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1 Supplementary Figure S PF (µm) U87 (EC 5 = 52.2 ± 8.8 µm) CMPD1 (µm) Primary GM (EC 5 = 1.55 ±.3 µm) U138 (EC 5 = 1.7 ±.8 µm) SW-188 (EC 5 =.93 ± 1.1 µm) NCI-H23 (EC 5 = 1.61 ±.2 µm) 549 (EC 5 =.99 ±.7 µm) HCT116 (EC 5 = 4.65 ± 1.3 µm) Cell line U138 SW-188 NCI-H HCT 116 Tumor type Glioblastoma strocytoma III Lung Lung Colon C CMPD1 (µm) D p-p38 Time (h) MK2 MK2 p-hsp27 p-hsp27 Hsp27 Hsp27 Supplementary Figure S1. () U87 cells were treated with PF (72 h) and lamarlue cell viability assay was performed. Data are expressed as mean ± SEM (n = 3). () Cancer cells were treated with CMPD1 (72 h) and lamarlue cell viability was performed. Data are expressed as mean ± SEM (n = 2). (C-D) U87 cells were incubated with CMPD1 for (C) 48 h or (D) 6-72 h and cell lysates were analysed by Western blotting with indicated antibodies. Representative blots of three independent experiments are shown.

2 Supplementary Figure S2 C % cells in cell cycle phases S2358 (µm) G2/M S G1 SubG1 % of cells with DN content > 4N *** *** Time (h) % cells in cell cycle phases Time (h) G2/M S G1 SubG1 D Time (h) p-cdc25c (S216) Cdc25c p-cdk1 (Y-15) Cdk1 cyclin p-hh3 (S28) GPDH Supplementary Figure S2. () U87 cells were treated with S2358 (48 h) and cell cycle distribution was analysed by flow cytometry. Data are expressed as the mean ± SEM (n=3). () U87 cells were treated with for indicated time and percentage of cells with DN content greater than 4n was determined by flow cytometry. Data are expressed as the mean ± SEM (n = 3; ***P <.1, 1-way NOV followed by Newman- Keuls post-test). (C) U87 cells were treated with for indicated time and cell cycle distribution analysed as in (). Data are expressed as the mean ± SEM (n = 3).(D) U87 cells were treated with for indicated time. Cell lysates were analysed by Western blotting using indicated antibodies. Representative blots of three independent experiments are shown.

3 Supplementary Figure S3 C nnexin-v positive cells (%) MK2i (µm) nnexin-v positive cells (%) S2358 (µm) MG132 (1 µm) Mcl-1 GPDH Supplementary Figure S3. (-) U87 cells were treated with MK2i () or S2358 () for 48 h and nnexin-v positive cell were analysed with flow cytometry. Data are expressed as mean ± SEM (n = 3). (C) U87 cells were pre-treated with proteasome inhibitor MG132 (1 µm; 1 h), then incubated with for 24 h. Cell lysates were analysed by Western blotting using indicated antibodies. Representative blots of three independent experiments are shown.

4 Supplementary Figure S4 C U251 U87-EGFRvIII U87 cells 48h post-transfection sirn MK2 sirn sirn MK2 sirn sirn MK2 sirn Supplementary Figure S4. () U251 cells and () U87-EGFRvIII cells were transfected with control sirn or sirns targeting MK2 (5 nm) overnight. lamarlue cell viability assay was performed 72 h post-transfection. Data are expressed as mean ± SEM (n = 3-5). (C) Representative image of U87 cells 48 h post-transfection with control sirn or MK2 sirn.

5 Supplementary Figure S5 U87 cells after 8h treatment with CMPD1 CMPD1 (.5 µm) 12h 16h 2h Nocodazole p-p38 (Thr334) MK2 p-hsp27 Hsp27 Supplementary Figure S5. () Representative images of morphological changes in U87 cells after treatment with CMPD1 (8 h). () U87 cells were treated with nocodazole (1 µg/ml) for indicated time. oth attached and floating cells were harvested at the 12 h time point, and only floating cells were harvested at the 16 h and 2 h time points. Cell lysates were analysed by Western blotting using indicated antibodies. Representative blots of three independent experiments are shown.

6 Supplementary Figure S6 strocytes after 48h treatment with CMPD1 CMPD1 (.5 µm) % of cells with DN content > 4N (fold change) *** ** CMPD1 (µm) strocytes U87 C strocytes after 1h U87 after 1h CMPD1 (.5 µm) Supplementary Figure S6. () Representative images of primary human astrocytes 48 h post-treatment with CMPD1. () U87 cells or primary human astrocytes were treated with CMPD1 (48 h) and percentage of cells with DN content greater than 4n was determined using flow cytometry. Data are expressed as mean ± SEM (n = 3; **P <.1, ***P <.1, 1- way NOV followed by Newman-Keuls post-test). (C) Representative images of morphological changes in U87 cells vs primary human astrocytes 1 h post-treatment with CMPD1.

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