1. Suppl. Figure 1 A C D B UAS-lz UAS-dPias-RNAi-#1 UAS-lz +hs
UAS-dPias
UAS-lz + UAS-dPias
UAS-Pias-C388A B
UAS-dPias-RNAi-#1
UAS-dPias-RNAi-#2
rp-49
UAS-dPias-C388A
UAS-lz + UAS-dPias-C388A

2. Figure S1. A large-scale genetic-modifier screen identified dPias as a regulator of lz/RUNXs
(A) UAS-lz and/or UAS-dPias were expressed in eye using the eye-specific driver GMR-gal4. Microscopic images (left) and scanning electron microscopic images (SEM, right) are shown. GMR-driven overexpression of either lz or dPias result in an mild rough-eye phenotype (UAS-lz and UAS-dPias). When both lz and dPias were overexpressed, the rough-eye phenotype was more severe (UAS-lz + UAS-dPias).
(B) dPias with a defect in the SUMO E3 ligase activity (UAS-dPias-C388A) was expressed with or without UAS-lz using the eye-specific driver GMR-gal4. Overexpression of dPias-C388A did not affect the eye-defect phenotype induced by lz.
(C) dPiasV29448 (UAS-dPias-RNA-i#1) and dPiasV31623 (UAS-dPias-RNAi-#2) bear UAS-dPias-RNAi. GMR-driven eye-specific knockdown of dPias upon expression of dPias-RNAi resulted in eye-defect phenotypes in both lines (left). The defective phenotype was rescued by overexpression of lz. Microscopic images are shown.
(D) UAS-dPias, UAS-dPias-C388A, UAS-dPias-RNAi-#1, and UAS-dPias-RNAi-#2 were crossed with the heat-shock driver (hs). The flies were cultured at either 25°C or 37°C (heat-shock) for 1 h, and the levels of dPias expression were measured by RT-PCR. rp49 was used as an internal control.

3. Figure S2. RUNX3 interacts with PIAS1 through the Runt domain.
Suppl. Figure 2
187
234
325
RX3-WT
Runt
451 RX
Runt RX
Runt
RX3-325
Runt
RX3-DRunt
1-187
1-234
1-325
DRunt WT
Myc-RX3
HA-PIAS1
IB : Myc
RX3/PIAS1
IP : HA
IB : HA
PIAS1
IB : Myc
RX3
Figure S2. RUNX3 interacts with PIAS1 through the Runt domain.
Schematic diagram showing the RUNX3 deletion constructs. Myc-tagged RUNX3-wild type (WT), RUNX3(1–187), RUNX3(1–234), RUNX3(1–325) and RUNX3-DRunt were coexpressed with HA-PIAS1, and the RUNX3-PIAS1 interaction was monitored by IP and IB.

4. Suppl. Figure 3
Figure S3. The crystal structure of Runt domain/CBFb/DNA (PDB accession code: 1H9D43). Amino acids in the Runt domain are labeled according to the primary sequence of RUNX3. K129 and K148 are exposed on the surface.

5. Suppl. Figure 4 A C
ERK1
ERK2
DRunt
FLAG-ERK
- - WT
187
234
325 WT
Myc-RX3 -
PIAS1/SUMO1
Myc-RX3
FLAG-AKT1
SUMO1-RX3
IP: FLAG
IB: Myc
RX3/AKT1
IB: Myc
RX3
IB: FLAG
ERK
IP: Myc
IB: Myc
IB: HA
PIAS1
RX3
IB: FLAG
SUMO1
IB: Tub
b-tubulin
IB: FLAG
AKT1
IB: Tub
b-tubulin B WT KD
FLAG-AKT1
- -
PIAS1/SUMO1
Myc-RX3
SUMO1-RX3
IB: Myc
RX3
IB: FLAG
AKT1
IB: HA
PIAS1
IB: FLAG
SUMO1
IB: Tub
b-tubulin
Figure S4. AKT1 pathway but not ERK pathway stimulates RUNX3 sumoylation
(A) Myc-RUNX3, HA-PIAS1, and FLAG-SUMO1 were coexpressed with ERK1 or ERK2 in HEK293 cells, and RUNX3 sumoylation was analyzed by IB.
(B) Myc-RUNX3, HA-PIAS1, and FLAG-SUMO1 were coexpressed with FLAG-AKT1-WT or FLAG-AKT1-KD (kinase-dead mutant) in HEK293 cells, and RUNX3 sumoylation was analyzed by IB.
(C) Myc-RUNX3-WT and serial deletion mutants were cotransfected along with FLAG-AKT1 into HEK293 cells, and the RUNX3-AKT1 interaction was monitored by IP and IB.