Supplementary information for the manuscript:
Phosphorylation of signal transducer and activator of transcription 1 (STAT1) reduces bortezomib-mediated apoptosis in cancer cells
C Kao1,2#, A Chao*1#, CL Tsai1, CY Lin1, WC Chuang1,2, HW Chen1,2, TC Yen3, TH Wang*1,2,4, CH Lai1, HS Wang1,5
1Department of Obstetrics and Gynecology, Linkou Medical Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333, Republic of China
2Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 333, Republic of China
3Department of Nuclear Medicine, Linkou Medical Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333, Republic of China
4Genomic Medicine Research Core Laboratory, Linkou Medical Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333, Republic of China
5Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 333, Republic of China
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Supplementary Figures
figure S1. Results of the reporter assays regulated by the treatment with bortezomib. (A) Ovarian cancer TOV112D cells were transfected with the indicated reporter constructs. Bortezomib decreased the activity of the NPM1/B23, E2F1, YY1, MMP-9, and HRE reporters. (B) In TOV21G cells, the STAT1 response element reporter was activated by bortezomib. However, both MG132 (a generic inhibitor that blocks proteasomal degradation) and paclitaxel (PTX) failed to activate the STAT1 response element reporter.
figure S2. HSP70 is involved in bortezomib-induced cytotoxicity. (A-D) Bortezomib increased HSP70 and caspase-3 cleavage in MDAH2774, TOV21G, BG1, and OVCAR3 cells. (E, G) The knockdown of HSP70 enhanced the activation of caspase-3 induced by bortezomib in MDAH2774 and TOV21G cells. The LDH assay showed that the knockdown of HSP70 enhanced the cytotoxicity of bortezomib in MDAH2774 (F) and TOV21G (H) cells. Actin was used as an internal control. The results are representative of at least three independent experiments. Abbreviation: BTZ, bortezomib.
figure S3. HSF-1 is involved in bortezomib-induced cytotoxicity. (A) We observed a dose-dependent activation of HSF1 in ovarian cancer cells exposed to bortezomib (0.05 or 0.25 μM) for 24 hours. (B) Results of western blot analysis showing the efficiency of shRNA for the knockdown of HSF-1. (C) The overexpression of either HSF1 or HSP70 attenuated the cytotoxic effects of bortezomib. Actin was used as an internal control. The results are representative of at least three independent experiments. Abbreviation: BTZ, bortezomib.
figure S4. Synergistic, cytotoxic effects of the combined treatment with cisplatin (CDDP) and bortezomib (BTZ) in SKOV3 and BR cancer cells. (A, B) SKOV3 and BR cells were treated with 5 μM of cisplatin and 10 μM of bortezomib for 24 h. The cytotoxic effects of the combined treatment with cisplatin (5 μM) and bortezomib (10 μM) were analyzed using the LDH assay. The results are representative of at least three independent experiments. Abbreviation: BTZ, bortezomib.
figure S5. S727E STAT1, which mimicks S727-phosphorylated STAT1, counteracts cell death induced by the combined treatment with bortezomib (BTZ) and JAK inhibitor (JAKi). (A) TOV112D cells were transfected with EGFP or EGFP-STAT1 (S727E), and then treated with bortezomib.TOV112D cells were immunostained with anti-cleaved-caspase 3 antibody and DAPI. Scale bars indicate 75μm. (B) TOV112D cells were treated with bortezomib alone or combined with JAKi for overnight. TOV112D cells were immunostained with anti-cleaved-caspase 3 antibody and DAPI. Scale bars indicate 100μm. (C) Expression of EGFP-STAT1 (S727E) was confirmed with western blot analysis.
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