2. Supplemental Figure S2. Location and H 2 O 2 -dependent binding of RBPs to biotinylated MKP-1 transcripts. (A) Schematic of the MKP-1 mRNA showing the coding region (CR) and the 3’- untranslated region (UTR). Biotinylated transcripts spanning the CR and full length (3’UTR) or partial (A, B, C) segments were prepared for biotin pulldown analysis. (B) Cytoplasmic lysates prepared from HeLa cells that were either left untreated or treated with H 2 O 2 (1 mM, 2.5 hr) were incubated with the biotinylated transcripts indicated. The ribonucleoprotein complexes were pulled down using streptavidin-coated beads and the presence of the RBPs indicated was tested by Western blot analysis (‘Beads’); aliquots of the ‘Reaction supernatant’ were examined in parallel. (C) The MKP-1 3’UTR was subdivided and biotin pulldown analysis performed to in order to test the relative binding of NF90 and HuR to each segment. As shown, both HuR and NF90 showed the greatest binding to biotinylated fragment C, both showed weak binding to transcript A and both showed potent H 2 O 2 -dependent binding to fragment B. The significance of this interaction pattern will be pursued elsewhere. 5’UTR 12492015 1352 CR3’UTR A B C CR MKP-1 HuR NF90 H 2 O 2 : - + - + - + - + - + RNA: 3’UTR A B C CR HuR AUF1 NF90 TIA-1 TIAR A Reaction supernatant RNA: CR 3’UTR GAPDH CR 3’UTR GAPDH Beads H 2 O 2 : - + - + - + - + - + - + Kuwano et al., Supplemental Figure S2 B C

3. 100 kDa - 50 kDa - - NF90 WB: phospho-Serine H 2 O 2 (h) 0 1 4 0 1 4 IP: IgG NF90 Kuwano et al., Supplemental Figure S3 Supplemental Figure S3. NF90 serine phosphorylation. HeLa cells were either left untreated or treated with H 2 O 2 (1 mM). (A) One hour later, the levels of total Akt and phosphorylated Akt were assessed by Western blot analysis using anti-Akt and anti-phospho (p)-Akt antibodies (Cell Signaling); GAPDH was measured as loading control. (B) Lysates were prepared 1 and 4 hr after H 2 O 2 treatment and used in IP reactions with IgG or anti-NF90 antibodies. Serine phosphorylation in the IP reaction products was detected by Western blot analysis of the IP reactions using anti-phospho-Serine antibody (Cell Signalling). p-Akt Akt GAPDH H 2 O 2 - + A B

4. MKP-1 mRNA in TAP-IP (relative levels) 0 2 4 6 Untr. H 2 O 2 A TAP HuR (WT) TAP HuR (S88A) TAP HuR (S100A) TAP HuR (T118A) TAP H 2 O 2 - + - + - + - + - + - + HuR 3’UTR siRNA Control siRNA TAP-HuR HuR  -Actin Supplemental Figure S4. Influence of HuR mutation on S88, S100, T118 on the association of HuR with MKP-1 mRNA. (A) HeLa cells were transfected with plasmids expressing either wild type (WT) HuR or HuR carrying mutations S88A, S100A or T118A (all TAP-tagged proteins). HeLa cells were simultaneously transfected with HuR siRNA targetting the HuR 3’UTR (to which the ectopic HuR-TAP transcripts were refractory). Forty-eight h after transfection, cells were treated with 1 mM H 2 O 2 and collected 3 h later. The association of each fusion protein to MKP-1 mRNA before and after H 2 O 2 treatment was tested by TAP-IP. (B) The levels of ectopic HuR-TAP proteins as well as endogenous HuR are shown. The siRNA targeting the HuR 3’UTR reduced HuR levels only modestly; therefore, the effect of expressing HuR mutants (S88A, S100A, T118A) on MKP-1 production could not be tested. Kuwano et al., Supplemental Figure S4 B TAP HuR (WT) TAP HuR (S88A) TAP HuR (S100A) TAP HuR (T118A) TAP

5. D A Supplemental Figure S5. Influence of MKP-1 on H 2 O 2 -induced apoptotic markers. (A, B) HeLa cells were transfected with control (Ctrl.), HuR-directed or MKP-1-directed siRNA; 48 h later, cells were either treated with H 2 O 2 (1 mM) or left untreated (-) and were collected for Western blot analysis at the times shown. The phosphorylation of ERK1/2, p38 MAPK, and JNK (A), as well as cleavage of caspase-9 and PARP signals were monitored thereafter (B). (C) HeLa cells were transfected with pHuR-TAP (26), treated with H 2 O 2 for the times shown, and HuR-TAP, HuR, as well as cleaved caspase-9 and PART were tested by Western blot analysis. (D) HeLa cells were transfected with plasmid pGFP-MKP-1 (obtained from Addgene), treated with H 2 O 2, and the levels of GFP-MKP-1 (GFP), cleaved caspase-9, and cleaved PARP were visualized by Western blot analysis. In all cases,  -Actin served as loading control. H 2 O 2 (h) 0 1 3 0 1 3 0 1 3 Ctrl. siRNA MKP-1 siRNA HuR siRNA p-JNK p-ERK p-38MAPK MKP-1 HuR  -Actin B H 2 O 2 0 3 6 0 3 6 Cleaved Caspase-9 Cleaved PARP  -Actin H 2 O 2 (h) 0 3 6 0 3 6 Ctrl. siRNA MKP-1 siRNA GFP Cleaved Caspase-9 Cleaved PARP  -Actin Vector pGFP- -MKP-1 H 2 O 2 (h) 0 3 12 0 3 12 pHuR- -TAP Tap-HuR HuR Cleaved Caspase-9 Cleaved PARP  -Actin Vector B Kuwano et al., Supplemental Figure S5

6. Supplemental Figure S6. Influence of MKP-1 on H 2 O 2 -reduced cell survival. HeLa cells were transfected as explained in Supplemental Figure S5, then treated with 0.5 mM H 2 O 2 and cell survival was measured by the MTT assay 24 h later. (A) Effect of overexpressing HuR in the presence of normal or reduced MKP-1 levels. (B) Effect of overexpressing MKP-1 in the presence of normal or reduced HuR levels. The data are the means and S.E.M. from three independent experiments. Vector pHuR- -TAP pHuR- -TAP Ctrl. siRNA MKP-1 siRNA A % Cell Survival (500  M H 2 O 2, 1 h) B 0 20 40 60 80 100 120 Ctrl. siRNA HuR siRNA % Cell Survival (500  M H 2 O 2, 1 h) 0 20 40 60 80 100 120 Untr. H 2 O 2 Kuwano et al., Supplemental Figure S6 Vector pGFP- -MKP-1 Vector pGFP- -MKP-1 Untr. H 2 O 2