2.8.1
Treatments
Cells were seeded into 6 cm dishes in duplicates. After 72h, at approximately 50% confluence, cells were treated with the corresponding inhibitors. Dilutions were made in OptiMEM if not stated otherwise. Total volume of the medium was 2 mL for incubation overnight. The application volume of the inhibitors was equalised to 2 μL per dish. If not stated otherwise, the reconstitution reagent was DMSO. An untreated and vehicle control were included.
2.8.2
Inhibitors of various pathway
In this section a variety of inhibitors (table 2.5) were used targeting several pathways. The aim was to perturb the expression of claudin1 to identify which pathway(s) are involved in ADAM15-mediated upregulation. The origin and mode of action will be explained for those inhibitors which helped identifying the underlying pathway of claudin1 regulation.
Table 2.5: Inhibitors used in this project.
Inhibitor Com- pany Cat. no. Stock conc. Final conc. Target Bim1 Millipore 203290 1 mM 1 μM Novel and
classical PKC FLLL31 Sigma F9057 5 mM 5 μM STAT3 Gefitinib see footnote1 500 μM 500 nM EGFR
Table 2.5 Continued: Inhibitors used in this project. Inhibitor Com- pany Cat. no. Stock conc. Final conc. Target G¨o6976 Merck/ Millipore 365250 1 mM 1 μM Ca2+– dependent PKCα and β Ku0063794 Sigma SML0382 1 mM 1 μM mTOR LY294002 CST #9901 50 mM 50 μM PI3K PD98059 Sigma P215 10 mM 10 μM MAPK PI-103 Selleck-
chem S1038 1 mM 1 μM PI3K PP2 Sigma P0042 100 μM 100 nM Src family kinase Rapamycin Merck/
Millipore 553210 100 μM 100 nM mTOR Rottlerin Merck/
Millipore 557370 5 mM 5 μM PKCδ SB203580 Sigma S8307 10 mM 10 μM p38MAPK
In the following section I am going to elaborate on the inhibitors which were of particular interest.
2.8.2.1 PI3K inhibitors
In this chapter two inhibitors were used to target the PI3K pathway - LY294002 and PI-103.
LY294002 was, along wortmannin, one of the first PI3K inhibitors. Since it binds to all class I variants it belongs to the category of “pan-inhibitors”. It is a synthetic compound based on the flavonoid quercetin448. LY294002 is widely used, due to its higher specificity and stability in solution, compared to wortmannin448. Although, both mode of action is to target the ATP binding pocket within the catalytic domain of PI3K, LY294002 is a reversible inhibitor while wortmannin is not.
LY294002 and wortmannin cannot be used in therapeutics, since they do not dis- tinguish between isoforms. Studies show that PI3K isoforms possess non redundant functions downstream of different receptors449. Therefore, there is still ongoing need for inhibitors which specifically inhibit each isoform.
One of the newer and more selective inhibitors is the synthetic pyridinylfuranopy- rimidine based inhibitor PI-103. It selectively inhibits the p110/α/β/γ/δ subunits in varying concentrations450. As LY294002 and wortmannin, PI-103 also competes for ATP and interacts with the ATP binding pocket of the p110 subunit451.
CHAPTER 2. GENERAL METHODOLOGY 105
2.8.2.2 Mammalian target of Rapamycin (mTOR) inhibitors
In this chapter two inhibitors were used to target the mTOR pathway - Rapamycin and Ku0063794.
Rapamycin was the first drug identified which targets mTOR. That is the reason why mTOR is named the mammalian target of rapamycin. Rapamycin was discovered in soil samples from the island of Rapa Nui452 (Easter Islands) and is produced by the bacterium Streptomyces hygroscopicus. It belongs to the group of macrolides and has a immunosuppressant mode of action. Its inhibitory behaviour results from the ability to bind FK506-binding protein 12 (FKBP12), a small protein which specif- ically associates with mTOR close to the kinase domain. Upon binding a complex is formed thus ceasing its activity337,453,454.
Another and rather recent inhibitor against mTOR is Ku0063794. It is small molecule which inhibits both mTORC1 and mTOR2455. The half maximal in- hibitory concentration (IC50) is approximately 10 nM. It is highly specific, since the activity of 76 other kinases as well as 7 lipid kinases is not affected even with 103-fold higher concentration455. Importantly, class 1 PI3Ks are not affected ei- ther. Ku0063794 can permeate the cell membrane and suppresses phosphorylation of Akt and S6K. Experiments show that Ku0063794 induces G1-cell-cycle arrest and
suppress cell growth.
2.9
Immunofluorescence
2.9.1
Pretreating of coverslips
If not stated otherwise, 3 x 16 mm glass cover slips were transferred into a 35 mm plastic dish and covered with growth medium without any antibiotic. The cover slips were incubated for at least 24h at 37◦C and 5% CO2.
2.9.2
Plating of cells
Before cells were seeded onto the coated cover slips the medium was changed cor- responding to the cell line described in subsection 2.2. Depending on the cell line between approximately 5 x 104 and 1.5 x 105 were seeded and grown at least 72h to assure a natural growth pattern.
2.9.3
Staining of IF slides
Cover slips were fixed in 4% (v/v) formaldehyde (FAH) for 20 min at RT, then washed once in PBS. After that the cells were permeabilised with 0.1% (w/v) saponin
in PBS at RT for 2 min. The cover slips were washed in PBS and blocked in 1% (w/v) BSA at RT for 30 min. The primary antibody probing was done in blocking buffer overnight at 4◦C. A list of used antibodies is shown in table 2.6. Following overnight incubation the cover slips were washed three times in blocking buffer and then incubated with AlexaFluor (AF) secondary antibody at RT for 1h in the dark. Coverslips were then washed three times with PBS and mounted on a microscope slide using Prolong mounting media (with DAPI). The slides were dried in the dark overnight at RT.
Table 2.6: Detailed information about antibodies used for Immunofluoerescence
Antibody Supplier Catalogue
number Source Dilution ADAM15 (ECD) R&D MAB935 Mouse 1:100 AF488 (α-mouse) Invitrogen A11001 Goat 1:1,000 AF488 (α-rabbit) Invitrogen A11008 Goat 1:1,000 AF568 (α-mouse) Invitrogen A11031 Goat 1:1,000 AF568 (α-rabbit) Invitrogen A11011 Goat 1:1,000 Claudin1 Invitrogen 37-4900 Mouse 1:100 Claudin1 Invitrogen 51-9000 Rabbit 1:100 Paxillin Abcam ab32084 Rabbit 1:250 Phalloidin1 Invitrogen A12379 Amanita
phalloides 1:40 V5 Invitrogen 46-0705 Mouse 1:500 Vinculin Santa Cruz sc-73614 Mouse 1:100 ZO1 CST #8193 Rabbit 1:200
2.9.4
Examination of IF slides
Coverslips were examined using a Leica SP5 confocal microscope. For AlexaFluor- 488 fluorescent dyes an argon laser with 488 nm excitation wavelength was used. For AlexaFluor-568 fluorescent dyes a helium-neon laser with 543 nm excitation wavelength was used. DAPI was examined with a 405 nm diode laser. If not stated otherwise acquisition of the images was performed at 400 Hertz scan speed with a resolution of 1024 x 1024 pixel.
CHAPTER 2. GENERAL METHODOLOGY 107
2.10
shRNA knockdowns
The production of lentiviral particles and generation of the knockdown cell lines were performed by Dr Zaruhi Poghosyan.
2.10.1
Production of lentiviral particles
293FT cells were cultured in DMEM medium supplemented with 10% FBS, 100 units/mL Penicillin, 100 μg/mL Streptomycin, 4 mM L-Glu, 1 mM MEM sodium pyruvate, 0.1 mM MEM non-essential amino acids (NEAA) and 500 μg/mL Ge- neticin. Cells were maintained in a humidified incubator at 37◦C with 5% CO2. A day prior to transfection 293FT cells were seeded into 10 cm dishes with 5 x 106 cells using growth medium without antibiotics which will be referred to as trans- fection medium. On the day of transfection the medium was replaced with Opti- MEM. Transfection was done according to manufucaturer’s protocol using Lipofec- tamine 2000 and ViralPowerTM packaging mix containing plP1, plP2, plP/VSVG and pLKO.1 expression constructs. An overview of the used plasmids is shown in table 2.7. A day after transfection the medium was replaced with transfection medium. After 48h the supernatants were collected and snap frozen in aliquots. Lentiviral particles were stored at -80◦C until transduction.
Table 2.7: shRNAs used for stable knock down cell lines
Supplier Catalogue number Target Description Sigma NM 003815.3-1890s21c15 ADAM15 Mission shRNA Sigma NM 003815.3-1361s21c1 ADAM15 Mission shRNA Sigma NM 003815.3-1736s21c1 ADAM15 Mission shRNA Sigma NM 003815.2-497s1c1 ADAM15 Mission shRNA Sigma NM 003815.2-1076s1c1 ADAM15 Mission shRNA Sigma NM 021101.3-902 CLDN1 Mission shRNA Sigma NM 021101.3-402 CLDN1 Mission shRNA Sigma NM 021101.3-305 CLDN1 Mission shRNA Sigma NM 021101.3-626 CLDN1 Mission shRNA Sigma NM 021101.3-627 CLDN1 Mission shRNA
2.10.2
Generation of stable knock down cells using shRNA
T47D or MDA-MB-231/ADAM15 A expressing cells were seeded into 35 mm dish with 2.5 x 105 cells each in complete growth medium without antibiotics. The fol- lowing day, shClaudin1 or shADAM15 containing lentiviral particles were added to
the cells. 8 μg/mL of Polybrene R (hexadimethrine bromide) was added to enhance transduction. A day after infection the medium was replaced with fresh transfec- tion medium. On the following day the medium was replaced with complete growth medium containing 1 μg/mL Puromycin for selection. Cells got expanded and knock- down efficiency determined by western blot analysis as described in section 2.7.