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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| Gene | Description1 | Secondary screen3 | Wound4 Image | Morphology5 | Time-lapse6 | ||
| 1 | AAK1 Entrez Gene: 22848 siRNA 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 |  Link |  Link | ||
| 2 | AATK Aliases: LMR1, AATYK, LMTK1 Entrez Gene: 9625 siRNA 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 |  Link |  Link | |
| 3 | ABI2 Aliases: ABI-2, ABI2B, AIP-1, AblBP3, SSH3BP2, argBPIA, argBPIB Entrez Gene: 10152 siRNA 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 |  Link |  Link | ||
| 4 | ABL1 Aliases: ABL, JTK7, p150, c-ABL, v-abl, bcr/abl Entrez Gene: 25 siRNA 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 |  Link |  Link | |
| 5 | ABL2 Aliases: ARG, ABLL Entrez Gene: 27 siRNA 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 | &fbin=No Phenotype&lib=Kinase) Link | &fbin=No Phenotype&lib=Kinase) Link | ||
| 6 | ACP1 Aliases: HAAP Entrez Gene: 52 siRNA 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 |  Link |  Link | ||
| 7 | ACP2 Entrez Gene: 53 siRNA 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 |  Link |  Link | |
| 8 | ACP5 Aliases: TRAP Entrez Gene: 54 siRNA 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 |  Link |  Link | Impaired D Link |
| 9 | ACP6 Aliases: LPAP, ACPL1, PACPL1 Entrez Gene: 51205 siRNA 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 |  Link |  Link | ||
| 10 | ACPL2 Entrez Gene: 92370 siRNA 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 |  Link |  Link | ||
| 11 | ACPP Aliases: PAP, ACP3, ACP-3 Entrez Gene: 55 siRNA 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 |  Link |  Link | ||
| 12 | ACPT Entrez Gene: 93650 siRNA 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 |  Link |  Link | ||
| 13 | ACTB Aliases: PS1TP5BP1 Entrez Gene: 60 siRNA 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 |  Link |  Link | Impaired unique Link |
| 14 | ACTG1 Aliases: ACT, ACTG, DFNA20, DFNA26 Entrez Gene: 71 siRNA 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 |  Link |  Link | |
| 15 | ACTN1 Entrez Gene: 87 siRNA 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 |  Link |  Link | ||
| 16 | ACTR3 Aliases: ARP3 Entrez Gene: 10096 siRNA 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 | &fbin=Discordant&lib=MAR) Link | &fbin=Discordant&lib=MAR) Link | |
| 17 | ACVR1 Aliases: FOP, ALK2, SKR1, ACTRI, ACVRLK2 Entrez Gene: 90 siRNA 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 |  Link |  Link | Accelerated unique Link |
| 18 | ACVR1B Aliases: ALK4, SKR2, ACTRIB, ACVRLK4 Entrez Gene: 91 siRNA 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 |  Link |  Link | ||
| 19 | ACVR1C Aliases: ALK7, ACVRLK7 Entrez Gene: 130399 siRNA 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 |  Link |  Link | ||
| 20 | ACVR2A Aliases: ACVR2, ACTRII Entrez Gene: 92 siRNA 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 |  Link |  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
