Download Supplementary Figure Legends

Supplementary Figure legends
Supplemental Figure S1. The effect of oncogenes on DX2 expression. (A) Expression of DX2 in
NSCLC and paired normal human lung tissues. Despite of expression of DX2 in all of tumors and
several kinds of cancer-adjacent normal tissues at transcription level (upper panels), protein of DX2
was detected only in 6 cases of tumor tissues. Emerin was used for loading control. N and T indicate
normal and tumor sample, respectively. (B) K-Ras status was tested by mutation specific PCR.
Her2/Neu expression was determined by IHC (data not shown). (C) Oncogenic K-Ras induces DX2
expression. HEK293 cells were co-transfected with Myc-tag AIMP2 or DX2 and oncogenic Ras genes
for 24 h. Actin was used for loading control. (D) Phosphatase inhibitor (Okadaic acid; OA, 10 M)
extends DX2 stability. DX2 or AIMP2 transfected HEK293 cells were incubated with OA for 3 h with
or without CHX. (E) Si-siah-1 increases DX2 expression. A549 cells were transfected with indicated
vectors for 24 h (F) Specific interaction of DX2 with siah-1. After transfection with indicated E3
ligases, HEK293 cells were harvested and mixed with His-DX2 protein. After IP with His Ab, coprecipitated proteins were analyzed with WB. (G) Siah-1 promotes ubiquitylation of DX2. In
HEK293 cells, ubiquitin-conjugated DX2 was increased by siah-1 transfection.
Supplemental Figure S2. Oncogenic property of DX2. (A) The effect of DX2 on cell proliferation.
MEF cells were incubated in complete medium for indicating time after seeding with 7x104 cells in
100 mm dish. (B) Cell growth in Adr-treated condition. After cell seeded, MEFs were incubated with
Adr (1 g/ml) for 24 h. (C) Ectopic expressed DX2 shows different localization in A549 and HCT116.
After transfection of Myc-DX2 into both cell lines, localization of DX2 was determined by IF staining
with Myc Ab. (D) Cellular localization of DX2 and AIMP2. Ectopic expressed DX2 (red) was located
in cytoplasm or nucleus, where AIMP2 was in cytoplasm (green). DAPI was used for DNA. HEK293
was transfected with DX2 or AIMP2 for 24 h. cells were fixed and stained with c-Myc.
Supplemental Figure S3. Small cell lung cancer in DX2 and DK transgenic model. (A) Basic
histology of K-RasLA2 and DK mice. In addition to ADC, DK mice possessed small cell mass in
central region, where blood vessel (BV) and large bronchiole (BR) were located. (B) IHC staining
with pro-surfactant C (SPC; marker of ADC), pan-keratin (marker of SQC), and NSE (marker of
SCLC). In tumors in DK mice, SPC and pan-keratin negative tumors were stained with NSE. (C)
Immunochemical staining with Neuron-specific enolase (NSE) in tumors of DX2 Tg mouse. Tumor
region of DX2 (6 month-old) was stained with NSE (enlarged picture in right panel). But, ADC,
occurred in K-RasLA2 (6 month-old), was not stained by NSE (lower panel). (D) H&E staining of old
DX2 Tg mouse lung tissues. In central region of lung, cell mass, composed of small cells, was
detected in aged DX2 mouse model. BV indicates blood vessel. (E) Increase of tumor area by aging.
Tumor area of DX2 was increased by aging. Tumor area indicated average of 5 sections of lung
tissues, obtained from two mice of each group. (F) Nuclear morphology of small cells in DX2 Tg
mouse. Comparing to adenocarcinoma (ADC) in K-RasLA2 (upper panel), small cells of DX2 Tg
mouse showed higher mitotic index (yellow arrows) and fine granule chromatin (red arrows), marker
of SCLC. (G) Example of Mixed type cancer in DK mouse (10 month-old). Region 1 was ADC.
However, region 2 and 3 were mixed with SCLC and ADC. (H) Detection of autoantibody against
DX2 in sera from SCLC patients. Recombinant AIMP2 and DX2 proteins were used for antigen.
Supplemental Figure S4. DX2 suppresses p14/ARF. (A) Si-DX2 can induce p14/ARF expression.
Elimination of DX2 using si-RNA could increase endogenous p14/ARF expression in several cell
lines. Although lung cancer cell lines (H1299, H358 and H23) showed the induction of p14/ARF in
response to si-DX2, 293 and L132 (human embryonic lung cell) did not respond to it. This result
indicates that endogenous DX2 is involved in p14/ARF reduction. In fact, two kinds of non-cancer
cell lines do not express DX2. Numbers below panels indicate comparative fold-change to control.
Band density was determined by image J. (B) Increase of p14/ARF in nucleoplasm by si-DX2. In
GFP-p14/ARF transfected A549, elimination of DX2 could increase p14/ARF expression. (C) Colocalization of DX2 with p14/ARF. In co-transfected HEK293, co-localization of DX2 (red) and
p14/ARF (green) in nuclear spot was detected. (D) IP analysis using GFP-p14/ARF. Cells were
incubated with GFP Ab and protein A/G agarose. Precipitated materials were dissolved in SDS-PAGE
and subjected into WB analysis with His-Ab. PPT indicated precipitated proteins with GFP-p14/ARF.
(E and F) To confirm the specific interaction of DX2 and p14/ARF, interaction of DX2 and unrelated
GST-proteins (Smad4; E and VHL; F) were tested. DX2 did not show interaction with both proteins,
whereas AIMP2 showed the interaction with them.
Supplemental Figure S5. Screening of specific inhibitor of p14/ARF and DX2. (A) Flow chart of
chemical screening. After collection of chemical libraries, we screened the binding inhibitor using
ELISA system. Based on the value of ELISA, we selected the chemicals (30 chemicals from about
9000 chemicals). We also excluded the chemicals, if it has been identified as a binding inhibitor of our
other kinds of chemical screening (excluded 16 chemicals at this step). Through specificity test, we
chose 5 chemicals as final candidates. Among them, 4 chemicals were originated from Korea
chemical bank and SLCB050 was isolated from synthetic library. (B) Diagram of ELISA based
screening method. (C) Example of ELISA based chemical screening. Every plate contained positive
(reaction without chemical; red line) and negative (reaction without GST-p14; yellow line) reaction.
We chose the candidate chemicals if it suppressed the interaction over than 70% (dashed line). Red
arrows indicate candidates. (D) Exclusion of non-specific inhibitor. Although SLC24 showed the
strong inhibition effect on p14/ARF-DX2 binding, since it has been isolated as inhibitor PAK1PUMA, we excluded it from candidates. (E) Checking the effect of chemicals on p14/ARF-DX2
binding through GST-pull down. (F) Negative selection. If chemicals can block the interaction of p53p14/ARF, we excluded them from candidates. For example, 886-D8 was excluded due to the strong
inhibition effect on p14/ARF-p53 binding. (G) Effect of SLCB050 on in vitro binding of p14/ARF
and DX2. Although SLC24 was also identified as candidate of the binding inhibitor, it has been also
detected as inhibitor of other binding pair. Thus, it is excluded from final candidates.
Supplemental Figure S6. SLCB050 as specific binding inhibitor of p14/ARF and DX2. (A)
Inhibitory effect of SLCB050 on p14/ARF and DX2. The binding between DX2 and GST-p14/ARF
recombinant protein was obviously diminished by SLCB050, but DMSO or SLC24. Actin was used
for negative control for binding, because DX2-transfected 293 was used for binding assay. (B) dosedependent effect of SLCB050 on DX2-p14/ARF binding. Following dosage of SLCB050 (from 1 to 5
g/ml), binding of DX2 and p14/ARF was reduced. (C) SLCB050 does not block the p53-AIMP2 or
p53-DX2 binding. GST-pull down assay using GST-p53 protein was performed with/without
SLCB050. However, this chemical did not alter the interaction of p53 and AIMP2 or DX2. (D)
SLCB050 does not inhibit p53-p14/ARF binding. These binding assays indicate that direct target of
SLCB050 was specific domain of DX2, which would be generated by junction of exon1 and exon3.
(E) The structures of SLCB050-related chemicals. (F and G) Specific inhibitory effect of chemicals
on DX2-p14/ARF. Newly synthesized chemicals were tested on p14/ARF-DX2 (F) and p14/ARF-p53
binding (G).
Supplemental Figure S7. The effect of SLCB050 on DX2. (A and B) Reduction of DX2 stability by
SLCB050, determined by Western blotting (A) and immunofluorescence staining (B). DX2transfected 293 cells were incubated with 10 M SLCB050 and CHX for the indicated periods. No
changes in DX2 (red arrow) or AIMP2 (black arrow) transcription were detected. (C) SLCB050
suppresses endogenous DX2, independently with p14/ARF. When cells were incubated with 10 M of
SLCB050 for 6 h, reduction of DX2 was detected in SCLC as well as p14/ARF-deficient NSCLC
(A549 and H460). (D) Soft agar colony formation assay. H128 cells (SCLC) were seeded in soft agar
plates and incubated with the indicated concentrations of SLCB050 for 48 h. Cells were visualized by
trypan blue staining after fixation. (E) The effect of SLCB050 related chemicals. Suppression of cell
viability was observed in SLCB050 and its closely related chemicals (HJH141204 and HJH141206).
(F) Significant enhanced effect of SLCB050 (10 M) with Adr (0.2 g/ml) in H446 cells for 48 h.
Before xenograft, responding of H446 cells to SLCB050 and combinational treatment were
investigated. (G) Isolated tumor mass from xenograft. Reduction of tumor volume by chemical
treatment was observed. (H) Graph of body weight. Obvious reduction of body weight by Adr-
treatment was observed. However, low dose of Adr with SLCB050 could keep body weight for
considerable period. * indicates expired time of Adr-injected mice. (I) The effect of SLCB050 on
normal lung epithelial cells. Differentially from Adr (0.2 g/ml), SLCB050 (20 M) did not reduce
the cell viability on L132 (embryonic lung epithelial cell) and WI-26 (lung epithelial cell). Cells were
incubated with indicated chemicals for 48 hr. Cell viability was determined by MTT assay. (J) To
know the effect of combination treatment of SLCB050 with Carboplatin (CPT; 1 M) and Taxol (2
M), H128 (left) and H69 (right) were incubated with indicated chemicals for 24 hr. N. S indicates
not significance.
Supplemental Figure S8. Anti-tumoral effects of SLCB050 in DK mice. (A) Diagram of
experimental schedule with DK mice. Concentration of Adr (1 mg/kg) was reduced due to toxicity. (B)
The body weight of DK mice did not change significantly during the experimental period. (C)
Hematoxylin-eosin staining of lung tumor tissues from Adr, SLCB050 or Adr+SLCB050 combined
treated DK mice. Combination treatment with SLCB050 and Adr obviously induced tumor regression.
SCLC (red letters) indicted small cell lung carcinoma region in DK lung tissues. (D) TUNEL staining
in DK mice after injection of indicating chemicals.
Supplemental Figure S9. Diagram for summary. DX2 blocked the oncogenic stimulation-induced
p14/ARF. SLCB050 inhibited DX2 and restored the tumor suppressive role of p14/ARF.