Inorganics 2017, 5, 12; doi:10.3390/inorganics5010012
S1 of S6
Supplementary Materials: A Cancer Stem Cell Potent
Cobalt(III)–Cyclam Complex Bearing Two
Tolfenamic Acid Moieties
Paul B. Cressey, Arvin Eskandari and Kogularamanan Suntharalingam
Table of Content
Figure S1
Figure S2
Figure S3
Figure S4
Figure S5
Figure S6
Figure S7
Figure S8
Figure S9
Figure S10
Figure S11
Figure S12
Figure S13
H NMR spectrum in DMSO-d6 of the cobalt(III)–cyclam complex, 3.
C NMR spectrum in DMSO-d6 of the cobalt(III)–cyclam complex, 3.
ESI-TOF mass spectrum (positive mode) of the cobalt(III)–cyclam complex, 3.
1H NMR spectrum in DMSO-d6 of tolfenamic acid.
UV–vis spectrum of 3 (25 μM) in PBS over the course of 24 h at 37 °C.
UV–vis spectrum of 3 (25 μM) in PBS in the presence of ascorbic acid (250 μM)
over the course of 24 h at 37 °C.
UV–vis spectrum of tolfenamic acid (25 μM) in PBS at 37 °C.
ESI-TOF mass spectrum (positive mode) of 3 (25 μM) in PBS, in the presence of
glutathione (250 μM) after 72 h.
Representative dose–response curves for the treatment of HMLER and HMLERshEcad cells with 3, after 72 h incubation.
Representative dose–response curves for the treatment of HMLER-shEcad
mammospheres with 3 after 5 days of incubation in the presence of CoCl2 (5 μM).
Quantification of mammosphere formation with HMLER-shEcad cells untreated
and treated with 3 (at the IC20 value, 5 days) in the presence of cobalt chloride (5 μM).
Representative bright-field images (×10) of HMLER-shEcad mammospheres
supplemented with cobalt chloride (5 μM) in the absence and presence of 3 (at the
IC20 value, 5 days).
Immunoblotting analysis of proteins related to the DNA damage and apoptosis
pathways.
1
13
Figure S1. 1H NMR spectrum in DMSO-d6 of the cobalt(III)–cyclam complex, 3.
Inorganics 2017, 5, 12; doi:10.3390/inorganics5010012
Figure S2. 13C NMR spectrum in DMSO-d6 of the cobalt(III)–cyclam complex, 3.
Figure S3. ESI-TOF mass spectrum (positive mode) of the cobalt(III)–cyclam complex, 3.
S2 of S6
Inorganics 2017, 5, 12; doi:10.3390/inorganics5010012
S3 of S6
Figure S4. 1H NMR spectrum in DMSO-d6 of tolfenamic acid.
1.2
0h
2h
4h
6h
8h
10 h
12 h
14 h
16 h
18 h
20 h
22 h
24 h
Absorption/ a.u.
1.0
0.8
0.6
0.4
0.2
0.0
300
400
500
600
700
Wavelength/ nm
Figure S5. UV–vis spectrum of 3 (25 μM) in PBS over the course of 24 h at 37 °C.
0h
2h
4h
6h
8h
10 h
12 h
14 h
16 h
18 h
20 h
22 h
24 h
Absorption/ a.u.
2.0
1.5
1.0
0.5
0.0
300
400
500
600
700
Wavelength/ nm
Figure S6. UV–vis spectrum of 3 (25 μM) in PBS in the presence of ascorbic acid (250 μM) over the
course of 24 h at 37 °C.
Inorganics 2017, 5, 12; doi:10.3390/inorganics5010012
S4 of S6
Absorption/ a.u.
0.4
0.3
0.2
0.1
0.0
300
400
500
600
700
Wavelength/ nm
Figure S7. UV–vis spectrum of tolfenamic acid (25 μM) in PBS at 37 °C.
Figure S8. ESI-TOF mass spectrum (positive mode) of 3 (25 μM) in PBS, in the presence of glutathione
(250 μM) after 72 h.
100
HMLER
HMLER-shEcad
% Cell viability
80
60
40
20
0
1E-3
0.01
0.1
1
Concentration/ microM
10
100
Figure S9. Representative dose–response curves for the treatment of HMLER and HMLER-shEcad
cells with 3, after 72 h incubation.
Inorganics 2017, 5, 12; doi:10.3390/inorganics5010012
S5 of S6
120
% Cell viability
100
80
60
40
20
0
0.1
1
10
Concentration/ micoM
100
Figure S10. Representative dose–response curves for the treatment of HMLER-shEcad
mammospheres with 3 after 5 days of incubation in the presence of CoCl2 (5 μM).
Figure S11. Quantification of mammosphere formation with HMLER-shEcad cells untreated and
treated with 3 (at the IC20 value, 5 days) in the presence of cobalt chloride (5 μM). Error bars represent
standard deviations and Student’s t-test, * p < 0.05.
Figure S12. Representative bright-field images (×10) of HMLER-shEcad mammospheres
supplemented with cobalt chloride (5 μM) in the absence and presence of 3 (at the IC20 value, 5 days).
Inorganics 2017, 5, 12; doi:10.3390/inorganics5010012
S6 of S6
Figure S13. Immunoblotting analysis of proteins related to the DNA damage and apoptosis
pathways. Protein expression in HMLER-shEcad cells following treatment with 3 (0.125, 0.25, and
0.5 μM), 2 (20 μM), and tolfenamic acid (20 μM) after 72 h incubation. Whole cell lysates were resolved
by SDS-PAGE and analyzed by immunoblotting against γH2AX, phos-CHK2, cleaved caspase 7,
cleaved caspase 3, and β-actin (loading control).