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UAS-dPias-C388A

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

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UAS-dPias-C388A

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  1. Suppl. Figure 1 A C UAS-lz UAS-dPias-RNAi-#1 UAS-lz + UAS-dPias-RNAi-#1 UAS-dPias UAS-dPias-RNAi-#2 UAS-lz + UAS-dPias-RNAi-#2 D - hs UAS-dPias UAS-Pias-C388A UAS-lz + UAS-dPias 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. Suppl. Figure 2 234 325 187 451 RX3-WT Runt RX3-1-187 Runt Runt RX3-1-234 RX3-325 Runt RX3-DRunt DRunt 1-234 1-325 1-187 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 C A ERK1 ERK2 DRunt - - 187 FLAG-ERK 234 WT 325 WT - Myc-RX3 - + + + + + PIAS1/SUMO1 - + + + + + + FLAG-AKT1 Myc-RX3 - + + + + + SUMO1-RX3 IP: FLAG IB: Myc RX3/AKT1 IB: Myc RX3 IB: FLAG ERK IP: Myc IB: Myc RX3 IB: HA PIAS1 IB: FLAG SUMO1 IB: FLAG AKT1 IB: Tub b-tubulin IB: Tub b-tubulin B WT KD FLAG-AKT1 - - PIAS1/SUMO1 - + + + + + Myc-RX3 + + + + + + SUMO1-RX3 IB: Myc RX3 IB: FLAG AKT1 PIAS1 IB: HA 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-AKT1intoHEK293 cells, and the RUNX3-AKT1 interaction was monitored by IP and IB.

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