Fluorogenic Probe for Constitutive Cellular Endocytosis

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Fluorogenic Probe for Constitutive Cellular Endocytosis Michael N. Levine, Trish T. Hoang, Ronald T. Raines Chemistry & Biology Volume 20, Issue 4, Pages 614-618 (April 2013) DOI: 10.1016/j.chembiol.2013.03.016 Copyright © 2013 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2013 20, 614-618DOI: (10. 1016/j. chembiol. 2013 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Structure and Function of Lipid 1 Fluorescence is unmasked by an esterase encountered upon endocytosis. Chemistry & Biology 2013 20, 614-618DOI: (10.1016/j.chembiol.2013.03.016) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Lipid 1 Reports on Endocytosis HeLa cells in serum-free Dulbecco’s modified Eagle’s medium (DMEM) were labeled for 3 hr at 4°C with lipid 1 (20 μM), washed with serum-free medium, and then incubated for 3 hr at (A) 37°C or (B) 4°C. Left: Confocal images. Right: Overlay of confocal and bright-field images. Blue dye, Hoechst 33342. Scale bars represent 20 μm. Chemistry & Biology 2013 20, 614-618DOI: (10.1016/j.chembiol.2013.03.016) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Lipid 1 Does Not Recycle to the Cell Surface (A–C) HeLa cells in serum-free DMEM were labeled for 3 hr at 4°C with lipid 1 (20 μM), washed with serum-free medium, and then incubated for 3 hr at 37°C. (A) Image after an Alexa Fluor 594 transferrin conjugate (1 μM) was added for the final 1 hr of a 3 hr incubation. (B) Image after a 24 hr incubation. (C) Image after LysoTracker red (50 nM) was added for the final 20 min of a 3 hr incubation. Blue, Hoechst 33342. Scale bars represent 20 μm. Chemistry & Biology 2013 20, 614-618DOI: (10.1016/j.chembiol.2013.03.016) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Time Course of Endocytosis by HeLa Cells HeLa cells in serum-free DMEM were labeled for 3 hr at 4°C with lipid 1 (20 μM), washed with serum-free medium, and incubated for various times at 37°C. Fluorescence was quantified by flow cytometry. Values in arbitrary units (AU) are the mean ±SD from assays of 10,000 cells. Chemistry & Biology 2013 20, 614-618DOI: (10.1016/j.chembiol.2013.03.016) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Rate of Endocytosis Is Greater in Cancerous Cells Matched breast cell lines HTB-125 (noncancerous) and HTB-126 (cancerous) in serum-free DMEM were labeled for 3 hr at 4°C with lipid 1 (20 μM), washed with serum-free medium, and incubated for 0 or 3 hr at 37°C. Fluorescence was quantified by flow cytometry. Values are the mean ±SD from triplicate assays of 2,000 cells. HTB-125 cells: 3.7 ± 0.7 and 9.4 ± 0.7 AU; HTB-126: 3.1 ± 0.6 and 18.6 ± 2.2 AU. Chemistry & Biology 2013 20, 614-618DOI: (10.1016/j.chembiol.2013.03.016) Copyright © 2013 Elsevier Ltd Terms and Conditions