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Table of Contents

Abstract... 3

List of Abbreviations ... 4

1. Introduction ... 5

1.1 The ErbB family ... 5

1.2 Regulation of the ErbB receptor family ... 5

1.3 The Non-Receptor Tyrosine Kinase ACK1 ... 7

1.4 Aim of the Study ... 8

2. Material and methods ... 9

2.1 Aim 1: Investigate if ACK1 depletion elevates ErbB3 protein levels ... 9

2.1.1 Cell treatment and transfection ... 9

2.1.2 Immunoblotting... 10

2.2 Aim 2: Investigate ErbB3 elevation is seen across different cell densities ... 10

2.2.1 Cell treatment and transfection ... 10

2.2.2 Immunoblotting... 11

3. Results ... 12

3.1 ACK1 depletion elevates ErbB3 protein levels in MCF10A cells ... 12

3.2 ACK1 regulation of ErbB3 is seen across different cell densities ... 12

4. Discussion ... 15

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Abstract

The ErbB family of receptors are involved in signalling relating to cell proliferation and differentiation through activation of pathways such as PI3K and MAPK. Their overexpression is often found in different cancer types and therefore, their expression is under tight regulation. The ErbB family includes, EGFR, ErbB2, ErbB3, and ErbB4 where all but ErbB3 has a kinase domain, making ErbB3 a pseudokinase. Upon activation of the receptors, they are endocytosed through the formation of a clathrin-coated pit and are degraded in the lysosome. Interestingly, researchers have found that newly synthesised ErbB3 can also be degraded in the proteasome by protein Nrdp1. Suggesting that ErbB3 might work in a ligand-independent manner and needs additional regulatory mechanisms. ACK1 is a non-receptor tyrosine kinase that has a reported effect on EGFR by promoting receptor degradation in the autophagosome. However, their role in EGFR regulation is still debated. Therefore, this information alludes to the fact that ACK1 might influence other ErbB family members as well.

This report aims to investigate whether ACK1 influences ErbB3 levels. Through RNAi mediated knockdown of ACK1 in MCF10A cells, a novel role of ACK1 acting as a regulator of ErbB3 is hinted at. Surprisingly, these results also show that ACK1 seems to act

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List of Abbreviations

ACK1 Activated Cdc42 kinase 1

Akt Protein kinase B

AP-2 Adaptor protein 2

AR Androgen receptor

AREG Amphiregulin

ATP Adenosine triphosphate

BTC Betacellulin

Cdc42 Cell division control protein 42 homolog c-Cbl Casitas B-lineage Lymphoma

CIE Clathrin-independent endocytosis CME Clathrin-mediated endocytosis CRIB Cdc42- and Ras-interacting domain

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

EPGN Epigen

ER Endoplasmic reticulum

ErbB2 Human epidermal growth factor receptor 2 ErbB3 Human epidermal growth factor receptor 3 ErbB4 Human epidermal growth factor receptor 4

EREG Epiregulin

ESCRT Endosomal sorting complex required for transport Grb2 Growth factor receptor-bound protein 2

GTP Guanosine triphosphate

HB-EGF Heparin-binding EGF-like growth factor

JAK Janus kinase

LRIG1 Leucine-rich repeats and immunoglobulin-like domains protein 1 MAPK Mitogen-activated protein kinase

MHR Mig6 homology region

MIG6 Mitogen-inducible gene 6 mTOR Mechanistic target of rapamycin

MVB Multivesicular body

Nrdp1 Neuregulin receptor degradation protein 1 NRG1-6 Neuregulin 1-6

PI3K Phosphoinositide 3-kinase

PLCγ Phospholipase C gamma 1

RTK Receptor tyrosine kinase

SAM Sterile alpha domain

SH3 Src homology 3 domain

TGFα Transforming growth factor TNK 1/2 Tyrosine kinase non-receptor 1/2 TRAIL TNF-related apoptosis-induced ligand UBA Ubiquitin association domain

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1. Introduction

1.1 The ErbB family

The ErbB family of receptor tyrosine kinases include epidermal growth factor receptor (EGFR), ErbB2, ErbB3 and ErbB4. Overexpression of these receptors is connected to malignant cancer progression due to their involvement in promoting cell proliferation and differentiation.[1]

All ErbB family members are transmembrane proteins made up of an extracellular region, a single transmembrane domain, and an intracellular region. The extracellular region is made up of four subdomains (I, II, III, IV) which are heavily glycosylated and contain the ligand-binding site. [2,3] The intracellular region is made up of a tyrosine kinase domain and a C-terminal

regulatory domain with multiple phosphorylation sites. The tyrosine kinase domain enables all but ErbB3 to exhibit an intrinsic kinase activity that is triggered upon ligand binding, causing dimerization and activation of the receptor.[4] ErbB3 has an impaired kinase activity since some residues in the dimerization interface are substituted, making the receptor a pseudokinase. [5,6]

The ligands for the family members vary slightly. The ligands for EGFR are EGF, TGF𝛂, HB-EGF, AREG, BTC, EREG, and EPGN. ErbB2 does not have any ligands. ErbB3 and ErbB4 both recognize NRG1 and NRG2, but ErbB3 also binds NRG6. Moreover, ErbB4 additionally binds NRG3, NRG4, NRG5, HB-EGF, BTC and EPGN. [7] Ligand-induced dimerization is facilitated by all receptors except ErbB2 through conformational changes in the extracellular region, revealing the dimerization interface. Research has shown that ligands may have different efficacy exposing the dimerization interface, possibly influencing whether homo- or heterodimers are formed.[8] Activation of the ErbB family has been proposed to occur through the asymmetric dimerization model, first introduced by Zhang et al. (2006). [4] This model suggests that dimerization of the ErbB family is asymmetric where one receptor acts as the activator by binding the N-lobe of the receiving monomer through their C-lobe, stabilizing the receiver’s active conformation. The receiver can then transphosphorylate the C-terminal tail of the activator, ultimately resulting in the recruitment of adaptor proteins to the C-terminal tails of the active dimer. Most combinations of ErbB dimers are possible, but some are more common than others. Interestingly, homodimers of ErbB3 are unstable due to their impaired kinase domain. Therefore, heterodimerization to other family members is required for its activation. [9.10] The most common heterodimer formation for ErbB3 is with either EGFR or

ErbB2. [11,12] Once heterodimerization occurs ErbB3 will act as the allosteric activator for the bound receiving receptor. [12] The main pathways activated by ErbB downstream signalling are the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, affecting differentiation, survival, and proliferation.[13] Additional pathways that can be activated through the ErbB family include Ras, Src, PLCγ, JAK, and mTOR. [1, 14, 15]

1.2 Regulation of the ErbB receptor family

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expression. One of these mechanisms involves closing the ligand binding site in a resting state. As mentioned above, ligand binding exposes the dimerization interface through conformational changes, in all ErbB receptors except ErbB2. [1] Interestingly, ErbB2 takes on a native conformation resembling a ligand-activated monomer. This allows the receptor to facilitate downstream signalling in a ligand independent manner by forming either homo- or heterodimers with its other family members. This introduces problems relating to their oncogenic potential, since ErbB2 can activate downstream signalling independently of ligands through formation of homodimers. [16] Moreover, this quality makes them a strong co-receptor to the other family members. [11]

Therefore, the cell needs additional regulatory mechanisms to control ErbB expression levels. Once a receptor is activated it needs a termination mechanism to avoid overactivation of downstream signalling. Upon activation, the receptors are internalised by the cell through either clathrin-independent endocytosis (CIE) or clathrin-mediated endocytosis (CME). [17-19] The CIE pathway is believed to be used in environments with high EGF levels when the CME pathway is occupied. [17,18] The CME pathway internalizes the phosphorylated receptor into a clathrin coated pit, containing the clathrin scaffold protein AP2 (or CLAPs for ErbB3) and clathrin.[20] Once bound, the receptor is mono- or polyubiquitinated through the recruitment of E3 ubiquitin ligase, c-Cbl, using scaffolding protein Grb2. Subsequently, the ubiquitinated receptor is recognized by Cbl-binding proteins, which induce phosphorylation and monoubiquitination of epsin. [21-23] As a result, the clathrin coated pit is released through dynamin and the receptor is internalized. Once endocytosis is complete, the receptor fuses with the early endosome where its ubiquitination status determines whether it enters the recycling- or lysosomal degradation pathway. [24] If the receptor enters the recycling pathway it gets shuffled back to the membrane. However, if the receptor enters the lysosomal degradation pathway it shuffles from the early endosome to the late endosome/multivesicular body (MVB) where it is either sorted through the Golgi apparatus or fuses with the autophagosome by interacting with the ESCRT machinery. Ultimately, the autophagosome degrades the receptor through lysosomal recruitment. [19]

Studies suggest that ErbB3 is exposed to both lysosomal and proteasomal degradation. [19, 25] Lysosomal degradation targets membrane bound ErbB3 while cytosolic ErbB3 is mainly subjected to proteasomal degradation. [26] Researchers have found that, in the absence of

ligands, ErbB3 remains resting in intracellular compartments where it is believed that E3 ubiquitin-protein ligase Nrdp1 plays an important role. [25] Nrdp1 marks this resting form ErbB3 for proteasomal degradation through ubiquitination. It is also believed that Nrdp1 targets newly synthesised ErbB3 in the endoplasmic reticulum (ER), resulting in receptors still located in the ER being targeted for degradation. [26] These findings allude to a possible tumor

suppressing role of Nrdp1, since overexpression of ErbB3 is connected to tumor progression.

[27] Moreover, no ER regulatory mechanism has been uncovered for any other ErbB receptor,

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An additional signal termination mechanism for all ErbB receptors involves interactions with the negative regulators LRIG1, MIG6, and ACK1. LRIG1 is a transmembrane protein that interacts with ErbB receptors and enhances c-Cbl recruitment, resulting in receptor endocytosis and degradation. [29] Both MIG6 and ACK1 act in the cytoplasm and are implicated in tumor regulation, except that MIG6 is a tumor suppressor while ACK1 promotes tumor progression.

[30,31] MIG6 binds ErbB receptors and blocks the dimer interface and thus prevents its

activation. Researchers have also found that MIG6 enhances endocytosis and degradation of EGFR. [32] ACK1 is a non-receptor tyrosine kinase whose role in EGFR endocytosis is debated. Some studies have shown that ACK1 acts to promote receptor degradation in the autophagosome. [33] While other studies have shown that it acts in the early endosome, determining whether the receptor enters the recycling- or lysosomal degradation pathway. [34]

Another mechanism that has a reported effect on EGFR levels is contact inhibition. [35] This is

a mechanism that allows cells to inhibit each other upon contact, which is an important mechanism in high cell density environments, both in vivo and in vitro. In the case of in vitro studies of receptors, where cells often grow in a monolayer, this mechanism becomes potentially complicated. Studies have found that EGFR is subjected to this type of inhibition but, so far, no such effect has been observed for the remaining receptors. [35]

1.3 The Non-Receptor Tyrosine Kinase ACK1

Activated Cdc42-activated kinase (ACK) is a family of non-receptor tyrosine kinases encompassing ACK1 and TNK1 in humans. [36] This report focuses on ACK1, which catalyses the transfer of phosphate groups to target tyrosine residues using ATP. ACK1 is ubiquitously expressed in many different cell types, and its amplification is strongly linked to cancer progression in triple negative breast cancer, and cancer of the lung, prostate, and pancreas. Their oncogenicity comes from their ability to remove tumor suppressors and activate target proteins, such as are pro-survival kinases. [37-40]

ACK1 is composed of eight different types of domain structures: a sterile alpha domain (SAM) by their N-terminal, followed by a large kinase domain, a SRC homology 3 domain (SH3), a Cdc42- and Ras- interacting domain (CRIB), two clathrin interacting domains, a PPXY motif/WW-binding domain, a MIG6 homology region (MHR), and a ubiquitin association domain (UBA) by their C-terminal end. [39]

Activation of ACK1 is triggered by many types of upstream events. This includes signalling from integrins, GPCRs, and RTKs. [41] Its activation is connected to increased cell growth and cell migration. The proteins involved in cell growth that are affected by ACK1 induce WWOX, androgen receptor (AR), and Akt. [37-39] ACK1 phosphorylates proapoptotic WWOX on Y287,

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interactions with Cdc42 and Wiliskott-Aldrich syndrome protein (WASP). [42,43] ACK1 can

bind to GTP-bound Cdc42 through CRIB, resulting in inhibition of Cdc42s GTPase activity and membrane ruffling. [44] WASP is a protein that is important for actin filament formation. Through phosphorylating Y256 and S242 on WASP, ACK1 induces actin polymerisation and cell migration. [42] Moreover, even though ACK1 presents high oncogenicity, it can also act in a pro-apoptotic way by inducing cell death through recruiting TNF-related apoptosis-induced ligand (TRAIL). [45] Another surprising ability of ACK1 is their ability to regulate EGFR levels.

[33] Studies have found that ACK1 can colocalize to the autophagosome where it interacts with

autophagy receptor sequestosome 1 (p62/SQSTM1) to ultimately direct EGFR for lysosomal degradation. [34] Other studies have also found that ACK1 interacts directly with EGFR, using MHR, to induce clathrin-mediated endocytosis. [43]

Since ACK1 is involved in many pathways it is crucial that it is tightly regulated. ACK1 auto-inhibits itself using a proline-rich amino acid insertion upstream of MHR. This proline-rich region has been suggested to interact with the SH3 domain which consequently orients MHR to inhibit the kinase domain, resulting in an auto-inhibited state of ACK1 in the absence of activating signals from receptors. An additional regulatory mechanism used by ACK1 is a sterile-alpha motif (SAM) that helps localize ACK1 to the membrane along with trans-phosphorylation and activation of ACK1, ultimately including an additional step to activating ACK1. [46]

1.4 Aim of the Study

This study aims to explore the effects ACK1, and cell density have on ErbB3 levels in the non-carcinogenic epithelial cell line, MFC10A. The following points are dealt with in this report:

1. Investigate if siRNA mediated knockdown of ACK1 results in elevated ErbB3 levels in MCF10A cells.

2. Investigate whether the elevated ErbB3 levels are indeed due to ACK1 depletion or an indirect effect, due to changes in cell density or cell-cell contacts.

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2. Material and methods

2.1 Aim 1: Investigate if ACK1 depletion elevates ErbB3 protein levels 2.1.1 Cell treatment and transfection

MCF10A cell line was cultured in a 10 cm cell culture dish using complete Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco) supplemented with 5% horse serum, 20 ng/ml EGF, 0.5mg/ml hydrocortisone, 100 ng/ml cholera toxin, 10µg/ml insulin, and 1% L-glutamine. Transfection experiment was performed using two different siRNA, where one was a non-targeting control siRNA (siCtr, ID AM4636, Ambion) while the other siRNA targeted ACK1 (siACK1 #1, ID s19850, Ambion). Once cells reached ~80 % confluency they were trypsinised and seeded onto two 10 cm cell culture dishes. 2000000 cells/plate were seeded for control siRNA while 2400000 cells/plate were seeded for siACK1. Lipofectamine™ RNAiMAX transfection reagent (Invitrogen) was used for siRNA mediated knockdown of ACK1, according to suppliers’ protocol. The media was replaced 8 h transfection, since long term exposure to lipofectamine is toxic to the cells. At 24 h post-transfection the cells were trypsinised again and seeded at 600000 cells/well into two 6-well cell culture plates, see schematic overview of experimental setup in Figure 1. The cells were left overnight to adhere to the cell dish. Cell starvation was performed 48-post transfection for six hours using deprivation DMEM/F12 media with 2% horse serum, 0.5µg/ml hydrocortisone, 100ng/ml cholera toxin and 1% L-glutamine. Subsequently, the ligand neuregulin-1β was added to the cells at time intervals: 0 min, 30 min, 60 min, and 120 min. The remaining two wells on each plate were empty. Cell lysates obtained using RIPA buffer (50mM Tris pH 7.5, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM EDTA) supplemented with PPase and protease HALT mix. Protein concentration of cell lysates was determined using Pierce™ BCA protein assay kit.

Figure 1: Schematic image of transfection setup. The left 6-well cell culture plate was treated with non-targeting control siRNA (siCtr) while the right plate was treated with ACK1 non-targeting siRNA (siACK1). The ligand for ErbB3, neuregulin-1β, was added at time intervals: 0 min, 30 min, 60 min, and 120 min. The wells

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2.1.2 Immunoblotting

Cells were lysed using SDS-sample buffer (120nM Tris-HCl pH 6.8, 3% SDS, 15% glycerol, 0.03% bromophenol blue, and 75 mM dithiothreitol) and RIPA buffer. Samples were then run on three 8% polyacrylamide gels at 100 V for 90 min followed by transfer to PVDF membranes (Hybond™-P) at 100 V for 90 min. Membranes were cut according to target protein size and their loading control, Akt. The blots were then blocked using 4% BSA or milk, diluted in TBST buffer (20mM Tris, 150mM NaCl, and 0.1% Tween 20) for 30 minutes. Subsequently, the primary antibodies anti-ErbB3, anti-phospho-ErbB3, anti-Ack1, and anti-Akt were added for four hours, see Table 1. The blots were then washed using TBST before adding the secondary antibodies HRP-conjugated goat anti-mouse or anti-rabbit (The Jackson Laboratory) for one hour. Membranes were developed using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific). Imaging was performed using ChemiDoc MP imaging system (Bio-Rad).

Table 1: Primary antibodies used during western blot experiment for Aim 1. The antibodies were targeting ErbB3, pErbB3, ACK1 and Akt. The brands and article numbers are found in the right column.

Antibodies Brand, article number

Anti-ErbB3 Cell Signalling Technology, #12708 Anti-phospho-ErbB3 Cell Signalling Technology, #4791

Anti-ACK1 Santa Cruz Biotechnology, sc-28336 Anti-Akt Cell Signalling Technology, #9272

2.2 Aim 2: Investigate if ErbB3 elevation is seen across different cell densities 2.2.1 Cell treatment and transfection

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Figure 2: schematic overview of transfection experiment in cell density experiment. The 12-well plates were transfected with the following siRNA: Plate 1 with control siRNA, plate 2 with siACK1 (#1), and plate 3 with siACK1 (#2). All plates were seeded with MCF10A cells in the ranges 50000-400000 cells/well.

2.2.2 Immunoblotting

The Western blot experiment was performed in the same way as for aim 1. However, antibodies anti-EGFR, anti-ErbB3, anti-Ack1, anti-Akt, and anti-GAPDH were used, see Table 2.

Table 2: Primary antibodies used during western blot experiment for Aim 2. The antibodies were targeting EGFR, ErbB2, ErbB3, ACK1, Akt and GAPDH. The brands and article numbers are found in the right column.

Antibodies Brand, article number

Anti-EGFR Cell Signalling Technology, #2232 Anti-ErbB2 Millipore, 06-562

Anti-ErbB3 Cell Signalling Technology, #12708 Anti-ACK1 Santa Cruz Biotechnology, sc-28336

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3. Results

3.1 ACK1 depletion elevates ErbB3 protein levels in MCF10A cells

Immunoblotting of MCF10A cells treated with siRNA targeting ACK1 showed increased levels of ErbB3, compared to the control sample. As seen in Figure 3, the levels of ErbB3 in the control siRNA sample expressed steady-state levels of ErbB3, since they remained unchanged over time. However, in the samples treated with siACK1 there was an increase in ErbB3 levels. There was a higher level of pErbB3 compared to ErbB3 in both siACK1 and siCtr samples. This makes sense since ligand stimulation causes phosphorylation of ErbB3. Moreover, the siACK1 samples showed significantly higher levels of pErbB3 than the control. However, it did not seem like ligand stimulation had a dramatic effect on ErbB3 and pErbB3 levels, since the levels remained similar across the different time intervals, with only a slight decrease in pErbB3 levels over time. The immunoblotting against ACK1 showed that the knockdown was successful, since the blot did not show any ACK1 in the siACK1 samples. Quantification was performed by normalising the band intensities against Akt, which acted as the loading control.

Figure 3: ACK1 depletion increases ErbB3 and P-ErbB3 levels in MCF10A cell line. Immunoblotting of MCF10A cells treated with neuregulin-1β at indicated time intervals in either control siRNA (siCtr) or ACK1 targeting siRNA (siACK1 #1, ID s19850). Immunoblotting performed against ErbB3, phospho-ErbB3 (pErbB3)

and ACK1, with Akt acting as loading control for the individual targets. Quantification of band intensity normalised against Akt, for both siCtr and siACK1.

3.2 ACK1 regulation of ErbB3 is seen across different cell densities

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Figure 4: Cell images of MCF10A cells. Row one show images of cells treated with control siRNA, row two show siACK1 (#1) treated cells while row three show siACK1 (#2) treated cells. The images aligned vertically

were chosen to visually correspond with each other regarding the number of cells.

Immunoblotting of chosen cell lysates showed a difference in expression when comparing ACK1 knockdown to control samples, see Figure 5. Quantification was performed by normalising the band intensities against Akt, which acted as the loading control. The results showed decreased or similar EGFR protein levels in siACK1 treated cells. The results for ErbB2 were not quantifiable, and therefore these results were not reliable. The results for ErbB3 showed a decrease in protein levels in the higher cell density samples of siACK1, compared to the control. The middle samples also showed a slight decrease in protein levels for siACK1 (#1) treated cells, however siACK1 (#2) showed no difference when compared to the control. Moreover, the only samples that showed an increase in ErbB3 protein levels for siACK1 treated cells were the ones with the lowest cell densities. The blot against ACK1 showed that the samples treated with siACK1 (#1 and #2) had a successful knockdown of ACK1.

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Figure 5: Immunoblotting of MCF10A cells in different cell densities. Samples were either treated with control siRNA (siCtr) or ACK1 targeting siRNA (siACK1 #1 or siACK1 #2). Cell samples range from

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4. Discussion

The results in Figure 3 suggests that ACK1 can act on ErbB3 in a ligand-independent manner, since ErbB3 upregulation is seen at both 0 h and 2 h in the ACK1 knockdown samples. Indeed, if ACK1 could regulate ErbB3 in a ligand-independent manner it would not be the first reported protein to do so. Research has shown that ErbB3 can be endocytosed in environments both lacking and containing ligands, where it is sometimes recycled to the membrane and other times it is degraded. [17-19] Moreover, even nascent ErbB3 is target for degradation by E3

ubiquitin-protein ligase Nrdp1. [25, 26] A few possible hypotheses to the mechanism of ACK1 include that ACK1 promotes ErbB3 endocytosis and turnover, that it regulates the endocytic recycling of ErbB3 or that ACK1 inhibits ErbB3 transcriptionally. All these hypotheses explain the elevated protein levels in ACK1 knockdown cells, but it is not known if ACK1 operates directly with ErbB3 or if it functions indirectly by acting upstream of ErbB3 regulatory mechanisms. Therefore, more research is needed to find the exact mechanism of ACK1. For example, using immunoblotting to investigate if RNAi-mediated depletion of ACK1 affects steady state or turnover of ErbB3 in MCF10A cells.

An interesting observation made during siRNA transfection of MCF10A cells was that the cells treated with control siRNA reached high confluency faster than cells treated with ACK1 targeting siRNA. This raises suspicion regarding the elevated ErbB3 protein levels in siACK1 treated cells. Mainly, whether these results are due to ACK1 depletion or due to an indirect effect from changes in cell density or cell-cell contacts. The results in this report cannot exclude the possibility of an indirect effect, caused by off target effects from ACK1 knockdown or from changes in cell density, causing elevated ErbB3 levels. However, that does not mean that ACK1 does not affect ErbB3. On the contrary, both ACK1 and the cell density seem to affect ErbB3. It is evident that high densities cause declined protein levels of ErbB3 as seen in Figure 5. This effect has been previously reported in the same cell line by Pilia et al., (2018), which strengthens the results in this report. [47]

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both high and low cell densities. By extending the research into the endocytosis and turnover of ErbB3 caused by ACK1 and cell density, this question could be clarified.

For future experiments, other cell lines, in addition to MCF10A, should be used to validate the results in this report. Additionally, the use of another loading control for the Western blots should be considered. In this report Akt was used as loading control, however there are some complications to consider when using this. Mainly, the fact that it is highly abundant in the cell. Moreover, Akt is activated through ACK1 and could therefore affect the reliability of the results, since some cells are depleted of ACK1. Some alternative proteins that can be used as a loading control are Hsp70, ALIX, or PARP-1. It is however difficult to find a good loading control since cell signalling is highly connected and cell treatment can have unexpected effects on many proteins. Additionally, immunoblotting is only regarded to be semi-quantitative in that it cannot measure the protein quantity, but rather provides a relative comparison of protein levels. Therefore, the use of an additional method to detect protein levels could be used to complement the results from immunoblotting.

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