8. Culture medium: This is as described in Aldaz et al. [14]. Supplement 5 ml of Shields and Sang M3 insect medium, with
25 μl of penicillin-streptomycin, 100 μl fetal calf serum, and
1 μl 1 mg/ml 20-hydroxyecdysone. Make fresh each time it is
required.
9. Phosphate buffered saline (PBS) 10Â Stock solution: Add
3.33 g NaH 2 PO 4 ∙2H 2 O + 22.4 g Na 2 HPO 4 to ~800 ml
dH 2 O, adjust pH to 7.4, add 102.2 g NaCl and adjust volume
to 1 L and store at room temperature.
Table 3
Fluorescent reporter stocks for EMT factors
EMT feature Stock
Comment
Source
Basement
membrane
Viking-GFP [34]
GFP exon trap inserted in Collagen IV
DGRC#110692
Adherens
junction
UAS-alpha-catGFP [35]
GFP fusion to the AJ protein alpha Catenin DGRC#109002
Adherens
junction
Ubi-p63E-DECadherin-GFP
[36]
See Note 1
GFP fusion to Epithelial Cadherin under the
control of the ubiquitin promoter
BDSC#58471
Myosin
sqh[AX3];P{sqhGFP.RLC}-42
[37]
GFP-tagged myosin regulatory light chain
under endogenous regulatory region, in
null background
BDSC#57144
F-actin
UAS-LifeAct-GFP,
UAS-LifeactRuby [38]
See Note 2
Actin-binding peptide fused to GFP and Ruby
(red fluorescence)
BDSC#35544,
#35545 and
others
JNK
pathway
puc
E69 -lacZ [39]
Enhancer trap in the JNK target gene puckered DGRC#109029
JNK
pathway
puc
A251.1F3 -lacZ
Enhancer trap in the JNK target gene puckered BDSC#11173
JNK
pathway
msn
06946 -lacZ [40] Enhancer trap in the Jun kinase kinase
kinase kinase, misshapen [41]
BDSC#11707
JNK
pathway
Tre-eGFP
Tre-DsRedT4 [42]
Synthetic JNK reporters with four binding
sites (Tre elements) for c-Jun, placed
upstream of a basal promoter
BDSC#59010,
BDSC#59011,
BDSC#59012
JNK
pathway
mRE-DsRedT4
[42]
Negative control line mRE-DsRedT4 in
which the consensus site is scrambled
BDSC#59013
Frazzled
Venus-frazzled
Venus-exon trap in frazzled, between the
signal sequence and the first extracellular
IgG domain [21]
DGRC#115148
Using Drosophila Wing Eversion to Study EMT
121
25 μl of penicillin-streptomycin, 100 μl fetal calf serum, and
1 μl 1 mg/ml 20-hydroxyecdysone. Make fresh each time it is
required.
9. Phosphate buffered saline (PBS) 10Â Stock solution: Add
3.33 g NaH 2 PO 4 ∙2H 2 O + 22.4 g Na 2 HPO 4 to ~800 ml
dH 2 O, adjust pH to 7.4, add 102.2 g NaCl and adjust volume
to 1 L and store at room temperature.
Table 3
Fluorescent reporter stocks for EMT factors
EMT feature Stock
Comment
Source
Basement
membrane
Viking-GFP [34]
GFP exon trap inserted in Collagen IV
DGRC#110692
Adherens
junction
UAS-alpha-catGFP [35]
GFP fusion to the AJ protein alpha Catenin DGRC#109002
Adherens
junction
Ubi-p63E-DECadherin-GFP
[36]
See Note 1
GFP fusion to Epithelial Cadherin under the
control of the ubiquitin promoter
BDSC#58471
Myosin
sqh[AX3];P{sqhGFP.RLC}-42
[37]
GFP-tagged myosin regulatory light chain
under endogenous regulatory region, in
null background
BDSC#57144
F-actin
UAS-LifeAct-GFP,
UAS-LifeactRuby [38]
See Note 2
Actin-binding peptide fused to GFP and Ruby
(red fluorescence)
BDSC#35544,
#35545 and
others
JNK
pathway
puc
E69 -lacZ [39]
Enhancer trap in the JNK target gene puckered DGRC#109029
JNK
pathway
puc
A251.1F3 -lacZ
Enhancer trap in the JNK target gene puckered BDSC#11173
JNK
pathway
msn
06946 -lacZ [40] Enhancer trap in the Jun kinase kinase
kinase kinase, misshapen [41]
BDSC#11707
JNK
pathway
Tre-eGFP
Tre-DsRedT4 [42]
Synthetic JNK reporters with four binding
sites (Tre elements) for c-Jun, placed
upstream of a basal promoter
BDSC#59010,
BDSC#59011,
BDSC#59012
JNK
pathway
mRE-DsRedT4
[42]
Negative control line mRE-DsRedT4 in
which the consensus site is scrambled
BDSC#59013
Frazzled
Venus-frazzled
Venus-exon trap in frazzled, between the
signal sequence and the first extracellular
IgG domain [21]
DGRC#115148
Using Drosophila Wing Eversion to Study EMT
121