Figure 2. TPD54-depleted cells have... | Rockefeller University Press

$Figure 2. TPD54-depleted cells have defective anterograde membrane traffic and cargo recycling. (A) Still confocal images of RUSH experiments. SBP-EGFP–E-cadherin localization in control (siCtrl) and TPD54-depleted (siTPD54) HeLa cells at the indicated times (minutes and seconds) after biotin treatment. Scale bar, 10 µm. (B) Single cell traces of the E-cadherin fluorescence ratio of a control (gray) or TPD54-depleted (blue) cell, fitted with a logistic function and a line. (C) Normalized fraction of total E-cadherin fluorescence at the Golgi as a function of time in control (gray) or TPD54-depleted (colored) cells. Results from three siRNAs are shown as indicated. Line and shaded area, mean ± SEM. ncell = 85 (siCtrl), 62 (siTPD54 #1), nexp = 2; ncell = 23 (siCtrl), 12 (siTPD54 #2), nexp = 1; ncell = 43 (siCtrl), 20 (siTPD54 #3), nexp = 1. (D) Western blot to assess the depletion of TPD54 by RNAi for three siRNAs. The protein level of TPD54 and α-tubulin (loading control) is shown. (E–G) Box plots showing the t1/2 of E-cadherin transport from ER-to-Golgi (E) and from ER-to-PM (F) in control and TPD54-depleted cells. (G) The difference in t1/2 represents intra-Golgi transport. Dots represent individual cells, boxes show interquartile range, bars represents the median, and whiskers show 9th and 91st percentiles. The P values are from Student’s t test with Welch’s correction. ncell = 57−82, nexp = 2. (H) Plot showing the uptake and recycling of transferrin in control and TPD54-depleted cells. Dots represent individual cells, lines represent the median value. Wilcoxon rank test. **, P < 0.01. ncell = 77−160, nexp = 3.$

Figure 2.

TPD54-depleted cells have defective anterograde membrane traffic and cargo recycling. (A) Still confocal images of RUSH experiments. SBP-EGFP–E-cadherin localization in control (siCtrl) and TPD54-depleted (siTPD54) HeLa cells at the indicated times (minutes and seconds) after biotin treatment. Scale bar, 10 µm. (B) Single cell traces of the E-cadherin fluorescence ratio of a control (gray) or TPD54-depleted (blue) cell, fitted with a logistic function and a line. (C) Normalized fraction of total E-cadherin fluorescence at the Golgi as a function of time in control (gray) or TPD54-depleted (colored) cells. Results from three siRNAs are shown as indicated. Line and shaded area, mean ± SEM. n_cell = 85 (siCtrl), 62 (siTPD54 #1), n_exp = 2; n_cell = 23 (siCtrl), 12 (siTPD54 #2), n_exp = 1; n_cell = 43 (siCtrl), 20 (siTPD54 #3), n_exp = 1. (D) Western blot to assess the depletion of TPD54 by RNAi for three siRNAs. The protein level of TPD54 and α-tubulin (loading control) is shown. (E–G) Box plots showing the t_1/2 of E-cadherin transport from ER-to-Golgi (E) and from ER-to-PM (F) in control and TPD54-depleted cells. (G) The difference in t_1/2 represents intra-Golgi transport. Dots represent individual cells, boxes show interquartile range, bars represents the median, and whiskers show 9th and 91st percentiles. The P values are from Student’s t test with Welch’s correction. n_cell = 57−82, n_exp = 2. (H) Plot showing the uptake and recycling of transferrin in control and TPD54-depleted cells. Dots represent individual cells, lines represent the median value. Wilcoxon rank test. **, P < 0.01. n_cell = 77−160, n_exp = 3.

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