1. 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 -

2. BP1-102 (µM) 0 5 15 0 5 15 pY705-Stat3 Stat3 MDA-MB-231 A B Stat3 0 1 3 10 1 3 10 SH4-54 SH5-07 (µM, 1 h) U251MGMDA-MB-231 0 1 3 10 1 3 10 SH4-54 SH5-07 pS727-Stat3 Fig. S2 1 h24 h C pY705-Stat3 Stat3 RCC1 Stat3:Stat3 EMSA WB - 5 µM SH4-54 5 µM BP-1-102 +- - - -- + 5 µM SH-5-07 -+-- i) ii) D 0 1 3 5 8 10 SH5-07 ( μ M, 1 h) pY705-Stat3 Stat3 RCC1 pY705-Stat3 Stat3 RCC1 MDA-MB-231 U251MG SH5-07 ( μ M, 1 h) 0 1 3 5 8 10 0 1 3 10 SH4-54 ( μ M, 1 h) 0 1 3 10 i)ii) E Stat3 pcDNA3 (μg) ST3-SH2 (μg) pY705Stat3 Lane Stat3-SH2 - 1 4 - - - - - - 1 2 4 Lane U251 Stat3:Stat3 pcDNA3 (μg) ST3-SH2 (μg) - 1 4 - - - - - - 1 2 4 i)ii) U251 GAPDH Stat3:Stat3 123456 123456

3. µM SH5-070358101 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 08 24 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

4. Fig. S4 β-actin pY1068-EGFR pY705-Stat3 Stat3 - - 0.08 0.5 3 24 - + + + + + EGF (100 ng/ml, 12 min) SH5-07 (10 µM, h) EGFR

5. 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)

6. 13 C Chemical Shift (ppm) 1 H Chemical Shift (ppm) 1.51.31.1 7 3 -9 -5 0.9-0.70.5 27 19 23 11 15 0.10.70.3-0.3-0.5-0.9 Fig. S5 B)

7. pY705-Stat3 NIH3T3/v-Src DU145 MDA-MB-231 NIH3T3 SF295 U87MG U373MGU251MG Cell line Fig. S6 Tubulin Stat3 3.1 4.3 1.4 1.6 1.0 3.8 4.0 4.3 pYStat3/Stat3 Panc-1 MCF-7 A2780S 1.6 0.1 0.3 pY705Stat3 Stat3 Cell line pYStat3/Stat3 β-actin

8. MDA-MB-231 SH5-07 (µM) 0 2 5 8 U251MG Fig. S7 (B) MDA-MB-231 Duration (h) 0 22 0 22 0 22 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) 0 22 0 22 0 22 DMSO (0.05%) SH4-54 (8 µM) # of viable cells (x 10 5 ) U251MG DU145 SH4-54 (µM, 22 h)

9. 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)