1. sh-luc shSCP1-1 * IB: c-Myc IB: Tubulin shSCP1-2 IB: p-Ser62 1.0 1.8 2.5 60KD 70KD 60KD 70KD 1 2 3 (abnova) sh-luc shSCP1-1 * IB: c-Myc IB: Tubulin shSCP1-2 IB: p-Ser62 1.0 3.1 3.3 60KD 70KD 60KD 70KD 1 2 3 IB: SCP1 (abcam) B C 60KD 70KD 60KD 70KD 1 2 3 4 5 6 7 8 9 10 11 12 NC shSCP1-1 shSCP1-2 * IB: c-Myc IB: Actin IB: p-Ser62 IB: SCP1 0 20 40 60 0 20 40 60 0 20 40 60 CHX(min) (abcam) 0 20 40 60 0 20 40 60 0 20 40 60 CHX(min) NC shSCP1-1shSCP1-2 * IB: c-Myc IB: Tubulin IB: p-Ser62 1 2 3 4 5 6 7 8 9 10 11 12 (abnova) Figure S1 A T58A S62A T88A/S62A IB: p-Ser62 (abcam) IB: p-Ser62 (abnova) IB: c-Myc WT IB: HA-GFP 1 2 3 4 HA-c-Myc Figure S1. Specificity of two c-Myc Ser62 phosphorylation antibodies from Abcam and Abnova. (A). Both phosphorylation antibodies recognize the phosphorylated Ser62. HEK293T cells were transfected and c-Myc were analyzed by Western Blotting. (B). HepG2 cells infected with retrovirus were lysed and the protein levels were analyzed using Western Blotting. * indicatded a non-specific protein band.. ( C) Knockdown of SCP1 prolonged c-Myc half-life. HepG2 cells were infected with retrovirus of sh-Luc and shSCP1. The half-life of endogenous c-Myc in control or SCP1 knockdown cells was detected using a CHX chase assay. * indicated a non-specific protein band.

2. D IB: c-Myc IB: SCP1 IB: SKP2 IB: Actin siNC siSKP2 SCP1 - + - + c-Myc protein expression siNC siFBW7 siNC siSKP2 1 2 3 4 IB: c-Myc IB: SCP1 IB: FBW7 IB: Actin SCP1 - + - + siNC siFBW7 Figure S2 Figure S2. SCP1 affects c-Myc stability dependent on FBW7. (A). SCP1 has no significant effect on PP2A protein level. HEK293T cells were transfected as indicated. PP2A were analyzed by Western Blotting using PP2A specific antibody (CST #4953) and (B) the different subunits of PP2A were analyzed by QPCR. (C). SCP1 increases c-Myc ubiquitination. HEK293T cells were transfected as indicated. Cells were treated with MG132 for 6 hours, and ubiquitination was analyzed using Ni-NTA-based pull-down assays. (D). Knockdown of FBW7, but not SKP2, blocked the effect of SCP1 on c-Myc stability. HEK293T were transfected with 40nM of control siRNA (siNC), siFBW7 or siSKP2 respectively. After 24h, FLAG-vector or FLAG-SCP1 were transfected into cells in the indicated combinations and analyzed by Western Blotting. C HA-c-Myc + + + + - - His-Ub - + + + + + FLAG-SCP1 - - WT DN WT DN Ni-NTA precipitated WCE IB: FLAG IB: c-Myc 1 2 3 4 5 6 Myc(Ub) n IB: c-Myc IB: PP2A B unit vector IB: Tubulin IB: SCP1 SCP1-WT SCP1-DN A B

3. Figure S3 Relative mRNA expression RhoA reporter Relative luciferase activity ctrl c-Myc WT c-Myc S62A c-Myc S62D ** IB:c-Myc RhoA ** Figure S3. The transcriptional activity of c-Myc wild-type and its mutant. (A). SCP1 has no significant effect on mRNA level of WDFY2 and PIP5K1A. HEK293T cells were transfected as indicated. The expression of WDFY2 and PIP5K1A mRNA were analyzed by Q-PCR. (B). c-Myc WT, S62A and S62D mediated RhoA reporter gene activity. HEK293T cells were transfected as indicated, the promoter activity were analyzed by Dual-Luciferase assay, c-Myc protein expression were detected by Western blot. (C,D). HEK293T were transfected as indicated, the RNA expression of RhoA or Bax were detected by Q-PCR. The data are means ± s.d. (** P<0.01, t-test; n=3) A C Relative mRNA expression Bax ** * D B

4. Figure S4 OD 490 (nm) HeLa HT29 IB:c-Myc IB: SCP1 IB: Actin sh-SCP1 - + - + sh-c-Myc - - + + IB:c-Myc IB: SCP1 IB: Actin sh-SCP1 - + - + sh-c-Myc - - + + A B 1 2 3 4 Figure S4. SCP1 affects cells proliferation in a c-Myc dependent manner. HeLa (A) or HT29 (B) were infected with retrovirus as indicated. The cell proliferation was detected using MTT assay, and knockdown of c-Myc and SCP1 were analyzed by Western Blotting