Fig. 5

BFA induces massive PIN1-GFP aggregations in cytoplasm in BY-2 line upon inducible overexpression of Ntgnl1a ^M683L mutant allele. a-j In vivo confocal microscopy of 3-day-old tobacco PIN1::PIN1:GFP cells transformed with Ntgnl1a ^M683L and NtGNL1a genes, non-induced (g, h, i, j) and induced with 3 μM β-estradiol (c, d, e, f), pre-treatment with mock DMSO (a, c, e, g, i) or 20 μM BFA (b, d, f, h, j) for 30 min. Confocal sections of perinuclear plane captured with confocal microscope (488 nm excitation). a Control (CTRL) cells with mock treatment (DMSO). Note the PIN1-GFP at the PM. b BFA (20 μM)-induced aggregations of PIN1-GFP in the perinuclear area. c, e, g, i Unchanged PM localization of PIN1-GFP in induced and non-induced Ntgnl1a ^M683L cells (c, g) and NtGNL1a cells (e, i). f, h, j BFA (30 min, 20 μM)-induced few PIN1-GFP aggregations in perinuclear area with PIN1-GFP signal remaining at the PM in non-induced Ntgnl1a ^M683L cells (h) and induced and non-induced NtGNL1a cells (f, j). Note the contrasting situation in induced Ntgnl1a ^M683L cells (d) with BFA-induced FM 4–64 compartments observed in perinuclear area. k Relative area of intracellular PIN1-GFP (expressed in ‰ - per mil of the total cell area). Values represent the means of the ratios between integral areas of the intracellular PIN1-GFP and the total area of the cell. Error bars represent SEM from three biological repetitions, n = 3