Figure 7. 2ME2 impairs the turnover and endocytosis of myc-TβRII-GFP.

A, ES-2 cells, stably expressing myc-TβRII-GFP, were treated with 2ME2 or vehicle. Arrested and cycling cells were pre-treated for 20 min with cycloheximide (300 µg/ml), in combination (or not) with proteasome inhibitors (MG132 and ALLN); followed by the addition of TGF-β1 (5 ng/ml, 3 h, in the same media). Cells were subsequently lysed and immunoblotted with α-myc antibodies. Bar graph depicts average ± SEM of the 3 h/0 h ratio of the relative myc-TβRII-GFP content, n = 3; p = 0.05, 2-tailed t-test. B, ES-2 cells grown on glass coverslips were treated as in (A), cooled to 4°C, stained with α-myc/Alexa-546-GαM/DAPI and imaged by confocal microscopy. Bar graph depicts average ± SEM of the 3 h/0 h ratio of the Alexa-546 staining normalized to the DAPI staining of 18 fields of cells (10 x magnification, ∼1400 cells) for each condition; p<0.01. C, Confocal micrographs of ES-2 cells, stably expressing myc-TβRII-GFP, treated with 2ME2, hypertonic medium (0.45 M sucrose, 30 min), or vehicle and submitted to endocytosis experiment (as described in the Materials and Methods). Bar, 10 µm. Specific signals were identified through intensity-based segmentation, and co-localized signal-positive pixels were determined with Slidebook™ and employed for quantification. D, Bar graph depicts the average fold change (± SEM) in the percent of 546-α-myc that did not co-localize with 647-GαM in all experimental conditions as compared to that of cells kept at 4°C. Cycling cells show a 1.82±0.11 fold increase in internalized TβRII (n = 35; p<0.01, 1-tailed t-test); no changes in the levels of internalized TβRII were observed upon hypertonic medium treatment (0.97±0.08 fold change, n = 8; p>0.8, 2-Tailed t-test) and a slight reduction of this parameter, which failed to reach statistical significance, was induced by arrest in mitosis with 2ME2 (0.8±0.12 fold change; n = 20 cells; p>0.16, 2-tailed t-test).