Supplementary figures and legends for

Transcription

1 Supplementary figures and legends for No evident dose-response relationship between cellular ROS level and its cytotoxicity a paradoxical issue in ROS-based cancer therapy Chunpeng Zhu, Wei Hu, Hao Wu, Xun Hu correspondence to: huxun@zju.edu.cn 1

2 Supplementary figure S1. Breast cancer cell Bcap37 growth inhibition or death is dissociated from ROS levels induced by exogenous ROS insults. (a) Cell growth inhibition or cell death. Bcap37 cells were treated with LA (20 mm), PEITC (10 μm), PL (10 μm), L-BSO (20 μm), hydrogen peroxide (100 μm), Dox (10 μg/ml), or ATO (10 μm). Cells were counted at indicated time intervals. (b d). Except L-BSO treatment, in which cellular ROS and GSH levels were determined 12 hours after L-BSO exposure, ROS and GSH levels in Bcap37 cells exposed to other agents were measured 4 hours after treatment. 2

3 Supplementary figure S2. Hela cell growth inhibition or death is dissociated from ROS levels induced by exogenous ROS insults. (a) Cell growth inhibition or cell death. Hela cells were treated with LA (20 mm), PEITC (10 μm), PL (10 μm), L-BSO (20 μm), hydrogen peroxide (100 μm), Dox (10 μg/ml), or ATO (10 μm). Cells were counted at indicated time intervals. (b-d). Except L-BSO treatment, in which cellular ROS and GSH levels were determined 12 hours after L-BSO exposure, ROS and GSH levels in Hela cells exposed to other agents were measured 4 hours after treatment. 3

4 Supplementary figure S3. HepG2 growth inhibition or death is dissociated from ROS levels induced by exogenous ROS insults. (a) Cell growth inhibition or cell death. HepG2 cells were treated with LA (20 mm), PEITC (10 μm), PL (10 μm), L-BSO (20 μm), hydrogen peroxide (100 μm), Dox (10 μg/ml), or ATO (10 μm). Cells were counted at indicated time intervals. (b-d). Except L-BSO treatment, in which cellular ROS and GSH levels were determined 12 hours after L-BSO exposure, ROS and GSH levels in HepG2 cells exposed to other agents were measured 4 hours after treatment. 4

5 Supplementary figure S4. The composition of ROS in Bcap37 cells induced by LA, PEITC, PL, L-BSO, hydrogen peroxide, or ATO. Bcap37 cells were treated with LA (20 mm), PEITC (10 M), or PL (10 M), hydrogen peroxide (100 M) for 4 hours, or L-BSO (20 M) for 12 hours. The ROS levels were measured by 5 probes, as described in Materials and Methods. Note that we did not include Dox, which has the similar excitation and emission wavelength to DHE, MitoSOX Red, and HPF. Experiments are repeated at least 3 times. Scale bar =50 m. ** p<0.01, ***, p<

6 Supplementary figure S5. The composition of ROS in Hela cells induced by LA, PEITC, PL, L-BSO, hydrogen peroxide, or ATO. Hela cells were treated with LA (20 mm), PEITC (10 M), or PL (10 M), hydrogen peroxide (100 M) for 4 hours, or L-BSO (20 M) for 12 hours. The ROS levels were measured by 5 probes, as described in Materials and Methods. Note that we did not include Dox, which has the similar excitation and emission wavelength to DHE, MitoSOX Red, and HPF. Experiments are repeated at least 3 times. Scale bar =50 m. ** p<0.01, ***, p<

7 Supplementary figure S6. The composition of ROS in HepG2 cells induced by LA, PEITC, PL, L-BSO, hydrogen peroxide, or ATO. HepG2 cells were treated with LA (20 mm), PEITC (10 M), or PL (10 M), hydrogen peroxide (100 M) for 4 hours, or L-BSO (20 M) for 12 hours. The ROS levels were measured by 5 probes, as described in Materials and Methods. Note that we did not include Dox, which has the similar excitation and emission wavelength to DHE, MitoSOX Red, and HPF. Experiments are repeated at least 3 times. Scale bar =50 m. ** p<0.01, ***, p<

8 Supplementary figure S7. The effect of LA, L-BSO, H 2 O 2, PETIC, PL, ATO, and Dox on antioxidative enzymes in 4T1 cells. 4T1 cells were treated with LA (20 mm), PEITC (10 M), or PL (10 M), hydrogen peroxide (100 M) for 4 hours, or L-BSO (20 M) for 12 hours. Enzyme activities were measured as described in Materials and Methods. Experiments are repeated 2 times. * p<

9 Supplementary figure S8. No evident dose response between cellular ROS and cell growth inhibition or cell death. (a-c) Bcap37, Hela, or HepG2 cell growth inhibition or death is plotted against cellular ROS level induced by the indicated agents, showing no correlation (left panel). Cell growth inhibition or death is plotted against cellular GSH level, showing no correlation (middle panel). Cellular GSH level is plotted against ROS level, showing no correlation (right penal). Experiments were performed at least 3 times, except that the cell growth inhibition or cell death experiments which were performed twice. 9

10 10

11 Supplementary figure S9. NAC fully reverses Dox-induced ROS to basal level but does not block cell death. (a) ROS levels (probed by DCFH-DA) in 4T1 cells treated with 10 μg/ml Dox in the presence or absence of NAC. (b) 4T1 cell growth or death. Cells were treated with 10 μg/ml Dox in the presence or absence of NAC. Cell counts were done at the indicated time intervals. *** p< Experiments were performed for 3 times. 11

12 Supplementary figure S10. NAC fully reverses ATO-induced ROS to basal level but does not block cell death. (a) ROS levels (probed by DCFH-DA) in 4T1 cells treated with 10 μm ATO in the presence or absence of NAC. (b) 4T1 cell growth or death. Cells were treated with 10 μm ATO in the presence or absence of NAC. Cells counts were done at the indicated time intervals. *** p< Experiments were performed for 3 times. 12

13 Supplementary figure S11. NAC fully reverses PEITC-induced ROS to basal level but does not block cell death. (a) ROS levels (probed by DCFH-DA) in Bcap37, Hela, or HepG2 cells treated with 10 μm PEITC in the presence or absence of NAC. (b) Cell growth or growth inhibition (lower growth rate) or death. Cells were treated with 10 μm PEITC in the presence or absence of NAC. Cell counts were done at the indicated time intervals. Experiments were performed 3 times.scale bar =50μm. ***, p<

14 Supplementary figure S12. NAC fully reverses Dox-induced ROS to basal level but does not block cell death. (a) ROS levels (probed by DCFH-DA) in Bcap37, Hela, or HepG2 cells treated with 10 μg/ml Dox in the presence or absence of NAC. (b) Cell growth or death. Cells were treated with 10 μg/ml Dox in the presence or absence of NAC. Cell counts were done at the indicated time intervals. Experiments were performed 3 times. Scale bar =50μm. ***, p<

15 Supplementary figure S13. NAC fully reverses ATO-induced ROS to basal level but does not block cell death. (a) ROS levels (probed by DCFH-DA) in Bcap37, Hela, or HepG2 cells treated with 10 μm ATO in the presence or absence of NAC. (b) Cell growth or death. Cells were treated with 10 μm ATO in the presence or absence of NAC. Cells counts were done at the indicated time intervals. Experiments were performed 3 times. Scale bar =50μm. ***, p<

16 Supplementary figure S14. NAC fully blocks PL-induced cell death but only partially reverses ROS level. (a) ROS levels (probed by DCFH-DA) in Bcap37, Hela, or HepG2 cells treated with 10 μm PL in the presence or absence of NAC. (b) Cell growth or growth inhibition (lower growth rate) or death. Cells were treated with 10 μm PL in the presence or absence of NAC. Cell counts were done at the indicated time intervals. Experiments were performed 3 times. Scale bar =50μm. ***, p<

17 Supplementary figure S15. PL, PEITC, and ATO kill primary human colon or rectal cancer cells but only moderately increase cellular ROS. (a) Comparison of cellular ROS induced by hydrogen peroxide, LA, PEITC, PL, or ATO. Cells were treated with hydrogen peroxide (100 μm), LA (20 mm), PEITC (10 μm), PL (10 μm), or ATO (10 μm) for 4 hours, followed by cellular ROS measurement, as reflected by DCFH-DA signals. (b) Cells were treated with hydrogen peroxide (100 μm), LA (20 mm), PEITC (10 μm), PL (10 μm), or ATO (10 μm) for 24 hours, subjected for cell counts and death rate calculation. (c) Representative photos of cells 24 hours after treatment. Scale bar =50μm. * p<0.05, ** p<0.01, ***, p<