2014). a prototypical type-I membrane glycoprotein, -amyloid precursor protein, is definitely improved rather than diminished. A closely related MOMIPP analog, which causes considerable vacuolization without reducing cell viability, also impedes cathepsin processing and autophagic flux, but has more modest effects on receptor degradation. A third analog, which causes neither vacuolization nor loss of viability, has no effect on endolysosomal trafficking. The results suggest that differential cytotoxicity of structurally related indole-based chalcones is definitely related, at least in part, to the severity of their effects on endolysosomal trafficking pathways. to construction (Trabbic et al. 2014) (Fig. 1b & c). By phase contrast microcopy, the vacuoles induced from the lethal MOMIPP and the nonlethal MOPIPP appeared generally related in size and amount per cell. To obtain a more quantitative assessment, we counted the number of phase-lucent vacuoles achieving or exceeding an arbitrary threshold of 3m diameter in images of 75 cells treated with each compound for 24 h. This analysis did not reveal a significant difference in the average quantity of vacuoles per cell (Fig 1d). It was not possible to accurately count the large number of vacuoles below the 3 m threshold, so it remains possible that variations exist at that level. Open in a separate window Fig. 1 Different biological activities of closely related indole-based chalcones in U251 glioblastoma cells. a) Cells were co-incubated with Dextran Alexa Fluor-568 and the indicated compounds (10 M). After 24 h, phase-contrast and fluorescent images HLI-98C of the live cells were acquired. The same field of cells is definitely depicted in the coordinating phase-contrast and fluorescent images. b) Cells HLI-98C were treated with compounds in the indicated concentrations for 48 h. Cell viability was assessed using the CellTiter Glo? ATP assay. Ideals are means ( SD) from four replicates. c) Phase-contrast images display the Rabbit polyclonal to Kinesin1 morphology of cells treated for 48 h with the indicated compounds at 10 M. Level bars in all of the images symbolize 20 m. d) Cells were treated for 24 h with 10 M MOMIPP or MOPIPP. For each group, digital images of 75 individual cells were by hand obtained for the number of phase-lucent vacuoles/cell. The threshold for counting vacuoles was arbitrarily arranged at a diameter of 3 m. The means ( SD) for the two groups were not significantly different (p 0.05) as determined by College student s t-test. Essentially all the larger vacuoles induced by MOMIPP and MOPIPP exhibited characteristics of late endosomes, including the presence of Light1 and GFP-Rab7 in their limiting membranes (Fig. 2 a & b). The vacuoles were distinct from adult lysosomes detected with the cathepsin-B substrate, Magic Red?, which appeared as smaller punctate constructions in areas between the vacuoles (Fig. 2c). Open in a separate windows Fig. 2 Localization of endolysosomal markers in U251 cells treated with different indole-based HLI-98C chalcones. a) Cells were treated for 24 h with the indicated compounds (10 M) or an comparative volume of DMSO (control) and then fixed and processed for immunofluorescence microscopy to localize LAMP1. b) U251 cells expressing EGFP-Rab7 were treated with compounds at 10 M and live-cell fluorescence images were obtained after 24 h. c) Cells were treated with compounds for 24 h and then incubated in medium with Magic Reddish? RR for 1 h prior to live-cell imaging. The scale bars for all panels are 20 m. Autophagosomes are double-membrane vesicles that develop from cup-shaped isolation membranes (phagophores), which surround regions of cytoplasm and organelles destined for degradation (Dunn 1994; Klionsky et al. 2014). The material of autophagosomes are degraded when these constructions merge with lysosomes to become autolysosomes (Gordon and Selgen 1988; Dunn 1990; Lawrence and Brown 1992). Microtubule-associated protein 1A/1B-light chain 3 (LC3) is the most widely used molecular marker for autophagosomes (Mizushima 2004). LC3 is present inside a cytosolic form (LC3I) and a form that is conjugated to phosphatidylethanolamine within the inner and outer autophagosome membranes (LC3II) (Kabeya et al. 2000). Immunostaining with an antibody against LC3 exposed poor diffuse staining in control and LC3I in cells treated with the vacuole-inducing compounds, compared to cells treated with vehicle or the inactive analog, MOMIPP-treated cells. Prior to addition of EGF, the EGFR in control cells was localized mainly within the cell surface and the juxtanuclear region (Fig. 5e). Within 1 h after adding the growth element, the fluorescence transitioned to a cluster of punctate Light1-positive structures, consistent with the expected trafficking of the receptor to late endosomes and lysosomes (Fig. 5e&f). In cells pre-treated with.