A B A Stat3:Stat3 Supershift  -ST3 Free probe BP1-102 (µM) 0 5 10 20 Stat3:Stat3 % of Cont 100 56 43 1 Supplementary Figures Fig. S1 -

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Presentation transcript:

A B A Stat3:Stat3 Supershift  -ST3 Free probe BP1-102 (µM) Stat3:Stat3 % of Cont Supplementary Figures Fig. S1 -

BP1-102 (µM) pY705-Stat3 Stat3 MDA-MB-231 A B Stat SH4-54 SH5-07 (µM, 1 h) U251MGMDA-MB SH4-54 SH5-07 pS727-Stat3 Fig. S2 1 h24 h C pY705-Stat3 Stat3 RCC1 Stat3:Stat3 EMSA WB - 5 µM SH µM BP µM SH i) ii) D SH5-07 ( μ M, 1 h) pY705-Stat3 Stat3 RCC1 pY705-Stat3 Stat3 RCC1 MDA-MB-231 U251MG SH5-07 ( μ M, 1 h) SH4-54 ( μ M, 1 h) i)ii) E Stat3 pcDNA3 (μg) ST3-SH2 (μg) pY705Stat3 Lane Stat3-SH Lane U251 Stat3:Stat3 pcDNA3 (μg) ST3-SH2 (μg) i)ii) U251 GAPDH Stat3:Stat

µM SH p-Src JAK2 pJAK2 ERK1/2 MAPK pERK1/2 MAPK Src β-actin MDA-MB-231 pY1068EGFR U251MG µM SH5-07 EGFR pShc Shc pAkt Akt A) B) p-Src JAK2 pJAK2 ERK1/2 MAPK pERK1/2 MAPK Src β-actin pY1068EGFR EGFR pShc Shc pAkt Akt 1 h h Treatment time 08 1 h 24 h 08 Treatment time µM SH4-54 p-Src JAK2 pJAK2 ERK1/2 MAPK pERK1/2 MAPK Src β-actin pY1068EGFR EGFR pShc Shc pAkt Akt 08 1 h 24 h 08 Treatment time C) D) µM SH4-54 p-Src JAK2 pJAK2 ERK1/2 MAPK pERK1/2 MAPK Src β-actin pY1068EGFR EGFR pShc Shc pAkt Akt 08 1 h 24 h 08 Treatment time U251MG MDA-MB-231 Fig. S3

Fig. S4 β-actin pY1068-EGFR pY705-Stat3 Stat EGF (100 ng/ml, 12 min) SH5-07 (10 µM, h) EGFR

When intended to make 200 uM concentration solution, precipitation was observed and the signal of approximately 20 uM compound was observed. Structure and predicted NMR chemical shifts of SH4-54 SH4-54 NMR Fig. S5 A)

13 C Chemical Shift (ppm) 1 H Chemical Shift (ppm) Fig. S5 B)

pY705-Stat3 NIH3T3/v-Src DU145 MDA-MB-231 NIH3T3 SF295 U87MG U373MGU251MG Cell line Fig. S6 Tubulin Stat pYStat3/Stat3 Panc-1 MCF-7 A2780S pY705Stat3 Stat3 Cell line pYStat3/Stat3 β-actin

MDA-MB-231 SH5-07 (µM) U251MG Fig. S7 (B) MDA-MB-231 Duration (h) DMSO (0.05%) DU145 # of viable cells (x 10 5 ) SH5-07 (8 µM) (i) (ii) U251MG SH4-54 (µM, 22 h) (A) MDA-MB-231 Duration (h) DMSO (0.05%) SH4-54 (8 µM) # of viable cells (x 10 5 ) U251MG DU145 SH4-54 (µM, 22 h)

Body weight (g ) U251MG Duration time (day) Blood cell (x 10 6 /μL ) Body weight (g ) Duration time (day) MDA-MB231 A Fig. S8 C ii)i) Control SH5-07 SH4-54 MDA-MB-231 xenografts Control SH5-07 SH4-54 U251MG xenografts B i) ii)