Journal: Oncoimmunology

Article Title: Mild microwave hyperthermia promotes mitotic catastrophe, induces time-delayed cGAS-STING activation and restores sensitivity to anti-PDL1 therapy in Pan02 pancreatic cancer model

doi: 10.1080/2162402X.2025.2602216

Figure Lengend Snippet: (A–I) Representative images of cGAS, STING, pSTAT1, IFNβ, CCL5, CXCL10, HMGB1, PD-L1, and γH2AX immunofluorescent or immunohistochemical stained tissue sections, respectively, of untreated, αPD-L1 mAb-treated, Ht-treated and Ht+αPD-L1 mAb-treated tumors (treatment side and abscopal side).

Article Snippet: The following antibodies were used for western blotting and immunostaining: anti-human cGAS (Cell Signaling), anti-mouse cGAS (SantaCruz), STING (Novus Biological), phospho-IRF3 Ser-386 (Cell Signaling), phospho-IRF3 Ser-396 (Cell Signaling), IRF-3 (SantaCruz), phospho-TBK1 Ser172 (Cell Signaling), TBK1 (SantaCruz), phospho-H2A.X Ser139 (Cell Signaling), PD-L1 (Cell Signaling), CD11c (Cell Signaling), CD8α (Cell Signaling), F4/80 (Proteintech), CD163 (Proteintech), CD86 (Novus Biologicals), FoxP3 (Cell Signaling), Gr1 (Invitrogen), GranzymeB (Invitrogen), Tim3 (Invitrogen), phospho-STAT1 Tyr701 (Cell Signaling), and IFNβ, CCL5, CXCL10, CD56, MICA, HMGB1, SUMO1, SUMO2/3, UBC9, SAE1, UBA2, SENP3, Beta-Actin (all from Proteintech).

Techniques: Immunohistochemical staining, Staining

Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: Stimulation of ISRE reporter by RTD-1. The activity of Lucia luciferase secreted in the medium was assayed and the luminescence units are plotted. (A) THP-1 Dual cells were stimulated with HOAc (vehicle) or increasing concentrations of RTD-1. Results are from 3 experiments. * P < 0.05, 2-tailed t test, comparison with HOAc-treated sample. Error bars indicate standard deviation. (B) THP-1 Dual cells were pretreated with DMSO or Ruxo as indicated. Assay done in triplicate. * P < 0.05 when compared with HOAc-treated control, 2-tailed t test. # P < 0.05 when compared with RTD-1 treatment. Error bars indicate standard deviation. (C) THP-1 Dual cells were transferred to RPMI + 1% HI FBS + P/S + normocin medium and either 2.5 μg human IgG1 or anti-IFNAR1 antibodies were added to the culture. Cells were incubated for 2 h, and RTD-1 or IFN-β was added as indicated and the incubation continued for 18 h. Assay was done in triplicate. * P < 0.05 when compared with HOAc-treated control, 2-tailed t test. Error bars indicate standard deviation. (D) THP-1 Dual cells were treated with RTD-1, IFN-β, or both as shown. Assay was done in triplicate. * P < 0.05, 2-tailed t test. Error bars indicate standard deviation.

Article Snippet: IFN-β was purchased from R&D Systems and human IgG1 and human anti IFNAR1 antibodies were purchased from Selleck USA.

Techniques: Activity Assay, Luciferase, Comparison, Standard Deviation, Control, Incubation

Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: ISRE stimulation activity is distinct from RTD-1–mediated inhibition of NF-κB activation. THP-1 Dual cells were treated with LPS, RTD-1, or IFN-β or vehicle as shown and the medium was assayed for ISRE reporter activity (top) and NF-κB reporter activity (bottom) using Quanti-Luc and Quanti-Blue assays, respectively. Assay done in triplicate. # P > 0.05 (nonsignificant) when compared with respect to LPS treated samples, 2-tailed t test. Error bars indicate standard deviation.

Article Snippet: IFN-β was purchased from R&D Systems and human IgG1 and human anti IFNAR1 antibodies were purchased from Selleck USA.

Techniques: Activity Assay, Inhibition, Activation Assay, Standard Deviation

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 decreased IFNAR1 protein levels to promote OC43 replication. a) Luciferase assay of ISRE element activity after PIM1/K67M overexpression (n = 3). b) Immunoblots of lysates from HEK293T cells transfected with vector/PIM1 plasmids for 48 h. c) Western blot for IFNAR1 in HEK293T cells transfected with vector/WT‐PIM1/PIM1‐K67M plasmids for 48 h. d) IFNAR1 expression level in PIM1 knockdown cells. e) Immunoblots analysis of RD cells infected with OC43 at an MOI of 10 for the indicated time. f) Expression of IFNAR1 in RD cells transfected with siPIM1 RNA, followed by OC43 infection. g–j) Western blot for IFNAR1 expression in HEK293T or HepG2 cells treated with PIM1 inhibitors (CX‐6528, SGI‐1776, and AZD‐1208) at the indicated concentration for 48 h. k) The IFNAR1 expression in RD cells pre‐treated with PIM1 inhibitor 2 for 2 h, followed by OC43 infection for 48h. l)The intracellular OC43 genomic RNA in HEK293T cells transfected with vector/PIM1/PIM1 with IFNAR1 followed by the 24 h challenging of OC43 at an MOI of 0.1. Statistical analyses were conducted with Student's t‐test. * : p < 0.05, ** : p < 0.01. Data are depicted as mean ±SD. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: Cell surface expression of IFNAR1 was quantified by flow cytometry using a phycoerythrin (PE)‐conjugated anti‐human IFNAR1 antibody (R&D Systems, Catalog # FAB245P).

Techniques: Luciferase, Activity Assay, Over Expression, Western Blot, Transfection, Plasmid Preparation, Expressing, Knockdown, Infection, Concentration Assay, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 promoted IFNAR1 degradation via ubiquitination. a) IFNAR1 mRNA level in HEK293T cells transfected with vector/PIM1 plasmids for 48 h. b) The mRNA level of IFNAR1 in RD cells transfected with scramble/PIM1‐specific siRNA for 48 h. c) The mRNA level of IFNAR1 in RD cells after challenging with/without OC43 at an MOI of 1 or 10. d) IFNAR1‐overexpressed HEK293T cells were transfected with scramble/PIM1‐specific siRNA, followed by 50 µ m of CHX treatment for the indicated periods. IFNAR1 protein level were determined by western blot. e) IFNAR1 expression level after PIM1/K67M overexpression followed by MG132 or NH 4 Cl treatment. f) Flow Cytometry of cell surface IFNAR1 after PIM1 overexpression, stained with IFNAR1‐PE (n = 3). g) IFNAR1 expression in cell membrane or cytosol fractions after PIM1/K67M overexpression. h) Immunoblots of IFNAR1 in HEK293T cells transfected with WT‐IFNAR1 or mutated IFNAR1 (S535A, S539A) with/without PIM1. i) Western blot analysis for ubiquitination and IFNAR1 in HEK293T cells co‐transfected with HA‐Ubiquitin, IFNAR1 and vector/PIM1/PIM1 K67M constructs. j) Immunoprecipitation of IFNAR1 and HA‐tagged ubiquitination from lysates of HEK293T cells co‐transfected with HA‐K48 ubi/HA‐K63 ubi, IFNAR1 and vector/PIM1 plasmids. Statistical analyses were conducted with Student's t‐test. * : p < 0.05, ** : p < 0.01. Data are depicted as mean ±SD. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: Cell surface expression of IFNAR1 was quantified by flow cytometry using a phycoerythrin (PE)‐conjugated anti‐human IFNAR1 antibody (R&D Systems, Catalog # FAB245P).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Expressing, Over Expression, Flow Cytometry, Staining, Membrane, Construct, Immunoprecipitation, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 promoted IFNAR1 degradation via β‐TrCP1. a) IFNAR1 protein level in IFNAR1‐expressed HEK293T cells transfected with diverse siRNAs. b) IFNAR1 protein level in IFANR1‐expressed HEK293T cells transfected with scramble/β‐TrCP1 specific siRNA with/without PIM1 plasmids. c) IFNAR1 protein level in IFNAR1‐expressed HEK293T cells with β‐TrCP1 and/or PIM1 siRNA knockdown. d) Viral genomic RNA of OC43 in RD cells transfected with scramble/β‐TrCP1 specific siRNA followed by infection at an MOI of 0.1 for 24 h (n = 4). e) Viral genomic RNA level of OC43 in HEK293T cells transfected with increasing amounts of β‐TrCP1 followed by the challenging of HCoV‐OC43 at an MOI of 0.1 for 24 h (n = 3). f) Viral genomic RNA level in HEK293T cells after β‐TrCP1 siRNA knockdown with or without ectopic expression of PIM1, followed by OC43 infection at an MOI of 0.1 for 24 h (n = 3). g) Viral genomic RNA level in RD cells treated with PIM1 inhibitor (CX6528=8 µ m ) with or without β‐TrCP1 inhibitor (GS143) at indicated concentration for 2 h, followed by OC43 infection at MOI of 0.01 for 72 h (n = 4). Statistical analyses were conducted with Student's t‐test. * : p <0.05, ** : p < 0.01. Data are shown as mean ±SD. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: Cell surface expression of IFNAR1 was quantified by flow cytometry using a phycoerythrin (PE)‐conjugated anti‐human IFNAR1 antibody (R&D Systems, Catalog # FAB245P).

Techniques: Transfection, Knockdown, Infection, Expressing, Concentration Assay, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: PIM1 promoted IFNAR1 and β‐TrCP1 interaction. a,b) Protein level of β‐TrCP1 in HEK293T cells transfected with vector/PIM1 construct or scramble/PIM1 specific siRNA. c) Immunoprecipitation of IFNAR1 and HA‐tagged β‐TrCP1 from lysates mixture of vector/PIM1/PIM1 mutant expressed HEK293T and IFNAR1 expressed HEK293T. d) IP blots for purified PIM1 post incubation with or without purified βTrCP1 in the IP buffer. e) Immunofluorescent assay of HEK293T cells co‐transfected with PIM1 and HA‐ β‐TrCP1 plasmids under 40× microscope. PIM1 was in Red and HA‐ β‐TrCP1 was in Green. Scale bar = 10 µ m . Line intensity profiles (indicated as white line) and Scatter plots (boxed ROI ) for PIM1 and β‐TrCP1 were analyzed by Image J software. Pearson's r = 0.62. f) Purified β‐TrCP1 incubated with/without recombinant PIM1 in a kinase assay buffer at 37 °C for 30 min. Lambda protein phosphatase (lambda PP) was added to one tube of the PIM1 and β‐TrCP1 mixture for another 30 min. The products were visualized post Phos‐tag PAGE separation, which separates the phosphorylated β‐TrCP1 as up‐shifted migration bands from the non‐phosphorylated ones. The densities of all proteins of interest were quantified using ImageJ and normalized to the respective control indicated beneath the bands.

Article Snippet: Cell surface expression of IFNAR1 was quantified by flow cytometry using a phycoerythrin (PE)‐conjugated anti‐human IFNAR1 antibody (R&D Systems, Catalog # FAB245P).

Techniques: Transfection, Plasmid Preparation, Construct, Immunoprecipitation, Mutagenesis, Purification, Incubation, Microscopy, Software, Recombinant, Kinase Assay, Migration, Control

Journal: Advanced Science

Article Title: PIM1 Attenuates Innate Immunity to Foster Coronavirus Replication through Ubiquitin Ligase β‐TrCP‐Mediated IFNAR1 Degradation

doi: 10.1002/advs.202503487

Figure Lengend Snippet: Phosphorylated β‐TrCP1 at S82 by PIM1 enhanced its interaction with IFNAR1. a) Predicted PIM1‐phosphorylation sites on β‐TrCP1. b) ISRE luciferase assays using HEK293T cells transfected with ISRE‐Luci and β‐TrCP1 / β‐TrCP1 mutants, followed by stimulation with 500U IFNα for 16 h (n = 3). c) Protein level of IFNAR1 in HEK293T cells ectopically expressed with WT‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A. d) Protein level of phosphorylated STAT1/2 (p‐STAT1/2) in HEK293T cells ectopically expressed with vector/WT‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A followed by the stimulation of IFNα for 16 h. e) Quantification of intracellular viral genomic RNA in HEK293T cells ectopically expressed with WT‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A followed by the infection with HCoV‐OC43 at an MOI of 1 for 36 h (n = 3). f) Western blot analysis of HEK293T cells transfected with WT/S82A/S521A β‐TrCP1 plasmids with/without PIM1. Blots of IP assay using p‐Ser/Thr antibody were shown in the bottom panel. g) Co‐IP assay of IFNAR1 from the cell lysate mixture of HEK293T cells transfected with IFNAR1/HA‐ β‐TrCP1 / β‐TrCP1 S82A/ β‐TrCP1 S521A separately.

Article Snippet: Cell surface expression of IFNAR1 was quantified by flow cytometry using a phycoerythrin (PE)‐conjugated anti‐human IFNAR1 antibody (R&D Systems, Catalog # FAB245P).

Techniques: Phospho-proteomics, Luciferase, Transfection, Plasmid Preparation, Infection, Western Blot, Co-Immunoprecipitation Assay