Regulation of TP53INP1β via SUMOylation Pathway in Cellular Signaling

1. B A GFP-TP53INP1βWT GFP-TP53INP1α GFP-TP53INP1βK113R GFP-TP53INP1β 6His-Flag-SUMO1 6His-Flag-SUMO1 kDa kDa 250 250 150 TP53INP1 SUMO1 (anti TP53INP1) TP53INP1β SUMO1 150 * 100 100 075 SUMO1 SUMO1 Ni2+ pull down Ni2+ pull down TP53INP1β 100 100 SUMO1 TP53INP1 β-Tubulin SUMO1 Input β-Tubulin Input C 6His-Flag- GFP-TP53INP1βWT GFP-TP53INP1βK113R kDa 250 150 Modified TP53INP1β * 100 SUMO1, Ub, Nedd8 Ni2+ pull down TP53INP1β 100 SUMO1, Ub, Nedd8 β-Tubulin Input Supplementary Figure 1

2. A GFP-TP53INP1αWT GFP-TP53INP1αWT kDa GFP-TP53INP1αK113R GFP-TP53INP1αK113R 250 GFP-TP53INP1βWT GFP-TP53INP1βWT 150 GFP-TP53INP1βK113R GFP-TP53INP1βK113R TP53INP1 SUMO1 100 6His-Flag-SUMO1 6His-Flag-SUMO1 075 SUMO1 Ni2+ pull down 100 TP53INP1 075 SUMO1 β-Tubulin Input B kDa 150 TP53INP1 SUMO1 100 075 SUMO1 Ni2+ pull down 100 TP53INP1 075 SUMO1 β-Tubulin Input Supplementary Figure 2

3. PonA TP53INP1α βTubulin U2OS Inducible U2OS Stable U2OS (WT) Stable U2OS (K113R) Inducible U2OS Supplementary Figure 3

4. TP53INP1 LC3 DAPI Merge TP53INP1αWT Untreated TP53INP1αK113R TP53INP1αWT Bafilomycin A1 TP53INP1αK113R Supplementary Figure 4

5. A PIAS4 PIAS2 RanBP2 PIAS3 PIAS1 CBX4 Scramble Scramble Ubc9 siRNA 6His-Flag-SUMO1 kDa 250 150 TP53INP1α SUMO1 100 175 SUMO1 Ni2+ pull down 175 TP53INP1α SUMO1 Input B 1.2 1.2 1.2 1.2 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 0.6 PIAS1 mRNA level PIAS2 mRNA level PIAS3 mRNA level PIAS4 mRNA level * 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 * * 0.2 0.2 0.2 0 * 0 0 0 Scramble Scramble Scramble Scramble SiRNA PIAS4 SiRNA PIAS2 SiRNA PIAS3 SiRNA PIAS1 1.2 1.2 1.2 1.0 1.0 1.0 0.8 0.8 0.8 CBX4 mRNA level RanBP2 mRNA level Ubc9 mRNA level 0.6 0.6 0.6 * 0.4 0.4 0.4 * 0.2 0.2 0.2 * 0 0 0 Scramble Scramble Scramble SiRNA RanBP2 SiRNA Ubc9 SiRNA CBX4 Supplementary Figure 5

6. Supplementary Table 1 Supplementary Table 2