concanamycin a  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: ERAD Component MoHrd3 Facilitates Pathogenicity and Establishes a Direct Regulation on Autophagy in Magnaporthe Oryzae

doi: 10.1002/advs.202520627

Figure Lengend Snippet: MoHrd3 interacts with MoAtg8 and is degraded through autophagy. (A) The split‐ubiquitin membrane yeast two‐hybrid system was used to test the interaction between MoHrd3 and MoAtg8. NubG/Cub co‐transformation was used as negative controls. (B) MoHrd3 interacted with MoAtg8 in a co‐IP assay. GFP‐MoAtg8 and MoHrd3‐HA plasmids were co‐transformed into P131. Total proteins were purified by GFP‐Nanoab‐Agarose beads and the products were detected using the indicated antibodies. (C) MoHrd3 associated with MoAtg8 in a BiFC assay. nYFP‐MoAtg8/MoHrd3‐cYFP and their related control pairs were co‐expressed in M. oryzae . Strains were cultured in liquid CM treatment with or without 1 µ m ConA for 4 h before observation. Scale bar, 10 µm. (D) MoHrd3‐GFP co‐localized with mCherry‐MoAtg8 in growth hyphae. MoHrd3‐GFP and mCherry‐MoAtg8 were co‐transformed into P131. The generated strains were cultured in liquid CM medium with or without 1 µ m ConA before observation. Scale bar, 10 µm. (E) MoHrd3‐GFP was co‐localized with mCherry‐MoAtg8 in hyphae. Strains were cultured in liquid MM‐N medium with or without 1 µ m ConA. Mycelia were stained with CMAC to indicate the vacuoles. Scale bar, 10 µm. (F) MoHrd3 was degraded through autophagy pathway. MoHrd3‐HA/P131 were treated with DTT (10 m m ), MG132 (100 µ m ), or 3‐MA (500 µ m ) for 10 h. The band intensity was calculated by Image J.

Article Snippet: The transformed strains were cultured in liquid CM medium for 36 h at 28°C, and then shifted to fresh liquid MM‐N medium with or without 1 μ m ConA (Concanamycin A, MedChemExpress) for 5 h before the observation.

Techniques: Ubiquitin Proteomics, Membrane, Transformation Assay, Co-Immunoprecipitation Assay, Purification, Bimolecular Fluorescence Complementation Assay, Control, Cell Culture, Generated, Staining

Journal: Advanced Science

Article Title: ERAD Component MoHrd3 Facilitates Pathogenicity and Establishes a Direct Regulation on Autophagy in Magnaporthe Oryzae

doi: 10.1002/advs.202520627

Figure Lengend Snippet: MoHrd3 interacts with MoYpt7 and enhances the association between MoYpt7 and MoAtg8. (A) The interaction between MoYpt7 and MoHrd3 was verified by yeast two‐hybrid. (B) MoHrd3 interacted with MoYpt7 in a co‐IP assay. MoHrd3‐HA and GFP‐MoYpt7 plasmids were transformed in P131 strain, respectively. The MoHrd3‐HA was detected following GFP‐MoYpt7 immunoprecipitation. (C) MoHrd3 associated with MoYpt7 by BiFC assays. nYFP‐MoYpt7/MoHrd3‐cYFP and their related control pairs were co‐expressed in M. oryzae . Strains were cultured in liquid CM for 36 h. Scale bar, 5 µM. (D) MoHrd3‐GFP co‐localized with mCherry‐MoYpt7 in growth hyphae. The strains co‐transformed with MoHrd3‐GFP and mCherry‐MoYpt7 were cultured in liquid CM medium with or without further treatment with MM‐N medium (with 1 µ m ConA) for 5 h before observation. Scale bar, 10 µm. (E) MoHrd3 enhanced the interaction between MoYpt7 and MoAtg8 in a co‐IP assay. GFP‐MoAtg8/∆ Mohrd3 , MoHrd3‐HA/∆ Mohrd3 and MoYpt7‐HA/∆ Mohrd3 strains were used in this assay. The MoHrd3‐HA and MoYpt7‐HA were detected following GFP‐MoAtg8 immunoprecipitation. (F) Deletion of MoHrd3 reduced the interaction between MoYpt7 and MoAtg8 in a co‐IP assay. GFP or GFP‐MoAtg8 plasmids were co‐transformed with MoYpt7‐HA plasmid in P131 or ∆ Mohrd3 strains respectively. The MoYpt7‐HA was detected following GFP‐MoAtg8 immunoprecipitation with or without MoHrd3‐HA. GFP: MoYpt7‐HA/P131 and GFP: MoYpt7‐HA/∆ Mohrd3 were used as negative controls. (G) The localization of GFP‐MoYpt7 and mCherry‐MoAtg8 in mycelium of P131 and ∆ Mohrd3 . Strains were cultured in liquid CM or MM‐N medium with ConA (1 µ m ). Mycelia were stained with CMAC to indicate the vacuoles. Scale bar, 10 µm.

Article Snippet: The transformed strains were cultured in liquid CM medium for 36 h at 28°C, and then shifted to fresh liquid MM‐N medium with or without 1 μ m ConA (Concanamycin A, MedChemExpress) for 5 h before the observation.

Techniques: Co-Immunoprecipitation Assay, Transformation Assay, Immunoprecipitation, Control, Cell Culture, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: Filopodia-mediated trans-endocytosis

doi: 10.64898/2026.02.09.703982

Figure Lengend Snippet: ( A-I ) DCIS.com cells expressing MYO10 HMM -GFP were mixed with parental cells and grown as a monolayer. After 24 h, cells were fixed, labelled to visualise the plasma membrane and nuclei (DAPI), and imaged using high-resolution Airyscan fluorescence microscopy and focused-ion-beam scanning electron microscopy (FIB-SEM). ( A ) Representative registration and alignment of the fluorescence and FIB-SEM datasets. ( B ) Examples of internalised MYO10-positive filopodia tips identified and manually segmented from the FIB-SEM volume. ( C ) Quantification of the volume of internalised filopodia tips based on manual segmentation (n = 47 internalised filopodia tips, two biological replicates). ( D ) Representative FIB-SEM image showing an internalised MYO10-positive filopodia tip displaying a double-membrane structure. ( E ) Pie chart quantifying the proportion of internalised filopodia tips exhibiting two clearly visible membranes and a discernible lumen (n = 47 internalised filopodia tips, two biological replicates). ( F ) Quantification of the percentage of internalised filopodia tips in contact with endoplasmic reticulum (ER) tubules or mitochondria, as assessed from segmented FIB-SEM volumes (n = 47 internalised filopodia tips, two biological replicates). ( G ) Three-dimensional rendering of an internalised filopodia tip (magenta) in close association with ER tubules (green). ( H ) High-resolution Airyscan image showing an internalised filopodia tip in proximity to ER structures. ( I ) Representative electron microscopy images illustrating the ultrastructural diversity of internalised MYO10-positive filopodia tips. ( J ) DCIS.com cells expressing MYO10 HMM -GFP were mixed with parental cells and cultured as a monolayer for 24 hours. Cells were then treated with concanamycin A (125 nM) for 24 hours. Subsequently, cells were fixed, stained for LAMP2, and imaged using an Airyscan confocal microscope. Representative images and quantification of the impact of lysosomal inhibition on the number of MYO10 HMM dots associated with recipient cells, as well as the percentage of MYO10 HMM dots overlapping with LAMP2 staining, are shown (4 biological replicates, 47 fields of view). Quantification was carried out per field of view, excluding the MYO10 HMM -GFP-expressing cell; only MYO10 HMM dots associated with neighbouring parental cells were analysed (see Methods). ( C, J ) Data are presented as boxplots, with whiskers extending from the 10th to the 90th percentiles. Boxes indicate the interquartile range, and the central line denotes the median. Data points outside the whiskers are shown as individual dots. The raw images used to generate this figure have been archived on Zenodo .

Article Snippet: Additional reagents used in this study include wheat germ agglutinin (WGA, ThermoFisher Scientific, W32466), ER-TrackerTM Red (ThermoFisher Scientific, E34250), SiR-lysosome (Spirochrome, SC012), Dyngo4a (Hydroxy-Dynasore) (Abcam, AB120689), DAPI (ThermoFisher Scientific, D1306), Alexa Fluor 647-conjugated phalloidin (Invitrogen, A30107), Poly-D-Lysine (GibcoTM, A3890401), Alexa Fluor 647-conjugated Concanavalin A (ThermoFisher Scientific, C21421), Concanamycin A (Santa Cruz Biotechnology, sc-202111A), and fibronectin (Sigma-Aldrich, 341631).

Techniques: Expressing, Clinical Proteomics, Membrane, Fluorescence, Microscopy, Electron Microscopy, Cell Culture, Staining, Inhibition

Journal: bioRxiv

Article Title: Filopodia-mediated trans-endocytosis

doi: 10.64898/2026.02.09.703982

Figure Lengend Snippet: ( A ) DCIS.COM cells expressing MYO10HMM–GFP were mixed with parental cells and cultured as a monolayer. After 24 hours, cells were fixed, labelled to visualise the plasma membrane and nuclei (DAPI), and imaged using high-resolution Airyscan confocal microscopy followed by focused ion beam–scanning electron microscopy (FIB–SEM). A MYO10HMM-GFP-positive punctum identified in the correlated EM volume is highlighted and displayed with its segmentation. Scale bars: 5 µm. ( B ) DCIS.com MYO10HMM-GFP and parental cells were mixed and imaged live after incubation with SiR-lysosome. A representative confocal image is shown. The white square indicates an ROI shown at higher magnification; yellow arrows highlight MYO10HMM–GFP-positive internalised filopodia tips overlapping with SiR-lysosome signal. Scale bars: main image, 25 µm; ROI, 5 µm. ( C ) DCIS.com MYO10HMM–GFP and parental cells were mixed and cultured in the presence of concanamycin A (125 nM, 24 hours). Cells were fixed, stained for LAMP2, and imaged using Airyscan confocal microscopy. Representative orthogonal views (xy, xz, and yz) are shown. Scale bar: 25 µm. The raw images used to generate this figure have been archived on Zenodo .

Article Snippet: Additional reagents used in this study include wheat germ agglutinin (WGA, ThermoFisher Scientific, W32466), ER-TrackerTM Red (ThermoFisher Scientific, E34250), SiR-lysosome (Spirochrome, SC012), Dyngo4a (Hydroxy-Dynasore) (Abcam, AB120689), DAPI (ThermoFisher Scientific, D1306), Alexa Fluor 647-conjugated phalloidin (Invitrogen, A30107), Poly-D-Lysine (GibcoTM, A3890401), Alexa Fluor 647-conjugated Concanavalin A (ThermoFisher Scientific, C21421), Concanamycin A (Santa Cruz Biotechnology, sc-202111A), and fibronectin (Sigma-Aldrich, 341631).

Techniques: Expressing, Cell Culture, Clinical Proteomics, Membrane, Confocal Microscopy, Electron Microscopy, Incubation, Staining