1. Volume 26, Issue 2, Pages 173-183 (January 2016)
USP9X Controls EGFR Fate by Deubiquitinating the Endocytic Adaptor Eps15 
Michol Giovanna Savio, Nadine Wollscheid, Elena Cavallaro, Veronica Algisi, Pier Paolo Di Fiore, Sara Sigismund, Elena Maspero, Simona Polo 
Current Biology 
Volume 26, Issue 2, Pages (January 2016)
DOI: /j.cub
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2. Current Biology 2016 26, 173-183DOI: (10.1016/j.cub.2015.11.050)
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3. Figure 1 EGFR Degradation Kinetics Measured after DUBs Knockdown
(A) HeLa cells transfected with scrambled oligo pool were stimulated with EGF (100 ng/ml) for the indicated time points. Total cell lysates were subjected to immunoblot (IB) and DELFIA analyses. The graph shows the quantification of EGFR degradation in control cells measured by IB and DELFIA.
(B) Scatterplot representing the degradation rate for each DUB. Each dot represents a single DUB whose x, y coordinates are obtained by the ranking position (from fastest to slowest) of the degradation rate as identified by IB (x axis) or by DELFIA (y axis) assays (Figures S1A and S1B). The dashed black line represents the bisector (y = x), and the red line represents the linear regression of the data. In bold are the 24 deconvoluted DUBs; in red are AMSH and USP8 as internal controls.
(C and D) Total cell lysates from HeLa cells transfected with the oligo pool for indicated DUBs were treated as in (A). USP25- and BRCC36-depleted cells show faster degradation rate (C), whereas USP9X and USP10 knockdown are examples of slower degradation rate (D).
See also Figure S1.
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4. Figure 2 USP9X Regulates EGFR Internalization and Degradation
(A) HeLa cells were transfected with the indicated siRNAs targeting USP9X or with a scrambled oligo (control). Cells were serum starved overnight and then stimulated with EGF (100 ng/ml) for the indicated time points. Lysates were analyzed by IB with the indicated antibodies. One representative experiment of three is shown.
(B) Quantitation of the IBs as in (A). Data are expressed as the mean ± SD of three independent experiments.
(C) Lysates as in (A) were subjected to DELFIA analysis. Data are expressed as the mean ± SD of three independent experiments.
(D and E) EGFR internalization rates were measured at low (1.5 ng/ml; D) or high (20 ng/ml; E) of 125I-EGF ligand. Results are expressed as fold change of the internalization rate Ke compared to control cells and represent the average of five independent experiments ± SD. ∗∗∗∗p <
See also Figure S2.
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5. Figure 3
Depletion of USP9X Alters EGFR Fate During the Endocytic Route
HeLa cells were transiently transfected with scrambled, USP9X, and USP8 RNAi oligos and stimulated with high dose of EGF for the indicated time points. Alexa Fluor 555-EGF (40 ng/ml, red) and anti-EGFR (13A9, green) are shown. In blue, DAPI staining is shown. The scale bar represents 10 microns. See also Figure S3.
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6. Figure 4 Depletion of USP9X Alters EGFR Trafficking
(A) HeLa control cells and USP9XKD were stimulated for 45 min with Alexa Fluor 555-EGF (40 ng/ml) at 16°C and then rinsed with mild acid wash to remove membrane-bound EGF (T0). Upon re-addition of culture medium at 37°C, cells were fixed at the indicated time points. In blue, DAPI staining is shown. The scale bar represents 10 microns. Representative confocal microscopy images are shown.
(B and C) Quantification of total EGF signal intensity expressed as a.u. (B) or of number of EGF-labeled vesicles (C). At least 35 cell profiles were counted; see Supplemental Experimental Procedures for details. Data are represented as mean of three independent experiments ± SD.
See also Figures S4 and S5.
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7. Figure 5
Depletion of USP9X Increases Eps15 and Epsin Monoubiquitination
(A) HeLa cells (control and USP9X-KD) were serum starved and stimulated with EGF (100 ng/ml) for the indicated time points. Total cell lysates were immunoprecipitated (IP) for EGFR and IB as indicated.
(B) Lysates as in (A) were IB with the indicated antibodies. Arrows indicate monoubiquitinated proteins.
(C–E) Lysates as in (A) were IP and IB with the indicated antibodies.
(F) Epitope-tagged full-length USP9X (Usp9X-V5) and Eps15 (FLAG-Eps15) were transiently expressed in HEK293T cells. IP and IB are as indicated.
(G) MEF Eps15 KO cells stably transfected with HA-Eps15 were stimulated with EGF (100 ng/ml). Lysates were IP with anti-HA and incubated with GST-UCH (ubiquitin C-terminal hydrolase) of USP9X, GST-UCH C1566S (C/S) USP9X mutant, or GST-AMSH for the indicated time points. IB is as indicated.
See also Figure S6.
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8. Figure 6 Eps15 Monoubiquitination Is Required for EGFR Internalization
(A) HeLa and triple-KD cells were transiently knocked down for USP9X, and EGFR internalization rates were measured using 125I-EGF ligand (1.5 ng/ml). Results are expressed as internalization rate constant Ke (right panel) and are the mean of three independent experiments ± SD. Total cell lysates from the indicated cell lines is shown (left panel). Panels of the left show the level of depletion achieved. IB is as indicated.
(B) A schematic representation of Eps15 structural domains is shown in the upper panel. Asterisks highlight the ubiquitination sites. In the lower panel, Eps15/Eps15R KD HeLa cells stably re-expressing Eps15 constructs were stimulated with EGF (100 ng/ml) for the indicated time points. IP and IB are as indicated.
(C) Eps15/Eps15R KD HeLa cells stably re-expressing wild-type HA-Eps15 were stimulated with low and high doses of EGF. Lysates were IP and IB as indicated.
(D) EGFR internalization rates were measured using 125I-EGF ligand (1.5 ng/ml) in Eps15/Eps15R KD HeLa cells stably re-expressing HA-Eps15 constructs and transiently silenced for Epsin or mock transfected with control oligo. Results are expressed as % of the internalization rate constant Ke compared to control cells and represent the average of three independent experiments (mean ± SD). ∗∗p < 0.01.
See also Figure S7.
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9. Figure 7
Impaired Eps15 Monoubiquitination Affects Eps15-EGFR Co-trafficking
(A) Gel filtration analysis of B82L/EGFR cells serum starved overnight and induced (+) or not (−) with EGF for 10 min. Fractions from 8.5 to 13.5 ml were separated by SDS-PAGE and IB as indicated. Monoubiquitinated Eps15 peaks at the same fractions of the unmodified Eps15 are shown, at a molecular weight higher than 670 KDa.
(B) Lysates (500 μg) as in (A) were IP and IB as indicated. No difference in the stoichiometry of the AP2 interaction is visible for the monoubiquitinated form of Eps15.
(C) Lysates (1 mg) as in (A) were pulled down with GST-α-adaptin and IB as indicated. Coomassie shows equal loading of GST proteins.
(D) Eps15/Eps15R KD HeLa cells stably re-expressing the indicated Eps15 constructs were serum starved and stimulated with Alexa Fluor 555-EGF for either 10 min or 20 min. Cells were stained with anti-Eps15 antibody (green). In blue, DAPI staining is shown. The scale bar represents 10 microns. Representative confocal microscopy images are shown. Magnification from merged images highlight colocalization (yellow) between EGF and Eps15.
(E) Quantification of colocalizing pixels. Data are expressed as percentage of EGF pixels colocalizing with Eps15 and represent the average of three independent experiments (mean ± SD).
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