Supplementary Materials: Identification of Selective ERRγ ... · Web viewFigure S7. Induction of dose-dependent apoptosis in CAL62 cells following DN200434 treatment. (A) CAL62 cells

Supporting Information S1 of S34

A novel orally active inverse agonist of estrogen-related receptor gamma (ERRγ), DN200434, a booster of NIS in anaplastic thyroid cancerThoudam Debraj Singh1,2,‡, Jaeyoung Song3,‡, Jina Kim3,‡, Jungwook Chin3,‡, Hyun Dong Ji4, Jae-Eon Lee5, Sang Bong Lee3, Heeseok Yoon3, Ji Hoon Yu3, Sang Kyoon Kim5, GhilSuk Yoon6, Hayoung Hwang3, Ho Won Lee4, Ji Min Oh4, Sang-Woo Lee2,4, Jaetae Lee4, Hueng-Sik Choi10, Soon-Young Na10, Won-Il Choi2, 11, Young Joo Park7, Young Shin Song7, Young-A Kim8, In-Kyu Lee2,9,*, Sung Jin Cho2,3,*, Yong Hyun Jeon2,5,*

1Department of Medical Oncology Laboratory, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, India; 2Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu, South Korea; 3New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, South Korea; 4Department of Nuclear Medicine, School of Medicine, Kyungpook National University Hospital, Daegu, South Korea; 5Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, South Korea; 6Department of Pathology, School of Medicine, Kyungpook National University Hospital, Daegu, South Korea; 7Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, South Korea; 8Department of Pathology, Borame Medical Center 20, Boramae-ro 5-gil, Dongjak-gu, Seoul, South Korea; 9Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, South Korea; 10National Creative Research Initiatives Center for Nuclear Receptor Signals, School of Biological Sciences and Technology, Chonnam National University, Gwangju, South Korea; 11Bio-Medical Research Institute, Kyungpook National University Hospital, Daegu, South Korea‡Authors contributed equally to this work.1To whom correspondence should be addressed. Email: [email protected], [email protected], [email protected]


NMR spectra and HPLC chromatograms

Figure S1. 1H-NMR spectrum


Figure S2. 13C-NMR spectrum


Figure S3. HRMS spectrum


Figure S4. HPLC spectrum


Figure S5. Immunoblotting analysis showing NIS and ERRγ expression levels in normal

thyroid and cancer tissues from ATC samples.


Figure S6. Cytotoxic effects of DN200434 on CAL62 cells using in vitro BLI



Figure S7. Induction of dose-dependent apoptosis in CAL62 cells following DN200434

treatment. (A) CAL62 cells were treated with DN200434 at different concentrations (0, 6,

and 12 μM) for 24 h. Equal amounts of total protein were analyzed by western blotting

using the indicated antibodies. β-actin was used as a loading control. Representative data

from three independent experiments are shown. (B) Flow cytometry analysis of CAL62

cells treated with different concentrations of DN200434 for 24 h, followed by staining with

Annexin V-FITC and PI. The apoptosis ratio increased in a DN434 dose-dependent

manner. (C) Cell cycle analysis of CAL62 cells treated with different concentrations of

DN200434 for 24 h. The sub-G0 phase of Cal62 was induced by DN434 in a dose-

dependent manner.


Figure S8. (A) Downregulation of ERRγ protein expression in DN200434-treated CAL62

cells and quantification of band intensity. (B) Upregulation of p-ERK1/2 levels by

DN200434 and quantification of band intensity. Band intensity of western blots was

determined using Image J. Data are presented as the means ± SD. *P < 0.05, **P < 0.01,

***P < 0.001.


Figure S9. (A) Inhibition of increased radioiodine uptake by the MAP kinase inhibitors

U0126 and PD98059. (B) Reversal of activated p-ERK1/2 and NIS protein by PD980519

(upper) and (C) quantification of band intensity (bottom). The band intensity of western

blots was determined using Image J. Data are presented as the means ± SD. *P < 0.05, **P

< 0.01, ***P < 0.001.


Figure S10. TG, TSHR, and TPO protein expression levels in DN200434-treated CAL62

cells quantified via western blotting. Band intensity was determined using Image J. Data

are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.


Figure S11. Increase in membranous and total NIS protein levels in DN200434-treated

CAL62 cells and quantification of band intensity. Band intensity of western blots was

determined using Image J. Data are presented as the means ± SD. *P< 0.05, **P < 0.01,

***P < 0.001.


Figure S12. Reversal of increased NIS protein levels in DN200434-treated CAL62 cells by

the MEP-specific inhibitors PD98059 and U0126.


Figure S13. Time-dependent changes in thyroid-regulating gene protein levels in DN200434-treated CAL62 cells and quantification of band

intensity. Band intensity of western blots was determined using Image J. Data are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P <

0.001.


Figure S14. Protein expression levels of GLUT1 and GLUT4 quantified via western blot

band intensity. Data are presented as the means ± SD. *P < 0.05



Figure S15. (A) Schematic illustrating the biodistribution study. (B) Radioactivity in

CAL62 tumors from mice treated with either 100 or 200 mg/kg DN200434. (C)

Radioactivity of all organs without tumors in 100 and 200 mg/kg-treated mice. CAL62

tumor-bearing mice received either vehicle or DN200434 (100 and 200 mg/kg) via oral

administration once a day for 6 days. Mice were sacrificed and all organs were excised,

followed by measurement of organ weight. Radioactivity of organs was measured with a

gamma counter. %ID/g: percentage injected dose per gram.


Figure S16. Enhanced cytotoxicity of 131I by DN200434 in CAL62 cells. (A and B)

Clonogenic assay of CAL62 cells treated with vehicle, DN200434 alone, 131I alone, and a

combination of DN200434 and 131I. N.S, not significant. ***P < 0.001.



Figure S17. In vivo antitumor effects of the DN200434 and radioiodine-131 combination

therapy in BHT101 tumor-bearing mice. (A) Schematic illustrating the in vivo therapy

design. BHT101 tumor-bearing mice were divided into four groups as follows, group 1,

vehicle-treated group; group 2, 131I-treated group; group 3, DN200434-treated group; and

group 4, DN200434+131I-treated group. Tumor volume measurement was conducted to

assess therapeutic response over time. DN200434 was orally administered once a day for 6

days. (B) Comparison of BHT101 xenograft growth curves of mice treated with either

single (DN200434 or 131I) or combination (DN200434 + 131I) therapy. Inset shows the

statistical comparison of therapeutic outcomes among groups at day 32 post-treatment. *P

< 0.05, **P < 0.01, ***P < 0.001.


a

b

Figure S18. Body weight measurement (A) CAL62 or (B) BHT101 tumor-bearing mice

during DN200434 and radioiodine-131 combination therapy.

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Supplementary Materials: Identification of Selective ERRγ ... · Web viewFigure S7. Induction of dose-dependent apoptosis in CAL62 cells following DN200434 treatment. (A) CAL62 cells