Coupling receptor activation to cytoskeleton changes 2

Chapter 2 – Material and Methods 2.1 Preparation of Competent Bacteria Cells for Transformation via Electrophoration E.coli of XL-1 Blue For DNA production or BL-21For protein productio

Chapter 2 – Material and Methods

2.1 Preparation of Competent Bacteria Cells for Transformation via Electrophoration

E.coli of XL-1 Blue (For DNA production) or BL-21(For protein production) strains

were streaked on plain LB agar plate under sterile condition and incubated overnight at 37 0C

A single colony was inoculated into 50ml of LB and incubated overnight at 37 0C using a shaker The culture was diluted 20x into 400ml of LB and incubated at 37 0C using a shaker for around 2 hrs till the OD660 was about 0.6 to 0.8 The cooled bacteria were spun down at

4000 rpm for 15 min at 4 0C The supernatant was discarded and the pellet was resuspended in ice cold deionized water for desalting This step was repeated and the bacteria pellet was resuspended in cold water with 10% glycerol and transferred to Falcon tube on ice The bacteria were spin at 2800 rpm for 15 min at 4 0C The supernatant was carefully removed and the bacteria were resuspended in 1ml of cold water with 10% glycerol The bacteria suspension were aliquoted into sterile eppendorf tube, snapped freezed and store at -80 0C

2.2 Preparation of Competent Bacteria Cells for Transformation via Heat Shock method

XL-1 Blue (For DNA production) or BL-21(For Protein production) bacteria were streaked on plain LB agar plate under sterile condition and incubated overnight at 37 0C A single colony was inoculated into 50ml of LB and incubated overnight at 37 0C using a shaker The culture was diluted 20x into 400ml of LB and incubated at 37 0C using a shaker for around 2 hrs till the OD660 was about 0.6 to 0.8 The cooled bacteria were spun down at 4000 rpm for 15 min at 4 0C The supernatant was discarded and the pellet was resuspended in 15ml of ice cold 0.1M CaCl2 and incubated in ice for 40min The bacteria were spun down at

4000 rpm for 15 min at 4 0C and the pellet was resuspended in 8ml 0.1M CaCl2 with 20% glycerol The bacteria were aliquoted into sterile eppendorf tube, snapped freeze and store

-800C

2.3 Cloning

The gene of interest was typically amplified via PCR with primers containing suitable restriction sites The amplified product of the correct size (analyzed using gel electrophoresis) was then digested overnight at 370C with the suitable restriction enzymes (NEB) Meanwhile, the desired vector was also digested for a few hours at 370C The digested samples were diluted with 6X DNA loading buffer (5mM Tris-Cl pH 8, 0.5mM EDTA, 50% glycerol, bromophenol blue) into a final concentration of 1X DNA loading buffer and the samples were ran in agarose gel electrophoresis (Invitrogen, Ultrapure Low melting point agarose) The band of the correct size was excised and melted at 70 0C for about 10 mins The vector and the insert were mixed at a ratio of 1:10 and incubated with T4 ligase (Fermentas) in T4 ligase buffer containing 10 mM ATP (New England BioLabs) and incubated at 16 0C overnight for ligation

After ligation, a few microliters of the samples were transformed into the E.coli

XL-1 blue strain via either electrophoration or heat shock method Briefly, for heat shock method, the thawed bacteria on ice were incubated with the 10ul of ligated mixture (diluted 2x with water) on ice for 30 min and immediately transferred into 42 0C water bath for 1 min The bacteria mixture were cooled immediately on ice for 2 min and allowed for recovery in SOC buffer (see below) at 37 0C for 1 hr before plating on antibiotic containing LB agar plate (Ampicillin 50ug/ml or Kanamycin 30ug/ml) For electroporation method, thawed bacteria on ice was mixed with 10ul of the ligated mixture (diluted 2x with water) and transferred into the provided electroporation cuvette The bacteria were electroporated using the Bio-Rad Micro-Pulser and allowed for recovery in SOC buffer and plated The plates were incubated overnight at 37 0C and a few colonies were inoculated in the antibiotic-containing LB broth (4ml) and incubated overnight at 37 0C in a shaker

The bacteria cultures were then spun at 3500rpm for 10min to collect the pellet and subjected to plasmid purification using the mini-prepation method (Qiagen) A small volume

of the purified plasmids were digested with suitable restriction enzymes and ran in agarose gel electrophoresis to diagnose whether the insert is successfully cloned in

Confirmation was done via DNA sequencing of the plasmid using designed primers Briefly, a few microliters of the plasmid was mixed with specific 5‟ end or 3‟ end primers and ABI big dye containing the DNA polymerase and tagged nucleotides The reactions were allowed to carry out at:

Step1: 96 0C -30s

Step2:96 0C -10s

Step3:50 0C - 5s

Step4:60 0C – 4min

Step5:4 0C - 

Repeat Step 2-4 for 30cycles

After the PCR, the reaction mix was then sent to a DNA sequencing unit for reading the sequences The sequences were aligned with the relevant sequences from Pubmed using the BLAST service and checked for proper reading frame and lack of mutation The bacteria containing the correct insert was inoculated into 200ml/400ml LB culture with antibiotic for subsequent purification of the plasmid in medium or large scale (midi-prep or maxi-prep) respectively

2.4 DNA purification

The desired plasmid transformed in bacteria was purified using Qiagen kit Briefly, the bacteria were lysed under alkaline condition and the DNA was specifically adsorbed onto the provided column under high salt conditions The plasmids were eluted under low salt conditions The eluted DNA was precipitated by isopropanol and the dried DNA pellet was dissolved in TE buffer or water

2.5 Electrophoresis

Agarose gel electrophoresis was performed at 100 volts or 90 volts (low melting point agar) for around 1 hr in 1x TAE buffer (see below) The agar gel was prepared with 1%

agarose in 1x TAE buffer with 5ug of Ethidium Bromide in 100ml of molten agar The molten agar was allowed to solidify in suitable cast

For SDS-PAGE analysis (Sodium dodecyl sulfate polyacrylamide gel electrophoresis), the polyacrylamide gel of the suitable percentage (usually 10%, see below) was prepared and ran using 1x SDS running buffer (see below) at 160 volts for at least 1 hr The gel was then stained using Coomassie blue and destained with 40% Methanol/10% Glacial Acetic Acid or transferred onto PVDF membrane (Polyvinylidene fluoride) using the „Wet transfer‟ method

in 1x Transfer buffer

2.6 Generation of RTK constructs

Constructs encoding the ORF of the kinases or complete EST clones were obtained

from Open biosystem The constructs were cloned into pcDNA3 with a C-terminal Flag-tag using standard PCR protocols The respective primers used are documented in Table 3

Table 3 Primers for generating RTK constructs

ALK 5' -TCCGAATTCATGGGAGCCATCGGGCTCCTG-3'

5' -CCGCTCGAGGGGCCCAGGCTGGTTCAT-3' Axl 5‟ –TTGAAGCTTATGGCGTGGCGGTGCCCCAGG-3‟

5‟ –AGGGGCGGCCGCGGCACCATCCTCCTGCCC-3‟

DDR1 5' -TCAGAATTCATGGGACCAGAGGCCCTGTCA-3'

5' -TGTCTCGAGCACCGTGTTGAGTGCATCCTC-3' EPHB4 5‟–GGCAAGCTTATGGAACTCCGGGTGCTGCTC-3‟

5‟–TCCTGCGGCCGCGTACTGCGGGGCCGGTCC-3‟

EPHA3 5‟ –ACCGGATCCATGGATTGTCAGCTCTCCATC-3‟

5‟ –CGTCTCGAGCACGGGAACTGGGCCATTCTT-3‟

FGFR1 5‟ –AGAGGATCCATGTGGAGCTGGAAGTGCCTC-3‟

5‟ – GGGTGCGGCCGCGCGGCGTTTGAGTCCGCC-3‟

v-fms 5‟ –GCGGGATCCATGGTCAGCTACTGGGACACC-3‟

5‟ –AGTCTCGAGATGTTTTACATTACTTTGTGT-3‟

LTK 5‟-ACCGGATCCATGGGCTGCTGGGGACAGCTG-3‟

5‟-AAGGCGGCCGCGGAGCGATAAGTGGGATT-3' NT3 5‟ –AATAAGCTTATGGATGTCTCTCTTTGCCCA-3‟

5‟ –GAGGGTACCAAAGCCATGACGTCCTTTGCT-3‟

PDGFR 5‟ –AAGGAATTCATGCGGCTTCCGGGTGCGATG-3‟

5' -CGAGCGGCCGCTTCAGGAAGCTATCCTCTG-3' RET 5‟ –GCAAAGCTTATGGCGAAGGCGACGTCCGGT-3‟

5‟ –ACAGGCGGCCGCGAATCTAGTAAATGCATG-3‟

TEK 5‟ –GAACTCGAGATGGACTTGATCTTGATCAAT-3‟

5‟ –TCTGGTACCGGCCGCTTCTTCAGCAGAACA-3‟

2.7 Generation of ACK1, MIG6 and Grb2 constructs

ACK T1 was constructed via PCR using the primers 5'–CTAGGATCCTGCCC GCCCTCCCTGGCGCAG-3' and 5'-CAGG TCGACTTAAGGCACAGGCAGGGGGGT-3',

which contain flanking BamHI or SalI restriction sites ACK T2 and T3, which were previously cloned into pXJ-Flag vector, were re-cloned into pXJ-GST via BamHI/HindIII

restriction sites MIG6 fragment was PCR using the following primers 5'-CACGGATCCGCTGGCTCCTTTA ACAAGCCA-3' and 5'-CGCCTCGAGCTAAG GAGAAACCACATAGGA-3' The amplified products were cloned into pXJ-GST via

BamHI/XhoI restriction sites Grb2 mutants were created via 2-steps PCR mutagenesis and

sequenced to confirm the mutations The mutated full length products were cloned into

pXJ-HA via BamHI/XhoI sites

2.8 Generation of CRMP, MAP6 and Stathmin constructs

Constructs encoding the ORF of rat CRMP1-4 and MAP6 were cloned into

pXJ40-HA via HindIII/XhoI restriction sites The respective primers used are documented in Table 1

(Supplementary experimental procedures) The C-terminal CRMP1(480-572),

CRMP1(491-572) and CRMP2(480-CRMP1(491-572) were cloned into pXJ-GST using PCR to introduce HindIII and

XhoI cloning sites Full length CRMP1 and CRMP2 were cloned into a modified pET21D

His6 vector for expression in bacteria CRMP1(491-572) was cloned into pGEX4T1 using

introduced 5'BamHI and 3'XhoI restriction sites Mouse stathmin was amplified from total cDNA and cloned via 5'BamHI and 3'XhoI restriction sites into pGEX4T1 The primers are

listed in the Table 4

Site-directed mutagenesis of CRMP1 at Ser-522, Ser-537, Ser-540, Ser-542 and

Thr-554 were mutated and cloned into pXJ-HA as for wildtype The primers employed are given

in Table 4.The mutants were created via 2-steps PCR mutagenesis and sequenced to confirm the mutations

Table 4 Primers for Cloning CRMP, CRMP mutants and Stathmin

CRMP1 Full

Length Primer

UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3' CRMP2 Full

Length Primer

UP: 5‟- GGA TCC AAG CTT ACC ATG TCT TAT CAG-3‟

LP: 5‟- CGG GGC CTC GAG TTA GCC CAG GCT GGT-3‟

CRMP3 Full

Length Primer

UP: 5' -TGT AAG CTT ATG TCC TTC CAA GGC AAG AAG -3' LP: 5' - ACC GGT ACC CTA GGA AAG AGA CGT GAT GTT -3' CRMP4 Full

Length Primer

UP: 5' - ATC AAG CTT ATG TCC TAC CAG GGC AAG AAG -3' LP: 5' - TGG CTC GAG TTA ACT CAG GGA TGT GAT GTT -3'

CRMP1 491-572 UP: 5' - GTT AAG CTT TTG CAT AGT GTC TCA AGG GGC -3'

LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 480-572 UP: 5' - CAC AAG CTT CAG CGT GTC AGG ATC AGG AGC AAG -3'

LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP2 480-572 UP: 5' - TTT AAG CTT AAA CGC ATC AAG GCA AGG AGC -3'

LP: 5‟- CGG GGC CTC GAG TTA GCC CAG GCT GGT-3‟

CRMP1 (1-490) UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

LP: 5‟ – GAC CTC GAG TCA CCC GAA AAC CTT GCT CCT -3‟ CRMP1 (1-520) UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

LP: 5' - AGA CTC GAG TTA CTT GGC AGA AGG AGC AGG - 3' CRMP1 (1-550) UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

LP: 5' - TGT CTC GAG TTA ATT GTT GTC ATC TAT CTG -3' CRMP1 (1-560) UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

LP: 5' - GCC CTC GAG TTA CGC CAC GAT GCG GTG -3' CRMP1 (1-565) UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

LP: 5' -GGT CTC GAG TTA GCG GCC ACC AGG GGG -3' CRMP1 S522A UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

ALP: 5' –CTG GTG TTT AGA AGG CGC GGA CTT GGC AGA AGG -3' AUP: 5‟ –CCT TCT GCC AAG TCC GCG CCT TCT AAA CAC CAG -3‟ LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 S522D UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

DLP: 5' –CTG GTG TTT AGA AGG ATC GGA CTT GGC AGA AGG -3' DUP: 5‟ –CCT TCT GCC AAG TCC GAT CCT TCT AAA CAC CAG -3‟ LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 S537A UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

ALP: 5' - TGA CAA GCT GAA GTT GGC CTG GTG GAG GTT-3' AUP: 5' - AAC CTC CAC CAG GCC AAC TTC AGC TTG TCA -3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 S537D UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

DLP: 5' - TGA CAA GCT GAA GTT GTC CTG GTG GAG GTT-3' DUP: 5' - AAC CTC CAC CAG GAC AAC TTC AGC TTG TCA -3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 S540A UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

ALP: 5' - GGC ACC TGA CAA GGC GAA GTT GGA CTG GTG-3' AUP: 5' - CAC CAG TCC AAC TTC GCC TTG TCA GGT GCC-3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3' CRMP1 S540D UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

DLP: 5' - GGC ACC TGA CAA GTC GAA GTT GGA CTG GTG-3' DUP: 5' - CAC CAG TCC AAC TTC GAC TTG TCA GGT GCC-3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3' CRMP1 S542A UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

ALP: 5' - TAT CTG GGC ACC TGC CAA GCT GAA GTT GGA-3' AUP: 5' - TCC AAC TTC AGC TTG GCA GGT GCC CAG ATA -3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 S542D UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

DLP: 5' - TAT CTG GGC ACC GTC CAA GCT GAA GTT GGA-3' DUP: 5' - TCC AAC TTC AGC TTG GAC GGT GCC CAG ATA -3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 T554A UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

ALP: 5' - GAT GCG GTG CCC TGC ACG CCT TGG ATT GTT -3' AUP: 5' - AAC AAT CCA AGG CGT GCA GGG CAC CGC ATC -3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

CRMP1 T554E UP: 5‟ –TCC GGA TCC AAG CTT ATG TCT CAT CAG -3‟

ELP: 5' - GAT GCG GTG CCC TTC ACG CCT TGG ATT GTT -3' EUP: 5' - AAC AAT CCA AGG CGT GAA GGG CAC CGC ATC -3' LP: 5'- CGT CTC GAG TCA ACC GAG GCT GGT GAT -3'

Stathmin BAMUP: 5' - CTG GGA TCC ATG GCA TCT TCT GAT ATT CAG -3'

XHOLP: 5' - CGG CTC GAG TTA GTC AGC CTC AGT CTC ATC -3'

2.9 Materials for Chapter 3

High Affinity Glutathione-Sepharose beads were from GeneScript Anti-ACK1 (A-11), anti-EGFR, anti-myc, anti-actin and anti-Cdc42 antibodies were from Santa Cruz Anti-Axl (N-terminal) and anti-Anti-Axl (C-terminal) were from R&D Systems and Santa Cruz respectively Anti-Flag (M2), anti-GST antibodies and Fibronectin solution were from Sigma Anti-Grb2, anti-phosphotyrosine (4G10), anti-Rac1 antibodies and EGF were from Millipore/Upstate Anti-HA antibody (12CA5) was from Roche Molecular Biochemicals Anti-pAkt (Ser473, 4060) and anti-pERK1/2 (Thr202/Tyr204, 9101) antibodies were from Cell Signaling Technology Transwell of 8m pore size and 6.5mm diameter was from Costar Secondary antibodies used for immuno-fluorescence (IF) were from Molecular Probes

2.10 Materials for Chapter 4

Anti-HA (Y-11) and anti--actin (C4) antibodies were from Santa Cruz Anti-CRMP2 (C-terminal region) was from ECMbiosciences Anti--tubulin, Anti--tubulin, Anti-polyhistidine and Epothilone B were from Sigma Anti-CRMP1 was generated previously in the lab The reverse transcriptase – MuLV RT enzyme was from NEB and the RNase inhibitor was from Roche Specific Cdk1 inhibitor –RO3306 and Taxol (Paclitaxel, semi-synthetic) were from Calbiochem CK1 inhibitor was from Tocris Purified Bovine Brain tubulin (TL238) was from Cytoskeleton Inc The full length or Kinase Domain (KIN) of ROK constructs were provided by a colleague HRP-coupled Secondary antibodies for Western blot were from Dako (Denmark) Secondary antibodies used for immunofluorescence (IF) were from Molecular Probes

2.11 Cell cultures

COS7, OLDN-93, NIH3T3 and N1E-115 cells were maintained in DMEM supplemented with

10 % Fetal Bovine Serum (FBS) in a 37 0C incubator with 5% CO2 DU145 cells were cultured using RPMI supplemented with L-glutamine and 10% FBS and incubated under the same condition as the other cell lines

2.12 DNA transfection and RNAi

COS7 cells were transfected at 80-90% confluency with Lipofectamine 2000

(Invitrogen) at a ratio of 1ug DNA to 3ul of reagent and in conditions according to manufacturer‟s instructions Cells were harvested at 24 hrs or 48 hrs post-transfection

For RNAi, the ratio of 20pmoles to 3ul of the transfection reagent was used The SiRNAs were synthesized by Invitrogen Cells were treated with control SiRNA and SiRNAs against ACK1, Grb2 and Cdc42 The SiRNA sequences were:

ACK siA (5'-AAGAUGGUGACAGAGCUGGCA-3'),

ACK siC (5'-GCCUGUCCCACUUUGAGUAdTdT-3'), and

Grb2 SiRNA (5‟–CAUGUUUCCCCGCAAUUAUdTdT-3‟)

Cells were typically transfected for 24hrs before serum-starvation for another 24hrs before harvest For Chapter 4 experiments, The SiRNAs were synthesized by Invitrogen Double stranded CRMP2 SiRNA sequences contained 3' dTdT overhangs:

CRMP2 SiA (5'ACUCCUUCCUCGUGUACAUdTdT-3') and

CRMP2 SiB (5'CCCACUCCAGAAUGGUGAUdTdT-3')

Cells were typically used 48hrs post-transfection

2.13 Synthesis of recombinant GAS6

cDNA of human GAS6 (Open biosystem) was amplified via PCR using the primers 5'-AATGGATCCACCATGGCCCCTTCGCTCTCG-3' and 5'-TAAGCGGCCGCAAGGC TGCGGCGGG-3' The amplified product was cloned into C-terminal His-tag pcDNA3 vector via BamHI/NotI sites The construct was transfected into HEK293 cells via Lipofectamine

2000 (Invitrogen) for 4hrs before changing into serum-free media containing 4M Menadione sodium bisulfite (an analog of Vitamin K) The transfected cells were allowed to express and secrete the recombinant GAS6 into the media for 48hrs The media with the secreted GAS6 was purified via nickel beads (Ni-NTA, Qiagen) based His-tagged purification method Briefly, the media was reconstituted into a final concentration of 50mM NaH2PO4, 500mM NaCl, 10mM imidazole, 0.1% Triton-X100 and adjusted to pH 8.0 The media was allowed to flow through the nickel beads column and washed extensively with washing buffer of similar salts concentrations Recombinant GAS6 was eluted from the beads with 300mM imidazole and dialyzed against 1xPBS The protein concentration was measured via BioRad protein assay

2.14 Synthesis of recombinant proteins

Bacteria BL-21 carrying the pGEX4T1 vector, pGEX4T1 CRMP1 491-572, or pGEX4T1 Stathmin plasmid were induced with 20mM IPTG for 4hrs at room temperature for protein expression The bacteria were collected at 6krpm for 10mins at 4 0C The pellet was resuspended in 1% Triton-X, 50mM Tris pH 8.0, 1mM EDTA, 0.1mM DTT and 10% glycerol with protease inhibitor cocktail and subjected lysis via 1mg/ml lysozyme at 4 0C for 30min Complete lysis was ensured via pulsed-sonication The released proteins were separated from the cell debris via 13.5 krpm centrifugation for 30min at 4 0C The supernatants were passed through a column containing equilibrated Glutathione sepharose beads (GenScript) and washed with the lysis buffer The GST proteins were eluted with Elution buffer of 20mM Hepes, 150mM NaCl, 1mM DTT and 0.6% reduced L-Glutathione at

pH 7.3 The eluted proteins were dialyzed against PEM buffer The protein concentration was measured via BioRad protein assay

The His6-CRMP1 and His6-CRMP1 constructs were transformed into BL21 bacteria and protein expression was induced as above The protein was purified via nickel beads (Ni-NTA, Qiagen) based His-tagged purification method Briefly, the bacteria pellet was resuspended in a buffer (pH 8.0) containing 50mM NaH2PO4, 500mM NaCl, EDTA-free protease inhibitor cocktail, 10mM imidazole, and 0.1% Triton-X100 Lysis was performed as above The supernatant containing the His6-CRMP1 was allowed to flow through the nickel beads column and washed extensively with washing buffer of similar salts concentrations Recombinant His6-CRMP1 was eluted from the beads with 300mM imidazole and dialyzed against PEM buffer The protein concentration was measured via BioRad protein assay

2.15 RO-synchronization with taxol and epothilone B treatment

NIH3T3 or OLDN-93 cells were synchronized overnight with 9 M of RO-3306 The synchronized cells at G2/M phase were released via three washes with media To determine the effect of Taxol/Epothilone B on mitotic spindle CRMP2, upon 25 min into release, 100

nM of Epothilone B or 200 nM Taxol were added to the media for 15 min till the 40 min time