Supplementary information. Discovery of Novel STAT3 DNA Binding Domain Inhibitors

Table S1

Description of synthesis

Spectra of synthesized compounds

Figure S1. Western blot gel for Compound 11c and 11d. MDA-MB-231 cell line treated by DMSO (0.1%), PYM 100µM, 11c (5 µM and 10 µM), 11d (5 µM and 10 µM) for 24 h. (a) gel of Actin, Bcl-2, and Bcl-xL. (b) Gel of Cyclin D1. (c) Gel for p-STAT3 and Actin. (d) Gel of T-STAT3 and Actin.

Figure S2. Western blot gel for Compound 11b and 15a. MDA-MB-231 cell line treated by DMSO (0.1%), PYM 100µM, 11b (5 µM and 10 µM), 15a (5 µM and 10 µM) in two separate experiments for 24 h. (a). gel of p-STAT3, GAPDH, Cyclin D1, Bcl-2, and Bcl-xL. (b). Gel of T-STAT3 and Actin.

Figure S3. Absorbance of NADPH without DHFR reduction.

Figure S4. Novel STAT3 inhibitor candidates do not inhibit DHFR activities. DHFR activity assay was performed with 1% provided hDHFR incubating with NADPH and DHFR substrate as the positive control. The treatment group was treated with 1% DMSO, or DMSO solution of PYM 500µM (2x IC50), 125 µM, 31.25 µM, 11b-d (10x IC50) and 15a (1x IC50) along with provided hDHFR, NADPH and DHFR substrate. DHFR activity was read at OD 340nm for NADPH absorbance with kinetic scanning (2-minute intervals from 0 to 28 minutes). The experiment was performed in duplicate data