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. 2010 May 12;5(5):e10597. doi: 10.1371/journal.pone.0010597

Figure 2. Effect of AP-2 depletion on the surface distribution of AP-2 and on the uptake of transferrin.

Figure 2

(A, B) Control (-μ2 RNAi) and treated (+ μ2 RNAi) HeLa and BSC1 cells stably expressing σ2-EGFP were incubated at 37°C with 10 µg/ml of Tf-A594 for 10 min, followed by a quick rinse at 4°C to remove unbound transferrin, fixed with 3.7% PFA, and imaged in 3D with wide-field illumination. Representative views of the plane closest to the coverslip (σ2-EGFP) and of an equatorial plane (Tf) show a decrease in punctate σ2-EGFP signals at the cell membrane and inhibition of transferrin uptake. Insets correspond to representative boxed regions. Arrowheads mark the few remaining AP-2 spots in Tf-negative cells. Bar, 20 µm. (C, D) Histrograms showing the amount, relative to controls, of internalized transferrin obtained from the total fluorescence signal of Tf-A594 in the 3D stacks. The remaining signals were 11.9±6.9% (p<0.0001, 10 control and 14 AP-2 depleted HeLa cells) and 15.8±2.5% (p<0.0001, 15 control and 22 AP-2 depleted BSC1 cells), respectively.