mutant cdnas  (TaKaRa)

Journal: mSphere

Article Title: TgJosephin and TgRad23 are important for anti-IFN-γ virulence via deubiquitination of SPM1 in Toxoplasma

doi: 10.1128/msphere.00137-26

Figure Lengend Snippet: SPM1 ubiquitination was increased in TgJosephin-deficient parasites. ( A ) Volcano plots of differentially ubiquitinated peptides between WT and ΔTgJosephin parasites extracted from MEFs (left) and Vero cells (right). Abundance of each peptide was determined with ubiquitin-targeted mass spectrometry across four or five biological replicates each. Each dot represents a T. gondii peptide. ( B ) Schematic representation of the T. gondii SPM1 protein domain structure predicted by SMART. Arrowheads: lysine residues detected as ubiquitination sites by ubiquitin-targeted mass spectrometry. Sites with higher ubiquitination rates in ΔTgJosephin parasites compared to WT ( P < 0.05, Student’s t -test) are marked in green. ( C ) Complementation of SPM1 in the UPRT locus of the Δspm1 strain. pTUB: alpha tubulin TUBA1 (TGME49_316400) promoter region. ( D ) Western blot analysis of HA-tagged SPM1 with anti- Toxoplasma antibody detection as a positive control. Parasite samples blotted with a mouse anti-HA antibody following immunoprecipitation with a rabbit anti-HA antibody. Bar graph: HA/Toxo signal intensity ratio is shown for each lane. ( E ) Survival analysis of WT mice infected with WT parasites, Δspm1 parasites, or Δspm1 parasites complemented with SPM1 or SPM1 KR . ( F ) Survival analysis of WT mice infected with ΔTgJosephin parasites or DKO parasites complemented with either SPM1 or SPM1 KR . *** P < 0.001. DKO: Δspm1ΔTgJosephin double knockout.

Article Snippet: A synthetic mutant SPM1 KR cDNA was ordered from FASMAC, in which 20 lysine residues detected as ubiquitination sites by mass spectrometry ( ) were substituted with arginine ( ).

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Western Blot, Positive Control, Immunoprecipitation, Infection, Double Knockout

Journal: Journal of Extracellular Biology

Article Title: Extracellular Vesicle‐Mediated Delivery of Constrained Peptides Disrupts the Pathogenic Interaction of LRRK2‐FADD in Parkinson's Disease

doi: 10.1002/jex2.70116

Figure Lengend Snippet: Characterisation of extracellular vesicles (EVs) isolated from HEK293T‐conditioned medium. (A) Schematic representation of EV‐based platform for FARM5 loading and delivery, designed to enhance BBB penetration, cellular uptake, and therapeutic efficacy in disrupting the pathogenic interaction between mutant LRRK2 and FADD in recipient cells. (B) Size distribution and concentration analysis of HEK293T‐EVs by NTA. (C) Western blot analysis confirming the presence of EV markers CD81 and Alix in HEK293T‐EVs, while cell‐specific markers (β‐tubulin and Calnexin) were not detected. (D) A representative transmission electron microscopy (TEM) image revealing the characteristic morphology of EVs, which displays a typical biconcave‐disk shape.

Article Snippet: Human mutant LRRK2 cDNA (#25045, Addgene) with an N‐terminal GFP epitope tag and full‐length V5‐tagged wild‐type (WT) FADD was used as described (Melachroinou et al. ).

Techniques: Isolation, Drug discovery, Mutagenesis, Concentration Assay, Western Blot, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: Extracellular Vesicle‐Mediated Delivery of Constrained Peptides Disrupts the Pathogenic Interaction of LRRK2‐FADD in Parkinson's Disease

doi: 10.1002/jex2.70116

Figure Lengend Snippet: EV‐FARM5 disrupts LRRK2‐FADD interaction. (A) HEK293T cells were transiently transfected with GFP‐tagged G2019S‐LRRK2 and V5‐tagged FADD. Thirty‐six hours post‐transfection, cells were treated with vehicle, free FARM5 or EV‐FARM5 at the indicated concentrations and incubated for an additional 8 h. After treatment, cells were washed and subjected to co‐immunoprecipitation using GFP‐Trap Magnetic Agarose. The eluates were analysed for precipitated LRRK2 (GFP) and co‐eluting FADD (V5). Both free FARM5 and EV‐FARM5 disrupted the LRRK2‐FADD interaction; however, EV‐FARM5 achieved this effect at lower doses than free FARM5. (B) Quantification of the FADD/LRRK2 Co‐immunoprecipitation (Co‐IP) ratio. Band intensities from (A) were measured using ImageJ, normalised to precipitated LRRK2 (GFP), and plotted relative to treatment dose. Each dot represents a biological replicate, which consists of three technical replicates. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. NS, not significant.

Article Snippet: Human mutant LRRK2 cDNA (#25045, Addgene) with an N‐terminal GFP epitope tag and full‐length V5‐tagged wild‐type (WT) FADD was used as described (Melachroinou et al. ).

Techniques: Transfection, Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Journal of Extracellular Biology

Article Title: Extracellular Vesicle‐Mediated Delivery of Constrained Peptides Disrupts the Pathogenic Interaction of LRRK2‐FADD in Parkinson's Disease

doi: 10.1002/jex2.70116

Figure Lengend Snippet: EV‐peptide enhances cell viability and reduces apoptosis in LRRK2‐G2019S‐expressing neural cells. Neuro‐2a cells were transiently transfected with either wild‐type (WT) or mutant (G2019S) LRRK2 and subsequently treated with EV‐FARM5 or free FARM5 at the indicated concentration for 16 h. (A) Cell viability was assessed using a CCK‐8 assay. Viability fold change was normalised to WT LRRK2‐expressing cells treated with vehicle control. (B) Apoptosis activity was measured using a Caspase‐Glo 3/7 assay. Both free FARM5 and EV‐FARM5 significantly enhanced cell viability (A) and suppressed apoptosis signaling activity (B) in LRRK2‐G2019S‐ expressing cells. However, EV‐FARM5 achieved these effects at lower doses than free FARM5. (C) Western blot analysis of caspase 3 and cleaved caspase 3 expression. EV‐FARM5 treatment reduced cleaved caspase‐3 levels compared to a higher dose of free FARM5, suggesting a more pronounced suppression of apoptotic signaling. Each dot represents a biological replicate, which consists of three technical replicates. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. NS, not significant.

Article Snippet: Human mutant LRRK2 cDNA (#25045, Addgene) with an N‐terminal GFP epitope tag and full‐length V5‐tagged wild‐type (WT) FADD was used as described (Melachroinou et al. ).

Techniques: Expressing, Transfection, Mutagenesis, Concentration Assay, CCK-8 Assay, Control, Activity Assay, Caspase-Glo Assay, Western Blot

Journal: Cell Death and Differentiation

Article Title: ABCC10-mediated cGAMP efflux drives cancer cell radiotherapy resistance

doi: 10.1038/s41418-025-01552-1

Figure Lengend Snippet: a Schematic of a metabolic in vitro CRISPR screen for identifying regulators of RT. b Scatter plot showing the top 10 negative regulatory genes in two rounds of the CRISPR-Cas9 screen using MAGeCK analysis. c Indication of the combined analysis of the two rounds of the functional screen. d Gene ontology (GO) analysis of molecular function (MF) for the intersection of 99 negative regulatory genes. e Survival analysis for BRCA patients was conducted separately within the high and low expression groups, comparing patients treated with RT and those who did not receive RT. f Immunoblot analysis of the protein expression levels of ABCC10 in parallel Patu8988T cells and radioresistant Patu8988T cells.

Article Snippet: Wild-type (WT) and mutant human ABCC10 constructs were generated using a human ABCC10 cDNA clone (Sino Biological HG19038-UT) as a template.

Techniques: In Vitro, CRISPR, Functional Assay, Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: ABCC10-mediated cGAMP efflux drives cancer cell radiotherapy resistance

doi: 10.1038/s41418-025-01552-1

Figure Lengend Snippet: a Immunoblot analysis of the protein expression levels of ABCC10 in Patu8988T and Calu-1 cells treated with radiotherapy (RT) (8 Gy) at the indicated time points. b Cell viability in Patu8988T and Calu-1 ABCC10-knockout cells treated with RT (8 Gy). Representative images ( c ) and quantification ( d ) of clonogenic survival analysis of Patu8988T and Calu-1 ABCC10-knockout cells treated with the indicated dose of ionizing radiation. e Cell viability in WT cells, ABCC10-knockout cells and ABCC10-knockout cells with re-expression of ABCC10 treated with RT (8 Gy). Representative images ( f ) and quantification ( g ) of clonogenic survival analysis of WT cells, ABCC10-knockout cells and ABCC10-knockout cells with re-expression of ABCC10 treated with the indicated dose of ionizing radiation. h Cell viability in Patu8988T and Calu-1 ABCC10-overexpressing cells treated with RT (8 Gy). Representative images ( i ) and quantification ( j ) of clonogenic survival analysis of Patu8988T and Calu-1 ABCC10-overexpressing cells treated with the indicated dose of ionizing radiation. k–n NXG mice were transplanted subcutaneously with doxycycline (DOX)-inducible ABCC10-knockdown Patu8988T cells and treated as indicated. A diagram of tumor growth delay experiments performed in vivo and the ionizing radiation fractionated treatment protocol is shown in ( k ); tumor volumes were calculated ( l ); tumor images were acquired as shown in ( m ); tumor weights ( n ) were measured. Experiments were repeated three times, and data are expressed as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Wild-type (WT) and mutant human ABCC10 constructs were generated using a human ABCC10 cDNA clone (Sino Biological HG19038-UT) as a template.

Techniques: Western Blot, Expressing, Knock-Out, Knockdown, In Vivo

Journal: Cell Death and Differentiation

Article Title: ABCC10-mediated cGAMP efflux drives cancer cell radiotherapy resistance

doi: 10.1038/s41418-025-01552-1

Figure Lengend Snippet: a Immunoblot analysis of the protein expression levels of γ-H2A.X in Patu8988T and Calu-1 ABCC10-knockout cells at the indicated times after RT (8 Gy). b Immunoblot analysis of the protein expression levels of γ-H2A.X in Patu8988T and Calu-1 ABCC10-overexpressing cells at the indicated times after RT. c Protein levels of γ-H2A.X in tumor tissues were detected. d Quantification of γ-H2A.X protein levels in in tumor tissues. e Immunoblot analysis of the protein expression levels of ABCC10 and γ-H2A.X in WT cells, ABCC10-knockout cells and ABCC10-knockout cells with re-expression of ABCC10 treated with RT (8 Gy) after 6 h. f Quantification of flow cytometry-based analysis of ROS levels in Patu8988T WT and ABCC10 KO cells at 24 h after RT. g Quantification of flow cytometry-based analysis of ROS levels in Patu8988T vector and ABCC10-overexpressing cells at 24 h after RT. h Quantification of flow cytometry-based analysis of ROS levels in WT and ABCC10 KO1 Patu8988T cells pretreated with or without NAC (1 mM) at 24 h after RT. i Immunoblot analysis of the protein expression levels of γ-H2A.X in WT and ABCC10 KO1 Patu8988T cells pretreated with or without NAC (1 mM) at 6 h after RT. j Cell viability of Patu8988T WT and ABCC10-knockout cells pretreated with or without NAC (1 mM) at 72 h after RT. Experiments were repeated three times, and the data are expressed as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Wild-type (WT) and mutant human ABCC10 constructs were generated using a human ABCC10 cDNA clone (Sino Biological HG19038-UT) as a template.

Techniques: Western Blot, Expressing, Knock-Out, Flow Cytometry, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: ABCC10-mediated cGAMP efflux drives cancer cell radiotherapy resistance

doi: 10.1038/s41418-025-01552-1

Figure Lengend Snippet: a A volcanic map was used to analyze differentially expressed transcriptome genes of ABCC10 KO1 cells treated with or without radiotherapy (RT) for 6 h. b Heatmap of STING pathway-related genes in ABCC10 KO1 cells compared to WT cells. c Gene Ontology (GO) molecular function enrichment analysis of upregulated genes from ABCC10 KO1 Patu8988T cells compared to WT cells treated with RT. d Western blot analysis of ABCC10, pTBK1, TBK1, pIRF3, and IRF3 protein levels in ABCC10 WT and KO Patu8988T cells. e Western blot analysis of FLAG, pTBK1, TBK1, pIRF3, IRF3, pSTING, and STING protein levels in vector and ABCC10-overexpressing Patu8988T STING-overexpression (OE) cells. f Quantification of flow cytometry-based analysis of reactive oxygen species (ROS) levels in Patu8988T WT and ABCC10 KO cells pretreated with H151 24 h after RT. g Western blot analysis of γ-H2A.X protein levels in Patu8988T and BxPC3 WT and ABCC10 KO cells pretreated with H151 (1 μM) 6 h after RT. h Quantification of clonogenic survival analysis of Patu8988T WT and ABCC10 KO cells pretreated with H151 (1 μM) after RT. Experiments were repeated at least three times, and the data are expressed as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Wild-type (WT) and mutant human ABCC10 constructs were generated using a human ABCC10 cDNA clone (Sino Biological HG19038-UT) as a template.

Techniques: Western Blot, Plasmid Preparation, Over Expression, Flow Cytometry

Journal: Cell Death and Differentiation

Article Title: ABCC10-mediated cGAMP efflux drives cancer cell radiotherapy resistance

doi: 10.1038/s41418-025-01552-1

Figure Lengend Snippet: a DNA was detected using PicoGreen dye in WT and ABCC10 KO1 cells. The nucleus was stained with DAPI dye. Fluorescence intensity of PicoGreen was calculated using ImageJ from three different areas, measuring the signal from PicoGreen in non-nuclear regions. b Cytoplasmic DNA from vector and ABCC10-overexpressing Patu8988T cell lysates was separated on an agarose gel. c Vector and ABCC10-overexpressing Patu8988T cells were treated with radiation (8 Gy) and measured for cGAMP in cell lysates and supernatants using an ELISA kit 8 h later. d WT and ABCC10-knockout Patu8988T cells were treated with radiation (8 Gy) and then measured for cGAMP in cell lysates and supernatants using an ELISA kit 8 h later. e 20-min vesicle transport assays using 293 T cell-derived vesicles expressing human ABCC10 or control vesicles with cGAMP (5 μM) in the presence of ATP or AMP. f Western blot analysis of vector and ABCC10-overexpressing cells treated with 2′3′-cGAMP after the indicated time points. g Western blot analysis of FLAG, ABCC10, pTBK1, TBK1, pIRF3, IRF3, pSTING, and STING expression in vector and ABCC10-overexpressing Patu8988T STING-OE cells. h qPCR analysis of IFNB1 mRNA expression in vector and ABCC10-overexpressing Patu8988T cells transfected with ctDNA at the indicated time points. i Western blot analysis of ABCC10 WT and KO Patu8988T cells transfected with ctDNA at the indicated time points. j qPCR analysis of IFNB1 mRNA expression in WT and ABCC10 KO1 Patu8988T cells transfected with ctDNA at the indicated time points. Experiments were repeated three times, and the data are expressed as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Wild-type (WT) and mutant human ABCC10 constructs were generated using a human ABCC10 cDNA clone (Sino Biological HG19038-UT) as a template.

Techniques: Staining, Fluorescence, Plasmid Preparation, Agarose Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Knock-Out, Derivative Assay, Expressing, Control, Western Blot, Transfection

Journal: Cell Death and Differentiation

Article Title: ABCC10-mediated cGAMP efflux drives cancer cell radiotherapy resistance

doi: 10.1038/s41418-025-01552-1

Figure Lengend Snippet: a Docking scores × (-1) of ABCC10 substrates. b 2′3′-cGAMP docked with the ABCC10-binding pocket. c Patu8988T cells transfected with empty vector, wild-type (WT), R545A, or R899A mutant ABCC10-overexpression lentivirus were harvested after stimulation with ctDNA for 4 h. The 2′3′-cGAMP level in cell lysates and supernatants were measured using an ELISA kit. d 20-min vesicle transport assays using 293 T cell-derived vesicles expressing human WT ABCC10, R545A mutant, R899A mutant or control vesicles with cGAMP in the presence of ATP. e Western blot analysis of Patu8988T cells transduced with WT, R545A, R899A mutant ABCC10, or control (empty vector) stimulated with ctDNA for 4 h. f Cell viability of Patu8988T cells transfected with empty vector, WT, R545A, or R899A mutant ABCC10-overexpression lentivirus was assessed 72 h after RT. g Quantification of clonogenic survival analysis of vector, WT, R545A, or R899A mutant ABCC10-overexpressing Patu8988T cells treated with RT. Experiments were repeated three times, and the data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Wild-type (WT) and mutant human ABCC10 constructs were generated using a human ABCC10 cDNA clone (Sino Biological HG19038-UT) as a template.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Over Expression, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, Control, Western Blot, Transduction

Journal: Cell Death and Differentiation

Article Title: ABCC10-mediated cGAMP efflux drives cancer cell radiotherapy resistance

doi: 10.1038/s41418-025-01552-1

Figure Lengend Snippet: a Docking scores × (-1) of ABCC10 inhibitors. b Nilotinib docked with the ABCC10-binding pocket. c 20-min vesicle transport assays using 293 T cell-derived vesicles expressing human WT ABCC10 and R545A mutant with cGAMP and nilotinib in the presence of ATP. d ELISA was performed to detect intracellular and extracellular cGAMP content in Patu8988T cells treated with nilotinib stimulated with ctDNA for 4 h. e Cell viability was assessed using CCK-8 in Patu8988T and KPC mouse cells treated with nilotinib at 72 h after RT. f Western blot analysis of γ-H2A.X in Patu8988T and KPC mouse cells pretreated with nilotinib at 6 h after RT. g Western blot analysis of pTBK1, TBK1, pIRF3, and IRF3 protein levels in Patu8988T and KPC cells pretreated with nilotinib at 24 h after RT. h Schematic diagram of in vivo tumor growth and fractionated treatment protocol with radiation. Tumor growth curves ( i ) and tumor weights ( j ) for tumors generated from KPC mouse cells implanted subcutaneously in C57BL/6 mice with the indicated treatments. k Representative photographs of isolated tumor tissues following the indicated treatments. l Immunohistochemical analysis of pSTING, ISG15, and γ-H2A.X protein levels in harvested tumor tissues. Experiments were repeated three times, and the data are expressed as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Data were analyzed using Student’s t-test or two-way analysis of variance. m A schematic model showing the role of ABCC10 in radioresistance. ABCC10 blocks the activation of the STING pathway by causing cGAMP efflux after RT. Moreover, nilotinib can overcome radioresistance by inhibiting ABCC10 activity, thereby activating the STING pathway and inducing DNA damage.

Article Snippet: Wild-type (WT) and mutant human ABCC10 constructs were generated using a human ABCC10 cDNA clone (Sino Biological HG19038-UT) as a template.

Techniques: Binding Assay, Derivative Assay, Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Western Blot, In Vivo, Generated, Isolation, Immunohistochemical staining, Activation Assay, Activity Assay