Supplementary Figures:
Figure S1. DNA damage-induced apoptosis is inhibited at high cell density in H1299
and U2OS cells a) H1299 cells were plated sparse or dense. Sixteen hours after seeding,
Abl inhibitor STI-571 was added at 10 or 20 M, as indicated. Cells were irradiated 2.5
hours later at 30 Gy, harvested 48 hours later, and analyzed by SDS-PAGE and
immunoblotting. b) H1299 cells were plated and treated as in (a). Cells were harvested
for FACS analysis 48 hours post-IR. Percentages of cells in the different fractions
(subG1, G1, S, and G2-M) are indicated. c) U2OS cells were plated sparse or dense, and
were irradiated at 20 Gy. Cells were harvested for FACS analysis 120 hours post-IR.
Percentages of cells in the different fractions (subG1, G1, S, and G2-M) are indicated.
d)U2OS cells were plated and treated as in (c). Cells were harvested for analysis by SDSPAGE and immunoblot 72 hours post-IR.
Figure S2. Knockdown of Yap1 reduces DNA damage-induced apoptosis. MCF10A
cells expressing control or Yap1 shRNA, were plated at different densities, irradiated at
20Gy, and harvested for FACS analysis 120 h post-IR. Percentages of cells in the
different fractions (subG1, G1, S, and G2-M) are indicated.
Figure S3. c-Abl is activated in response to DNA damage in sparse cells. a,b) mRNA
levels of transfected WW45 and Mst1. mRNA levels of duplicate plates from the
experiment shown in Fig. 3a were analyzed by qPCR and normalized by GFP mRNA. (a)
WW45, and (b) MST1 mRNA levels are shown. c) Caspase 3 activation and c-Abl
autophosphorylation in sparse NIH3T3 cells. NIH3T3 cells stably expressing empty
pBABE vector were plated sparse and dense. Cells were irradiated at 20 Gy and collected
1
3 days later. Upper panels: Cells were photographed (Scale bar = 0.1 mm) prior to
irradiation. Lower panels: c-Abl was immunoprecipitated from cell extracts using the
anti-cAbl K12 antibody, and analyzed for autophosphorylation using the anti-cAbl
pTyr412 antibody. Note that Yap pSer127, indicating Hippo activation, appears also in the
sparse unirradiated lane. This is because cells filled the plate during the 3 day incubation.
Activated caspase 3 is seen only in sparse cells with IR. d) c-Abl autophosphorylation
in MCF10A cells. MCF10A cells were plated at low and high density. Cells were
irradiated at 20Gy, and were collected 24 hours later. Upper panels: Cells were
photographed as in (c). Lower panels: c-Abl was immunoprecipitated from cell extracts
using the anti-cAbl K12 antibody, and active Abl was detected with the anti cAbl pTyr245
antibody. e) Overexpression of Lats2 and upstream Hippo kinase Mst1/WW45
inhibits c-Abl phosphorylation of p73. HEK293 cells were transfected with the
plasmids indicated. Cells were harvested 24 hours post-transfection, and were analyzed
by SDS-PAGE and immunoblotting with the indicated antibodies. PY20 detects
phosphotyrosine. P73 pTyr99 detects phospho p73 (Tyr99). Co-transfection of Mst1 and
WW45 along with Lats2 led to further inhibition of c-Abl activity.
Figure S4. Lats2 activated by Mst1/WW45 phosphorylates Yap1 and c-Abl in vitro.
a) HEK293 cells were transfected separately with myc-Lats2 (alone, lanes 2, or with
untagged Mst1 and WW45, lanes 3), Flag-cAbl 1-81, Flag-Yap, or empty plasmids
(lanes 1). Cells were collected, and proteins were immunoprecipitated with anti-myc
(control and Lats2) or anti-Flag. The Flag proteins were eluted with Flag peptide. In vitro
kinase assays were performed by adding the Flag-eluted proteins or buffer control to the
Lats2/control beads. “0” is a buffer control. Reaction products were analyzed by SDS-
2
PAGE followed by immunoblotting with the indicated antibodies. To avoid problems due
to incomplete stripping of membranes, Flag images were generated by loading identical
samples on separate gels. The results show that Lats2 phosphorylated Yap1 in vitro, as
indicated by the increase in pSer127. Activation of Lats2 by co-expression with
Mst1/WW45 increased the Yap Ser127 phosphorylation. Phosphorylation of c-Abl at
Thr197 is seen minimally with myc-Lats2 alone, but is increased with activated mycLats2. b) Kinase assay was performed as in (a), but myc-Lats2 kinase dead (KD) was
included, where indicated.
Figure S5. Knockdown of Lats2. a) HEK293 cells were transfected with Lats2 KD
shRNA or control plasmids. The next day, cells were replated dense, and 24 h later were
irradiated at 40 Gy. Duplicate samples were harvested 3 days later for analysis of RNA
and protein. Left panel, immunoblot results; right panel, qPCR analysis of Lats2 mRNA.
b) qPCR analysis of the H1299-based cell lines used in Fig 7d, demonstrating knockdown
of Lats2 to 42% of the control. c) Immunoblot analysis of the cells used in (b).
Figure S6. Expression of c-Abl W118A mutant rescues DNA damage-induced
apoptosis at high cell density. a) H1299 cells stably overexpressing wild-type c-Abl ,
the W118A mutant, or the empty plasmid were plated dense and irradiated the following
day at 25 Gy. Cells were harvested for FACS analysis 72 hours post-IR. Percentages of
cells in the different fractions (subG1, G1, S, and G2-M) are indicated. b) H1299 cells
expressing wt c-Abl, c-Abl W118A, c-Abl T197A, or empty pBABE vector were
analyzed by SDS-PAGE and immunoblotting.
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