Fig. 5

Increased numbers of MLF vesicles and levels of MLF protein and ROS production by nocodazole, DTT, and G418 treatment. A, B, C MLFVs can be induced by nocodazole, DTT, and G418 treatment. The wild-type non-transfected WB cells were cultured in growth medium with (A) 5 μM nocodazole, (B) 5 mM DTT, and (C) 217 μM G418, or the same volume of solvent (H2O or Me2SO) for 24 h and then subjected to immunofluorescence assay using anti-MLF antibody for detection. D Quantification of MLFVs in nocodazole, DTT, and G418 treated cells during vegetative stage was performed using Imaris software. *, p < 0.05. **, p < 0.01 (n = 200–300 cells/condition). E Nocodazole, DTT, and G418 treatment increased ROS production. The wild-type non-transfected WB cells were cultured in growth medium with 5 μM nocodazole, 5 mM DTT, and 217 μM G418, or the same volume of solvent (H2O or Me2SO) for 24 h and then subjected to ROS measurement. Fold change is calculated as the ratio of the difference between the treatment group and control group, to which a value of 1 was assigned. F, G, H Nocodazole, DTT, and G418 treatment increased the levels of MLF protein. The wild-type non-transfected WB were cultured in growth medium containing (F) 5 μM nocodazole, (G) 5 mM DTT, and (H) 217 μM G418, or the same volume of solvent (H2O or Me2SO) for 24 h and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-MLF and anti-RAN antibodies, respectively. SDS-PAGE with Coomassie Blue staining is included as a control for equal protein loading. I The band intensity from triplicate Western blots was quantified using Image J as described in Fig. 1E. **, p < 0.01