FIG 1.

MK2206 is an autofluorescent small molecule whose accumulation in cellular cytoplasmic compartments is sensitive to endocytic pH. (A) Absorbance spectra, fluorescence emission spectra, and total fluorescence of MK2206 at different pHs. The absorbance peaks are at 259 nm and 354 nm; the shorter peak corresponds to the excitation of individual phenyl rings, while the longer peak possibly corresponds to the system of three conjugated rings. The emission has a rather broad spectrum with a half-width of about 100 nm, possibly reflecting the emission input from excitation of different rings of the compound. (B) MDCK cells were treated with 10 μM MK2206, fixed at 24 h, and imaged. Scale bars, 10 μm. (C) MDCK cells were treated with increasing concentrations of MK2206, fixed after 4 h, and imaged with ScanR. The autofluorescent vesicles visible in the DAPI channel were counted, and their area, circularity, and mean intensity were measured. (D) Cells were treated with 10 μM MK2206 or its combination with 20 mM NH₄Cl, 0.4 μM SaliPhe, or 0.4 μM obatoclax (or remained nontreated), fixed at the indicated time points, and imaged with a ScanR. The points in panels A, C, and D are mean values, the number of observations used to derive the values is 2, and error bars represent the standard deviation (SD).