scale bar, 50 μm. ( B ) Lipid peroxidation was monitored by BODIPY 581/591 C11 staining and flow cytometry analysis. ( C - D ) The levels of MDA and GSH were detected using commercial kits. MDA or GSH level was normalized to total protein concentration determined by BCA protein assay. ( F ) Cell apoptosis was assessed by Annexin-V-APC/PI staining and flow cytometry analysis. **, P < 0.01; ***, P < 0.001

Article Snippet: Immortalized human pancreatic duct epithelial cell line hTERT-HRNE cells and human PDAC cell lines BxPC-3, PANC-1, MIA Paca-2 and CFPAC-1 were from ATCC (Manassas, VA, USA), and cultured in RPMI1640, DMEM or IMDM medium containing 10% FBS (Gibco, Grand Island, NY, USA).

Techniques: Staining, Flow Cytometry, Analysis, Protein Concentration, Bicinchoninic Acid Protein Assay

Journal: Journal of Translational Medicine

Article Title: PAFAH1B1 stabilization, through PUM2-mediated competitive binding with miRNAs on its’ 3’-UTR, takes active actions on ferroptosis to overcome the radioresistance to 125 I radioactive particles of pancreatic ductal adenocarcinoma

doi: 10.1186/s12967-026-08948-8

Figure Lengend Snippet: PAFAH1B1 weakens radioresistance by fostering radiation-induced ferroptosis. BxPC-3-P and PANC-1-P cells were stably transduced with AAV-shNC or AAV-shPAFAH1B1, and BxPC-3-RR and PANC-1-RR cells were stably transfected with vector alone and PAFAH1B1 overexpression plasmid. ( A ) Lipid peroxidation was monitored by BODIPY 581/591 C11 staining and flow cytometry analysis. ( B - C ) The levels of MDA and GSH were detected using commercial kits. MDA or GSH level was normalized to total protein concentration determined by BCA protein assay. ( D ) Intracellular iron was monitored by FerroOrange staining. Red, FerroOrange. Scale bar, 50 μm. ( E ) The protein levels of GPX4, SLC7A11 and ACSL4 in PDAC cells were detected by western blot. BxPC-3-RR and PANC-1-RR cells were transfected with vector alone or PAFAH1B1 overexpression construct in the absence or presence of Fer-1. ( F ) Cell viability of PDAC cells was assessed by CCK-8 assay. ( G ) Colony formation in PDAC cells was monitored by colony formation assay. BxPC-3-P and PANC-1-P cells were stably transduced with AAV-shNC or AAV-shPAFAH1B1, followed by Erastin treatment. ( H ) Cell viability of PDAC cells was assessed by CCK-8 assay. ( I ) Colony formation in PDAC cells was monitored by colony formation assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Immortalized human pancreatic duct epithelial cell line hTERT-HRNE cells and human PDAC cell lines BxPC-3, PANC-1, MIA Paca-2 and CFPAC-1 were from ATCC (Manassas, VA, USA), and cultured in RPMI1640, DMEM or IMDM medium containing 10% FBS (Gibco, Grand Island, NY, USA).

Techniques: Stable Transfection, Transduction, Transfection, Plasmid Preparation, Over Expression, Staining, Flow Cytometry, Analysis, Protein Concentration, Bicinchoninic Acid Protein Assay, Western Blot, Construct, CCK-8 Assay, Colony Assay

Journal: Journal of Translational Medicine

Article Title: PAFAH1B1 stabilization, through PUM2-mediated competitive binding with miRNAs on its’ 3’-UTR, takes active actions on ferroptosis to overcome the radioresistance to 125 I radioactive particles of pancreatic ductal adenocarcinoma

doi: 10.1186/s12967-026-08948-8

Figure Lengend Snippet: PUM2 is identified as a key regulator for increasing PAFAH1B1 expression via directly binding to its 3′UTR. ( A ) The mRNA level of PUM2 in PDAC tumors (n = 74) and matched adjacent normal pancreatic tissues (n = 74) was detected by qRT-PCR. ( B ) The correlation between PUM2 and PAFAH1B1 was analyzed by Spearman correlation analysis. ( C ) The mRNA level of PUM2 in BxPC-3-RR, PANC-1-RR and their parental cells was detected by qRT-PCR. BxPC-3-RR and PANC-1-RR cells were transduced with Lenti-NC or Lenti-PUM2. The mRNA levels of PUM2 ( D ) and PAFAH1B1 ( E ) were detected by qRT-PCR. ( F ) The protein levels of PUM2 and PAFAH1B1 were detected by western blot. ( G ) The mRNA stability of PAFAH1B1 in BxPC-3-RR and PANC-1-RR cells was monitored by RNA stability assay. ( H ) The luciferase activities of PAFAH1B1 promoter was measured. ( I ) 5’ UTR, CDS and 3’ UTR in BxPC-3-RR and PANC-1-RR cells were detected by dual luciferase reporter assay. ( J ) The interaction between PUM2 and PAFAH1B1 3’ UTR was assessed by RIP assay. Normal IgG served as a negative control. **, P < 0.01; ***, P < 0.001

Article Snippet: Immortalized human pancreatic duct epithelial cell line hTERT-HRNE cells and human PDAC cell lines BxPC-3, PANC-1, MIA Paca-2 and CFPAC-1 were from ATCC (Manassas, VA, USA), and cultured in RPMI1640, DMEM or IMDM medium containing 10% FBS (Gibco, Grand Island, NY, USA).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Analysis, Transduction, Western Blot, Stability, Luciferase, Negative Control

Journal: Journal of Translational Medicine

Article Title: PAFAH1B1 stabilization, through PUM2-mediated competitive binding with miRNAs on its’ 3’-UTR, takes active actions on ferroptosis to overcome the radioresistance to 125 I radioactive particles of pancreatic ductal adenocarcinoma

doi: 10.1186/s12967-026-08948-8

Figure Lengend Snippet: Overexpression of PUM2 competitively binds to PAFAH1B1 3′ UTR with miR-421 and miR-301a-3p. ( A ) Bioinformatics analysis predicted the putative binding sites between miR-421/miR-301-3p and PAFAH1B1 3’UTR. HKE293T cells were co-transfected with Luc-S-3’ UTR and mimics control (NC)/miR-421 mimics/miR-301a-3p mimics. ( B ) The luciferase activity of Luc-S-3’ UTR was detected by dual luciferase reporter assay. ( C ) The enrichments of miR-421 and miR-301a-3p were detected by Ago2-RIP assay. Normal IgG served as a negative control. HKE293T cells were transfected with miRNA mimics or inhibitor. ( D ) The mRNA level of PAFAH1B1 in BxPC-3-RR cells was detected by qRT-PCR. ( E ) The protein level of PAFAH1B1 in BxPC-3-RR cells was detected by western blot. Lentivirus-transduced PANC-1-RR cells were transfected with mimics control (NC)/miR-421 mimics/miR-301a-3p mimics. ( F ) The mRNA level of PAFAH1B1 was detected by qRT-PCR. ( G ) The protein level of PAFAH1B1 was detected by western blot. **, P < 0.01; ***, P < 0.001

Article Snippet: Immortalized human pancreatic duct epithelial cell line hTERT-HRNE cells and human PDAC cell lines BxPC-3, PANC-1, MIA Paca-2 and CFPAC-1 were from ATCC (Manassas, VA, USA), and cultured in RPMI1640, DMEM or IMDM medium containing 10% FBS (Gibco, Grand Island, NY, USA).

Techniques: Over Expression, Analysis, Binding Assay, Transfection, Control, Luciferase, Activity Assay, Negative Control, Quantitative RT-PCR, Western Blot

Journal: Journal of Translational Medicine

Article Title: PAFAH1B1 stabilization, through PUM2-mediated competitive binding with miRNAs on its’ 3’-UTR, takes active actions on ferroptosis to overcome the radioresistance to 125 I radioactive particles of pancreatic ductal adenocarcinoma

doi: 10.1186/s12967-026-08948-8

Figure Lengend Snippet: PUM2 enhances radiation-induced ferroptosis in a PAFAH1B1-dependent manner. BxPC-3-RR and PANC-1-RR cells were transduced with lentivirus and AAV. ( A ) Cell viability of PDAC cells was assessed by CCK-8 assay. ( B ) Colony formation in PDAC cells was monitored by colony formation assay. ( C - D ) The levels of MDA and GSH were detected using commercial kits. MDA or GSH level was normalized to total protein concentration determined by BCA protein assay. **, P < 0.01; ***, P < 0.001

Article Snippet: Immortalized human pancreatic duct epithelial cell line hTERT-HRNE cells and human PDAC cell lines BxPC-3, PANC-1, MIA Paca-2 and CFPAC-1 were from ATCC (Manassas, VA, USA), and cultured in RPMI1640, DMEM or IMDM medium containing 10% FBS (Gibco, Grand Island, NY, USA).

Techniques: Transduction, CCK-8 Assay, Colony Assay, Protein Concentration, Bicinchoninic Acid Protein Assay