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Genetic Activities/Screening Approaches

siRNA migration screen using a wound healing approach

The data in this website is published information; when you reference any part of this work, please cite:
KJ Simpson, LM Selfors, J Bui, A Reynolds, D Leake, A Khvorova and JS Brugge. (2008) "Identification of genes that regulate epithelial cell migration using an siRNA screening approach." Nature Cell Biology 10:1027-1038 PubMed

See footnotes below for experiment description and category definitions.
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  GeneDescription1    MCF-10A phenotype2    Secondary screen3Wound4 ImageMorphology5Time-lapse6
1 AAK1
Entrez Gene: 22848
RNA catalog
AP2 associated kinase 1
mRNA: NM_014911
Library: Kinase
Classification: Undetermined
Final bin: Discordant
SMARTpool bin:   No change
Avg area: 1.69
Avg Alamar: 0.81
Knockdown %: 40-50
 AAK1
Link
AAK1
Link
 
2 AATK
Aliases: LMR1, AATYK, LMTK1
Entrez Gene: 9625
RNA catalog
apoptosis-associated tyrosine kinase
mRNA: XM_375495
Library: Kinase
Classification: Ser/Thr Kinase
Final bin: Discordant
SMARTpool bin:   Impaired *
Avg area: 1.67
Avg Alamar: 0.93
Knockdown %: 90-100
ERBB2: No Change
AATK
Link
AATK
Link
 
3 ABI2
Aliases: ABI-2, ABI2B, AIP-1, AblBP3, SSH3BP2, argBPIA, argBPIB
Entrez Gene: 10152
RNA catalog
abl interactor 2
mRNA: NM_005759
Library: MAR
Classification: cytoskeletal
Final bin: No Phenotype
SMARTpool bin:   No change
Avg area: 1.20
Avg Alamar: 1.01
Knockdown %: not done
 ABI2
Link
ABI2
Link
 
4 ABL1
Aliases: ABL, JTK7, p150, c-ABL, v-abl, bcr/abl
Entrez Gene: 25
RNA catalog
v-abl Abelson murine leukemia viral oncogene homolog 1
mRNA: NM_007313
Library: Kinase
Classification: Tyr Kinase
Focal Adhesion Related: Yes
Final bin: Discordant
SMARTpool bin:   Impaired *
Avg area: 1.98
Avg Alamar: 0.90
Knockdown %: 60-70
ERBB2: No Change
ABL1
Link
ABL1
Link
 
5 ABL2
Aliases: ARG, ABLL
Entrez Gene: 27
RNA catalog
v-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related gene)
mRNA: NM_005158
Library: Kinase
Classification: Tyr Kinase
Final bin: No Phenotype
SMARTpool bin:   No change
Avg area: 1.27
Avg Alamar: 0.98
Knockdown %: 60-70
 ABL2
Link
ABL2
Link
 
6 ACP1
Aliases: HAAP
Entrez Gene: 52
RNA catalog
acid phosphatase 1, soluble
mRNA: NM_004300
Library: Phosphatase
Classification: Phosphatase
Final bin: Discordant
SMARTpool bin:   No change
Avg area: 1.79
Avg Alamar: 0.99
Knockdown %: 90-100
 ACP1
Link
ACP1
Link
 
7 ACP2
Entrez Gene: 53
RNA catalog
acid phosphatase 2, lysosomal
mRNA: NM_001610
Library: Phosphatase
Classification: Phosphatase
Final bin: Accelerated - MC
SMARTpool bin:   Accelerated *
Avg area: 0.87
Avg Alamar: 1.05
Knockdown %: 80-90
ERBB2: Accelerated
ACP2
Link
ACP2
Link
 
8 ACP5
Aliases: TRAP
Entrez Gene: 54
RNA catalog
acid phosphatase 5, tartrate resistant
mRNA: NM_001611
Library: Phosphatase
Classification: Phosphatase
Final bin: Impaired - HC
SMARTpool bin: Impaired
Avg area: 2.46
Avg Alamar: 1.01
Knockdown %: failed
ERBB2: No Change
ACP5
Link
ACP5
Link
Impaired D
ACP5
Link
9 ACP6
Aliases: LPAP, ACPL1, PACPL1
Entrez Gene: 51205
RNA catalog
acid phosphatase 6, lysophosphatidic
mRNA: NM_016361
Library: Phosphatase
Classification: Phosphatase
Final bin: Discordant
SMARTpool bin:   No change
Avg area: 1.17
Avg Alamar: 1.04
Knockdown %: 80-90
 ACP6
Link
ACP6
Link
 
10 ACPL2
Entrez Gene: 92370
RNA catalog
acid phosphatase-like 2
mRNA: NM_152282
Library: Phosphatase
Classification: Phosphatase
Final bin: No Phenotype
SMARTpool bin:   No change
Avg area: 1.93
Avg Alamar: 0.85
Knockdown %: 50-60
 ACPL2
Link
ACPL2
Link
 
11 ACPP
Aliases: PAP, ACP3, ACP-3
Entrez Gene: 55
RNA catalog
acid phosphatase, prostate
mRNA: NM_001099
Library: Phosphatase
Classification: Phosphatase
Final bin: No Phenotype
SMARTpool bin:   No change
Avg area: 1.96
Avg Alamar: 0.98
Knockdown %: failed
 ACPP
Link
ACPP
Link
 
12 ACPT
Entrez Gene: 93650
RNA catalog
acid phosphatase, testicular
mRNA: NM_033068
Library: Phosphatase
Classification: Phosphatase
Final bin: Discordant
SMARTpool bin:   No change
Avg area: 1.80
Avg Alamar: 0.95
Knockdown %: failed
 ACPT
Link
ACPT
Link
 
13 ACTB
Aliases: PS1TP5BP1
Entrez Gene: 60
RNA catalog
actin, beta
mRNA: NM_001101
Library: MAR
Classification: cytoskeletal
Focal Adhesion Related: Yes
Final bin: Impaired - HC
SMARTpool bin: Impaired
Avg area: 4.06
Avg Alamar: 0.92
Knockdown %: not done
ERBB2: No Change
ACTB
Link
ACTB
Link
Impaired unique
ACTB
Link
14 ACTG1
Aliases: ACT, ACTG, DFNA20, DFNA26
Entrez Gene: 71
RNA catalog
actin, gamma 1
mRNA: NM_001614
Library: MAR
Classification: cytoskeletal
Final bin: Discordant
SMARTpool bin:   Low Alamar
Avg area: 3.89
Avg Alamar: 0.73
Knockdown %: not done
ERBB2: Impaired
BCL2: Low Alamar
ACTG1
Link
ACTG1
Link
 
15 ACTN1
Entrez Gene: 87
RNA catalog
actinin, alpha 1
mRNA: NM_001102
Library: MAR
Classification: cytoskeletal
Focal Adhesion Related: Yes
Final bin: Discordant
SMARTpool bin:   No change
Avg area: 1.79
Avg Alamar: 0.96
Knockdown %: not done
 ACTN1
Link
ACTN1
Link
 
16 ACTR3
Aliases: ARP3
Entrez Gene: 10096
RNA catalog
ARP3 actin-related protein 3 homolog (yeast)
mRNA: NM_005721
Library: MAR
Classification: cytoskeletal
Final bin: Discordant
SMARTpool bin: Impaired
Avg area: 2.51
Avg Alamar: 0.84
Knockdown %: not done
ERBB2: No Change
ACTR3
Link
ACTR3
Link
 
17 ACVR1
Aliases: FOP, ALK2, SKR1, ACTRI, ACVRLK2
Entrez Gene: 90
RNA catalog
activin A receptor, type I
mRNA: NM_001105
Library: Kinase
Classification: Ser/Thr Kinase
Final bin: Accelerated - HC
SMARTpool bin: Accelerated
Avg area: 0.18
Avg Alamar: 1.03
Knockdown %: 80-90
ERBB2: Accelerated
ACVR1
Link
ACVR1
Link
Accelerated unique
ACVR1
Link
18 ACVR1B
Aliases: ALK4, SKR2, ACTRIB, ACVRLK4
Entrez Gene: 91
RNA catalog
activin A receptor, type IB
mRNA: NM_004302
Library: Kinase
Classification: Ser/Thr Kinase
Final bin: No Phenotype
SMARTpool bin:   No change
Avg area: 1.57
Avg Alamar: 0.93
Knockdown %: 60-70
 ACVR1B
Link
ACVR1B
Link
 
19 ACVR1C
Aliases: ALK7, ACVRLK7
Entrez Gene: 130399
RNA catalog
activin A receptor, type IC
mRNA: NM_145259
Library: Kinase
Classification: Ser/Thr Kinase
Final bin: Discordant
SMARTpool bin:   No change
Avg area: 1.60
Avg Alamar: 0.90
Knockdown %: <20
 ACVR1C
Link
ACVR1C
Link
 
20 ACVR2A
Aliases: ACVR2, ACTRII
Entrez Gene: 92
RNA catalog
activin A receptor, type IIA
mRNA: NM_001616
Library: Kinase
Classification: Ser/Thr Kinase
Final bin: No Phenotype
SMARTpool bin:   No change
Avg area: 1.14
Avg Alamar: 1.05
Knockdown %: 40-50
 ACVR2A
Link
ACVR2A
Link
 

Notes:

A description of the screen design according to MIARE guidelines (v. 0.6.5; www.miare.org) can be found here.

(1) We screened 3 separate human siRNA libraries. The protein and phosphoinositidyl kinases totalled 576 genes, the phosphatases totalled 192 genes and the Migration and Adhesion related (MAR) custom designed library contained 313 genes and includes previously uncharacterized members of protein families known to regulate migration or adhesion.

(2) MCF-10A Phenotypes

Final bin - The final hit call for each gene was based on both the numerical and visual scores after screening the SMARTpool siRNAs and subsequent validation by screening two and four individual sequences and ON-TARGETplus reagents. Each gene was annotated within the appropriate hit bin, Accelerated migration, Impaired migration, Low Alamar (impaired migration and decreased Alamar Blue reduction) and No Phenotype (no migration phenotype) as High Confidence (HC; 3/4 or 4/4 concordance with SMARTpool), Moderate Confidence (MC; 2/4 or 2/2) or Discordant (0/4, 1/4, 0/2, 1/2). The genes annotated with HC# originally scored with MC but were upgraded to HC based on validation with ON-TARGETplus SMARTpools. The Discordant category was created to define those siRNAs that did not validate with confidence based on differences between the SMARTpools and individual siRNA sequences or reproducibility between replicates. This is distinct from those siRNAs that induced a No Change phenotype with validated concordance of 2/2, which we termed No Phenotype.

SMARTpool bin - The initial classification of hits after the SMARTpool screen. Accelerated migration, Impaired migration, Low Alamar (impaired migration and decreased Alamar Blue reduction) and No Change (no migration phenotype). siRNAs that had the numerical score over-ridden by the visual score are indicated with an asterisk.

Avg area - The SMARTpool area score averaged over replicate experiments. The Accelerated migration bin had an area <0.75, the Impaired migration bin had an area score >2.0.

Avg Alamar - The SMARTpool Alamar Blue score averaged over replicate experiments. The Low Alamar bin was defined as an Alamar Blue score <0.8.

Knockdown - Knockdown was performed using bDNA for the kinase and phosphatase SMARTpool siRNAs. The data is reported within percentile ranges. Those that weren't done or failed are indicated.

ON-TARGETplus - phenotypic classification of the HC Accelerated genes using the standard criteria after screening using ON-TARGETplus reagents. A weak phenotype is indicated.

(3) Secondary Screen

ERBB2 - the Low Alamar, Impaired and Accelerated migration hit bins were screened in the ERBB2-MCF-10A cells. The phenotypic classification using the standard criteria is indicated.

BCL2 - the Low Alamar hit bin was screened in BCL2-MCF-10A cells and the phenotypic classification using the standard criteria is indicated.

(4) Wound Image - representative phalloidin-stained wound healing image beside mock-transfection control.

(5) Morphology: - an individual panel from the representative wound healing image. Phalloidin (green) and DAPI (blue).

(6) Migration time-lapse: - Cells were transfected with SMARTpool siRNA reagents at 50 nM in 24 well dish format. Cells were wounded at 52 hours post-transfection and imaged using a 20X ELWD objective on a Nikon TE2000E automated inverted microscope at 37oC in the presence of C02. Images were captured at 6 minute intervals for 20 hours. Migration patterns were analyzed relative to controls and similar phenotypes grouped for the Accelerated and Impaired migration bins. Six controls were imaged and one representative is shown (see link for more details).

Accelerated -
A- minimal adhesion, front-rear polarity
B- transient adhesion, front-rear polarity
C- minimal adhesion, erratic migration, poor or non-existent front-rear polarity
D- adhesive, large protrusions
E- adhesive, collective, medium protrusions
F- adhesive, collective, small compact cells
G- not significantly distinguishable from control

Impaired -
A- weak adhesion, erratic migration, unpolarized
B- dynamic adhesions, vertical migration
C- dynamic adhesion, larger cells
D- cells become stretched during migration
E- not significantly distinguishable from control