Supplemental Information
The Tumor Suppressor Caliban Regulates DNA Damage Induced Apoptosis
Through p53-Dependent and –Independent Activity
Yajie Wang , Zhe Wang, Bharat H. Joshi, Raj Puri, Brian Stultz, Qing Yuan, Yujie Bai,
Pingkun Zhou, Zengqiang Yuan, Deborah A. Hursh, and Xiaolin Bi
Supplemental Data
Figure S1. Targeted deletion of clbn locus.
(A) The ends-out targeting scheme for clbn. A targeting vector was constructed
containing a 3 kb homologous fragment upstream of the start codon, including the
Mocs2 and part of the vig2 genes, and a 3 kb fragment downstream of stop codon of
clbn gene region, including part of the Bili gene, flanking the armadillo
promoter-driven EYFP reporter gene (arm-EYFP). A standard “Ends-out” targeting
scheme was followed. The clbn genomic region was replaced with arm-EYFP after
targeting.
(B) Western blot analysis showing that wild-type (wt) flies express both Clbn and
Beta-actin protein but that caliban knockout flies (clbn) do not express Clbn protein.
Figure S2. Clbn regulates p53-independent cell death
Time course of irradiation-induced cell death in wild-type (wt) and mutant imaginal
wing discs was monitored by Acridine orange (AO). Third instar larvae were exposed
to 4,000 rad gamma radiation and wing discs were dissected and stained with AO at
the indicated times following treatment.
(A) Fluorescent photomicrographs of representative wing discs of the indicated
genotypes.
(B) AO staining was quantified at the indicated time points. The data was derived
from the averaged of at least five discs for each time point and three independent
experiments. Error bar represents the standard error of mean.
Figure S3. p35 rescues eye phenotype induced by clbn expression
The anti-apoptotic protein p35 from baculovirus was induced by GMR-gal4 together
with clbn in flies eye. Expression of p35 rescues eye phenotype induced by clbn
overexpression.
Figure S4. p35 suppresses clbn expression induced apoptosis
The anti-apoptotic protein p35 from baculovirus was induced by GMR-gal4 together
with clbn in flies eye. Expression of p35 suppresses apoptosis induced by clbn
overexpression.
Supplemental Experimental Procedures
Constructs for clbn targeting and rescue
A 3 kb genomic DNA fragment upstream from the clbn start codon was PCR
amplified and inserted into XbaI site of pBS-armEYFP vector (Gong et al 2005) , a 3
kb downstream fragment was inserted into HindIII site, respectfully. Primer pairs
were as follows: caliban-Xba-up-A, 5`-GC TCT AGA TTG ACG AGT GGA AGG
CTT TG-3`; caliban-Xba-up-B, 5`-GC TCT AGA GGT TCT TTA TGG AAA GTT
GAC G-3`; caliban-Hind-do-A, 5`-GGA TCC AAG CTT TGT ATG CAA AAT GTG
TTG AGC C-3`; caliban-Hind-do-B, 5`-GGA TCC AAG CTT TGC CAA CAT CAC
TTG CGA TGG-3`. The new plasmid was named pBS-armEYFP-clbnΔ.
pBS-armEYFP-clbnΔ was digested with Acc65I, filled 3`-overhang with Klenow
large fragment (New England Biolab), digested again with NotI. The 8.5kb targeting
fragment was recovered with gel purification, subcloned into StuI/NotI sites of pW30
plasmid (Gong and Golic 2003), the targeting plasmid was named
pW30-armEYFP-clbnΔ. To make the rescue transgenic flies, clbn cDNA was PCR
amplified, cloned into pUAST, and injected into w1118.
Clbn protein expression and antibody production
Clbn antigenic motif was analyzed with DNA Star software (Lasergene). A fragment
encoding clbn C-terminal AA 657-992 was PCR amplified with the following primer
pairs: clbn-FP, 5`-CGG GAT CCG GAG GAT AGC TTC ATT GAG CG-3`; clbn-RP,
5`-GGA ATT CTT ATT TGT GAT ACT TCT GAA GTT GCG-3`. Clbn cDNA
fragment was cloned into BamHI/EcoRI sites of pRSETb plasmid DNA (Invitrogen).
The His6 tagged Clbn fragment was expressed in BL21 (DE3) and purified with
HIS-Select Magnetic Agarose Beads (Sigma) following the manufacture`s
instructions. Purified Clbn fusion protein was used to immunize rabbits.
Western blotting
Five third instar larvae were homogenized in 20 μl protein loading buffer, denatured
at 95°C for 10 min. Ten microliter from each sample was subjected to SDS-PAGE
electrophoresis and transferred to nitrocellulose. Protein was detected with rabbit
anti-Clbn antibody (1:8,000) and anti-Beta-actin.
AO staining
Third-instar larvae were collected and irradiated with 4,000 rad gamma radiation.
After recovered at 25℃ for 1.5 hr, wing discs were dissected in 1×PBS, incubated in
PBS containing 0.5 mM acridine orange (AO, Sigma) at room temperature for 5 min,
washed three times with PBS, mounted in PBS, and imaged immediately.
References:
Gong M, Bi X, Rong Y (2005). Targeted mutagenesis of Drosophila atm and mre11 genes. Dros Inf
Serv 88: 79-83.
Gong WJ, Golic KG (2003). Ends-out, or replacement, gene targeting in Drosophila. Proc Natl Acad
Sci U S A 100: 2556-2561.