Journal: Pharmacology Research & Perspectives

Article Title: Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

doi: 10.1002/prp2.756

Figure Lengend Snippet: C‐Cbl knockout in hTCEpi cells reduces ligand‐mediated EGFR ubiquitylation. c‐Cbl was knocked out of hTCEpi cells using CRISPR/CAS9 technology (see Materials and Methods). (A) Cell lysates were prepared from hTCEpi cells expressing either only CAS9, or CAS9 and a human c‐Cbl specific gRNA. Equivalent amounts of cell lysate (20 μg) were resolved by 7.5% SDS‐PAGE, transferred to nitrocellulose, and immunoblotted for c‐Cbl, Cbl‐b, Cbl‐3, Nedd4, or α‐tubulin. Shown is a representative blot from at least three experiments. Indistinguishable results were observed with other clones. (B) CAS9 or c‐Cbl knockout cells were serum‐starved and then treated with 50 ng/ml of EGF for 0, 2, 10 min. Cell lysates were prepared, and the EGFR was immunoprecipitated (Ab‐1, EMD Millipore). Immunoprecipitates were divided into thirds, resolved by 7.5% SDS‐PAGE, and immunoblotted using antibodies against ubiquitin (Ub), phosphorylated EGFR (pY1068), and EGFR. Shown is a representative experiment repeated at least three times with multiple clones

Article Snippet: For internalization assays, 35 mm dishes of hTCEpi cells were incubated with 1 ng/ml of 125 I‐EGF (catalog number NEX160; PerkinElmer Life Sciences; specific activity 150–200 μCi/μg) for 2 h on ice to achieve steady‐state binding.

Techniques: Knock-Out, CRISPR, Expressing, SDS Page, Clone Assay, Immunoprecipitation

Journal: Pharmacology Research & Perspectives

Article Title: Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

doi: 10.1002/prp2.756

Figure Lengend Snippet: The EGF internalizes in the absence of c‐Cbl. CAS9 expressing or c‐Cbl knockout hTCEpi cells were plated on glass coverslips and grown to ~80% confluence. Cells were serum‐starved and then incubated with 2 μg/ml of Texas Red‐EGF (Invitrogen) for 10 min at 37°C to allow the internalization of ligand:receptor complexes. Extracellular fluorescently labeled ligand was removed by ice‐cold citric acid washes. Cells were re‐equilibrated in a neutral buffer and returned to 37°C growth media and 37°C. At the indicated time points, cells were fixed and processed for indirect immunofluorescence using a mouse monoclonal antibody against EEA1 (#C45B10, Cell Signaling) and an Alexa488 goat anti‐mouse secondary antibody. Cells were mounted on microscope slides using Prolong with DAPI (Thermo Fisher Scientific). The micrographs indicate the localization of Texas Red‐EGF (Red), EEA1 (green), and the nucleus (blue). Size bar = 10 μm. Shown is a representative experiment repeated at least three times

Article Snippet: For internalization assays, 35 mm dishes of hTCEpi cells were incubated with 1 ng/ml of 125 I‐EGF (catalog number NEX160; PerkinElmer Life Sciences; specific activity 150–200 μCi/μg) for 2 h on ice to achieve steady‐state binding.

Techniques: Expressing, Knock-Out, Incubation, Labeling, Immunofluorescence, Microscopy

Journal: Pharmacology Research & Perspectives

Article Title: Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

doi: 10.1002/prp2.756

Figure Lengend Snippet: EGFR distribution is altered by the knockout of c‐Cbl. CAS9 expressing or c‐Cbl knockout hTCEpi cells were plated on glass coverslips and grown to ~80% confluence. Cells were stimulated with 10 ng/ml of EGF for 0–60 min. At the indicated time point, cells were fixed and processed for indirect immunofluorescence using a mouse monoclonal antibody against the EGFR (Ab‐1, EMD Millipore) and an Alexa488 goat anti‐mouse secondary antibody. Coverslips were mounted on microscope slides using Prolong with DAPI (Thermo Fisher Scientific). Micrographs indicate the subcellular localization of the EGFR (green) and nucleus (blue). Size bar = 10 μm. Shown is a representative experiment repeated at least three times

Article Snippet: For internalization assays, 35 mm dishes of hTCEpi cells were incubated with 1 ng/ml of 125 I‐EGF (catalog number NEX160; PerkinElmer Life Sciences; specific activity 150–200 μCi/μg) for 2 h on ice to achieve steady‐state binding.

Techniques: Knock-Out, Expressing, Immunofluorescence, Microscopy

Journal: Pharmacology Research & Perspectives

Article Title: Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

doi: 10.1002/prp2.756

Figure Lengend Snippet: 125 I‐EGF:EGFR endocytic trafficking is slowed in c‐Cbl knockout cells. CAS9 expressing or c‐Cbl knockout hTCEpi cells were plated in 35 mm dishes and grown to ~80% confluence. 125 I‐EGF was used to monitor A–B) internalization, (C) recycling, and (D) degradation as described in Materials and Methods ( n = 3). (A) The average (±SD) ratio of internalized 125 I‐EGF/surface 125 I‐EGF. (B) The average (±SD) calculated internalization constant (Ke) for each cell line. Data were analyzed using a paired t ‐test. (C) The average (±SD) percentage of total bound 125 I that was secreted into the media. Data were analyzed using an ANOVA with a Holm‐Sidak post‐analysis ( α = 5.000%). (D) The average (±SD) percentage of intact 125 I‐EGF. Data were analyzed using an ANOVA with a Holm‐Sidak post‐analysis ( α = 5.000%). * p < 0.10; ** p < 0.05

Article Snippet: For internalization assays, 35 mm dishes of hTCEpi cells were incubated with 1 ng/ml of 125 I‐EGF (catalog number NEX160; PerkinElmer Life Sciences; specific activity 150–200 μCi/μg) for 2 h on ice to achieve steady‐state binding.

Techniques: Knock-Out, Expressing

Journal: Pharmacology Research & Perspectives

Article Title: Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

doi: 10.1002/prp2.756

Figure Lengend Snippet: Knockdown of c‐Cbl slows EGFR degradation and sustains ligand‐mediated phosphorylation. Control (Cas‐9) and c‐Cbl knockout hTCEpi cells were serum‐starved and treated with 10 ng/ml of EGF for the indicated amounts of time. (A) Cell lysates were prepared and equivalent amounts of protein (30 μg) were resolved by 7.5% SDS‐PAGE. The resulting gel was transferred to nitrocellulose and immunoblotted for EGFR, phosphoEGFR (pY1068), c‐Cbl and α‐tubulin. Shown are representative immunoblots from an experiment that was performed three times using multiple c‐Cbl knockout clones. (B and C) Immunoblots were quantified using NIH ImageJ. (B) The average (±SD) percentage of EGFR relative to α‐tubulin and normalized to unstimulated levels ( t = 0). (C) The average (±SD) percentage of phosphorylated EGFR (EGFR pY1068) relative to α‐tubulin and normalized to the maximal phosphorylation ( t = 15 min). Data were analyzed by an ANOVA with a Sidak‐Bonferroni post‐test analysis ( α = 5.000%). * p < 0.05; ** p < 0.01)

Article Snippet: For internalization assays, 35 mm dishes of hTCEpi cells were incubated with 1 ng/ml of 125 I‐EGF (catalog number NEX160; PerkinElmer Life Sciences; specific activity 150–200 μCi/μg) for 2 h on ice to achieve steady‐state binding.

Techniques: Knock-Out, SDS Page, Western Blot, Clone Assay

Journal: Pharmacology Research & Perspectives

Article Title: Knockout of c‐Cbl slows EGFR endocytic trafficking and enhances EGFR signaling despite incompletely blocking receptor ubiquitylation

doi: 10.1002/prp2.756

Figure Lengend Snippet: Knockdown of c‐Cbl sustains EGFR‐mediated MAPK phosphorylation. CAS9 expressing or c‐Cbl knockout hTCEpi cells were serum‐starved and treated with 10 ng/ml of EGF for the indicated amounts of time. (A) Cell lysates were prepared and equivalent amounts of protein (30 μg) were resolved by 10% SDS‐PAGE. The resulting gel was transferred to nitrocellulose and immunoblotted for phosphoMAPK and total MAPK. Shown are representative immunoblots from an experiment that was performed three times using multiple c‐Cbl knockout clones. (B) Immunoblots were quantified using NIH ImageJ. The average (±SD) relative phosphoMAPK activity (plotted as a ratio of phosphoMAPK/total MAPK). Levels are normalized to the maximal phosphorylation ( t = 15 min). Data were analyzed with an ANOVA with a Sidak‐Bonferroni post‐test analysis ( α = 5.000%, * p < 0.05; ** p < 0.01)

Article Snippet: For internalization assays, 35 mm dishes of hTCEpi cells were incubated with 1 ng/ml of 125 I‐EGF (catalog number NEX160; PerkinElmer Life Sciences; specific activity 150–200 μCi/μg) for 2 h on ice to achieve steady‐state binding.

Techniques: Expressing, Knock-Out, SDS Page, Western Blot, Clone Assay, Activity Assay

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet: EGF Internalization in AP2-KO MEFs or upon AP2-KD in Different Cell Contexts (A) 125 I-EGF (left) and 125 I-Tf (right) internalization in AP2-WT and AP2-KO MEFs in the presence or absence of clathrin KD. Internalization constants ( K e ) are the mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test ( ∗∗∗ p < 0.001). (B) Analysis of the impact of AP2μ KD versus clathrin KD in different cell lines as indicated. The number of EGFRs per cell in the different cell lines was measured using 125 I-EGF saturation binding assay. Kinetics of 125 I-EGF (1.5 ng/mL) were measured and are reported as internalization constants ( K e ). The percentage of AP2-independent EGFR internalization was calculated from the residual K e in AP2-KD cells relative to the K e in control cells (after subtracting the residual internalization in clathrin KD cells; Sigismund et al., 2013 ). (C) TEM analysis of EGFR internalization. AP2-WT and AP2-KO MEFs expressing EGFR under a doxycycline-inducible promoter (AP2-EGFR) were induced with doxycycline. Cells were then subjected to in vivo immunolabeling with anti-EGFR 13A9 antibody and 10 nm protein A-gold, stimulated 5 min with EGF (30 ng/mL), and fixed in the presence of ruthenium red, to distinguish PM-connected CCPs (ruthenium red positive) and internalized CCVs (ruthenium red negative) structures. Bar, 100 nm. (D) Morphometrical analysis of (C). Left: CCP number was normalized for the difference in PM length between AP2-KO MEFs versus control (∼1.5-fold increase; see Figure S3 A and ) and expressed as relative to control cells. Right: number of gold particles per CCS (CCPs + CCVs). N represents the numbers of random images (left) and CCSs (right) analyzed. Data are expressed as mean ± SEM. p values were calculated using two-tailed Student’s t test ( ∗ p < 0.05 and ∗∗ p < 0.01).

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Saturation Assay, Expressing, In Vivo, Immunolabeling, Two Tailed Test

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet: Mechanism of AP2-Independent EGFR-CME: Role of eps15/L1 and epsin1 (A) Transient KD of the μ subunit of the indicated AP complexes was performed in AP2-KO MEFs, and K e of 125 I-EGF internalization was calculated. Data are mean ± SD (two replicates). (B) K e of 125 I-EGF internalization in stable eps15/eps15L1-KD HeLa cells transiently depleted of epsin1, alone or in combination with AP2μ, in comparison with AP2-KD and clathrin-KD HeLa cells. Results are mean ± SD of two to eight independent experiments. p values were calculated using each pair Student’s t test ( ∗∗ p < 0.01 and ∗∗∗ p < 0.001). (C) AP2-WT and AP2-KO MEFs were transiently depleted as indicated. K e values of 125 I-EGF internalization are shown as mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test ( ∗∗ p < 0.01 and ∗∗∗ p < 0.001). (D) TEM analysis of CCSs in AP2-WT-EGFR MEFs, control or triple KD for eps15, eps15L1, and epsin1, induced with doxycycline (to allow EGFR expression) and stimulated with EGF (30 ng/mL). Morphometrical analysis was performed on EGFR gold-positive structures. Left: CCP number was normalized for the difference in PM length between eps15/eps15L1/epsin1 KD MEFs versus control (∼0.8 decrease; see Figure S3 A and ) and expressed relative to control cells. Right: mean number of gold particle per CCS (CCPs + CCVs). N represents the numbers of random images (left panel) and numbers of CCSs (center and right panels) analyzed. Data are expressed as mean ± SEM. p values were calculated using each pair Student’s t test ( ∗ p < 0.05). (E) Automated analysis of clathrin-coated structure formation at the plasma membrane from 196 traces containing CLTA-TagRFP and AP2σ-EGFP (clathrin+/AP2+; left panel), 200 traces devoid of AP2σ-EGFP (clathrin+/AP2−; middle panel) from 10 MEF AP2-WT/triple-KD, or 154 traces from 10 MEF AP2-KO/triple-KD cells (right panel).

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Expressing

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet: EGF-Dependent Signaling and Migration in AP2-Depleted Cells (A) HeLa cells were subjected to AP2 KD or eps15/L1/epsin1 KD followed by 125 I-EGF degradation assay at low EGF concentration (1.5 ng/mL; see ). At the indicated time points, (1) degraded EGF (top) represents the TCA soluble fraction of 125 I-EGF recovered in the medium and intracellularly, and (2) recycled EGF (bottom) represents the TCA insoluble fraction of 125 I-EGF recovered in the medium. Results are mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test ( ∗ p < 0.05 and ∗∗ p < 0.01). (B) Top: AP2-WT and AP2-KO MEFs were stimulated with low dose EGF (1.5 ng/mL) for the indicated times. Lysates were subjected to IB with the indicated antibodies. Bottom: quantitation of phosphoAKT signal normalized to total AKT (pAKT/AKT) and represented as percentage of signal in WT cells at 5 min of EGF stimulation. Results are mean ± SD of three independent experiments. p values were calculated using two-tailed Student’s t test ( ∗ p < 0.05 versus WT at each time point). (C) Left: HeLa cells were subjected to AP2 KD and/or eps15/L1/epsin1 KD in different combinations, followed by stimulation with low dose EGF (1.5 ng/mL) for the indicated times. IB was as shown. Right: quantitation of phosphoAKT signal normalized to total AKT (pAKT/AKT) and represented as percentage of signal in control cells at 5 min of EGF stimulation. Results are mean ± SD of three independent experiments. p values were calculated using two-tailed Student’s t test. ∗ p < 0.05 versus control at each time point. (D) Transwell migration assay of AP2 KD and eps15/L1/epsin1 KD HeLa cells under serum-starved (SS) conditions or in the presence of low-dose EGF (1.5 ng/mL) or 10% serum as indicated. Right: quantitation of migrating cells per field. Results are the mean ± SD of two to four independent experiments. p values were calculated using two-tailed Student’s t test ( ∗ p < 0.05 versus control in each condition).

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Migration, Degradation Assay, Concentration Assay, Quantitation Assay, Two Tailed Test, Transwell Migration Assay

Journal: Cell Reports

Article Title: Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

doi: 10.1016/j.celrep.2019.05.017

Figure Lengend Snippet:

Article Snippet: 125 I-EGF , PerkinElmer , Cat#NEX428.

Techniques: Transduction, Recombinant, Electron Microscopy, TaqMan Assay, Plasmid Preparation, Software