rapamycin analog  (TaKaRa)

Journal: Nature Communications

Article Title: TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport

doi: 10.1038/s41467-024-52706-7

Figure Lengend Snippet: a Representative images of cells cultured in biotin-free media expressing 3xFKBP-TDP43-mNG, 3xFRB*-Halo-SEC16A and RUSH-TNFα without or with AP21967 (AP) treatment for 6-10 hr. Arrowheads and triangles indicate TDP43-ERES and ordinary ERES, respectively. In “Zoom out”, yellow boxes mark the regions displayed in zoom-in views, and cyan dashes demarcate the nuclei. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. b Quantification of the experiments as in ( a ) and Supplementary Fig. g showing the volume percentage of SEC16A inclusions that enriched TDP43 under the indicated conditions. n = 45 cells examined over 3 independent experiments. The values of individual cells are plotted as dots, and the mean values of cells in the same experiment as horizontal segments, the median of which is elongated and thickened. Different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. c SEC24C or SEC31A immunostaining in cells with AP-induced TDP43/SEC16A coaggs. Scale bars except in “Zoom Out” represent 1 μm, and represent 5 μm in “Zoom Out”. Representative images of 3 independent experiments are shown. d Selected frames from a timelapse recording of an AP-induced TDP43-ERES after photobleaching. Insets show individual channels. 3xFKBP-TDP43-mNG was entirely photobleached whereas 3xFRB*-(JF646)-Halo-SEC16A was partially bleached. The scale bar represents 0.5 μm. A representative timelapse of 3 independent experiments are shown. e Selected frames of a representative timelapse recording of an AP-induced TDP43-ERES during RUSH-TNFα assay. The scale bar represents 1 μm. f Quantification of RUSH-TNFα and SEC16A intensities over time (normalized to their respective max intensities) in the TDP43-ERES tracked in ( e ). g Quantification of the percentage of AP-induced TDP43(-mNG)-ERES, mNG-ERES and ERES without TDP43 (in cells without AP treatment) that retained RUSH-TNFα in the experiments as in ( e ) and Supplementary Fig. . n = 9 cells examined over 3 independent experiments. Each dot represents one cell, and different colors denote different experiments. The asterisk and “ns” respectively stand for significant (p-value ≤ 0.05) and non-significant (p-value > 0.05) in two-sided t-tests. h Working model of TDP43 aggregation at ERES causing retention of transport cargos. TDP43-ERES also exert a dominant effect over ordinary ERES in the same cells possibly through sequestration of essential transport factors. The ER-to-Golgi transport defect caused by TDP43-ERES co-aggregation may further induce ER stress.

Article Snippet: 1 μM rapamycin analog AP21967 (AP, Takara 635056) was added ∼18 hr post-transfection to treat the cells for 6–10 hr to induce co-aggregation.

Techniques: Cell Culture, Expressing, Immunostaining

Journal: Nature Communications

Article Title: Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies

doi: 10.1038/s41467-023-38651-x

Figure Lengend Snippet: a Schematic illustration of the split T7 inducible expression cassette inserted into Thymidine Kinase (TK) open reading frame. The FKBP linked N-terminal portion of the T7 RNAP and the FRB linked C-terminal portion of the T7 RNAP were expressed under continuous vaccinia virus promoters. A Fusion protein consisting of GFP, and firefly luciferase (GFPLuc) was incorporated under T7 promoter and induced by the addition of Rapalogs. mCherry fluorescent protein is expressed from the virus to detect virus infection in cells. b , c Representative fluorescent images and quantitation of luciferase signal (RLU) of the T7 inducible system from U2OS cells 24 h after infection with VV-ST7-iGFPLuc in the presence or absence of 10 nM rapamycin. mCherry indicates virus infection. d Comparison of the luciferase signal from various cell lines 24 h after infection at MOI 0.1 with VV-ST7-iGFPLuc in the presence or absence of 10 nM rapamycin. e Multistep growth curve of VV-ST7-iGFPLuc compared to the control vaccinia virus at different time points from Hela cells infected at MOI 0.01 in the presence or absence of 10 nM rapamycin. f Representative fluorescent images of the T7 inducible GFP system from U2OS cells 24 h after infection with VV-ST7-iGFPLuc at MOI 0.1 in the presence of different Rapalogs at 10 nM concentration. g Relative luminescence emitted after 24 h from U2OS cells infected with VV-ST7-iGFPLuc at MOI 0.1 in the presence of different concentrations of Rapalogs. h Comparison of vaccinia virus growth in the presence of Rapalogs at 10 nM in U2OS and HELA cells 24 h after infection at MOI 0.1. i , j IVIS imaging of HT-29 tumors in CD-1 nude mice. Tumors were injected with VV-ST7-iGFPLuc (1E7 PFU/tumor) when they reached ~150 mm 3 in size. Different Rapalogs were administered at 1 mg/kg by their preferred route. After 24 h, luciferase signal was measured, and the control group received rapamycin intraperitoneally. Luciferase signal was measured in all groups again at 72 h. Scale bars = 40 μm in ( b , f ). Data indicate means ± SD of three ( c – e , g , h ) to four ( j ) biological replicates. ns P > 0.05, * P < 0.05 ** P < 0.003841, *** P < 0.000125, **** P < 0.001 in unpaired two-samples t -test. Source data are provided as a Source Data file.

Article Snippet: Cumate solution (QM150A-1) was purchased from System Biosciences, doxycycline (D9891-25G) was purchased from Sigma-Aldrich, and Rapamycin analogs were purchased from Selleck Chemicals.

Techniques: Expressing, Virus, Luciferase, Infection, Quantitation Assay, Comparison, Control, Concentration Assay, Imaging, Injection

Journal: Nature Communications

Article Title: Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies

doi: 10.1038/s41467-023-38651-x

Figure Lengend Snippet: a Schematic illustration of the ST7-Tet double inducible expression cassette inserted into TK locus. The TetR and split T7 fragment proteins were expressed under continuous vaccinia virus promoters and a GFPLuc fusion protein was incorporated under T7 promoter preceding a TetO element which binds TetR protein. The binding of split T7 fragments in the presence of Rapalogs and dissociation of the TetR from TetO upon Dox administration leads to initiation of transcription and gene expression. b , c Representative fluorescent images and quantitation of luciferase activity from U2OS cells 24 h following infection with VV-ST7-TetR-iGFPLuc in the presence or absence of Dox and rapamycin. mCherry signal indicates virally infected cells. d Schematic illustration of the ST7-Cumate double inducible expression cassette inserted into the same locus as described in A. The CymR and split T7 fragment proteins were expressed under continuous vaccinia virus promoters and GFPLuc was incorporated under T7 promoter preceding a CuO element which binds CymR protein. The binding of split T7 fragments in the presence of Rapalogs and dissociation of the CymR from CuO upon cumate administration leads to initiation of transcription and gene expression. e , f Representative fluorescent images and quantitation of luciferase activity from U2OS cells 24 h following infection with VV-ST7-CymR-iGFPLuc in the presence or absence of cumate and rapamycin. g Schematic illustration of the Cu-Tet double inducible expression cassette inserted into the same locus as described in A. The CymR and TetR proteins were expressed under continuous vaccinia virus promoters and GFPLuc was incorporated under different vaccinia virus promoters preceding a CuO element which binds CymR protein, and a TetO element which binds TetR protein. Dissociation of the CymR from CuO and TetR from TetO upon cumate and Dox administration leads to initiation of transcription and gene expression. h Luciferase signal (in RLU) was quantified 24 h after infection of U2OS cells with viruses expressing GFPLuc under the control of CuTet and various native and synthetic vaccinia promoters in the presence or absence of 100 µg/ml cumate and 100 ng/ml Dox. i , j Representative fluorescent images and quantitation of luciferase activity from HEK293T, Hela, Vero, HT-29, 786-O cells infected with VV-CymR-TetR-iGFPLuc in the presence or absence of cumate and Dox after 24 h. IRF signal indicates virally infected cells. k Schematic illustration of the CuTet double inducible expression cassette inserted in the intergenic location between UL26 and UL27 open reading frames of the HSV-1 genome. The CymR-T2A-rtTA3 fusion protein was expressed under the UBC promoter. For expression of the GFPLuc fusion protein, TetO followed by a minimal CMV promoter and CuO was used. In the presence of Dox, rtTA3 binds to the TetO and enhances CMV promoter activity which will be at maximum activity if CymR is also dissociated from CuO upon cumate administration. IRF is continuously expressed from the virus, to monitor viral infection. l , m Representative fluorescent images and quantitation of luciferase activity from U2OS cells infected with VV-CymR-TetR-iGFPLuc in the presence or absence of cumate and Dox after 24 h. IRF signal indicates virally infected cells. Scale bars = 40 μm in ( c , f , i , m ). Data indicate means ± SD of three ( b , h , j , l ) or four ( e ) biological replicates. ns P > 0.05, * P < 0.05 ** P < 0.003841, *** P < 0.000125, **** P < 0.001 in unpaired two-samples t -test. Source data are provided as a Source Data file.

Article Snippet: Cumate solution (QM150A-1) was purchased from System Biosciences, doxycycline (D9891-25G) was purchased from Sigma-Aldrich, and Rapamycin analogs were purchased from Selleck Chemicals.

Techniques: Expressing, Virus, Binding Assay, Gene Expression, Quantitation Assay, Luciferase, Activity Assay, Infection, Control