Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition Yogesh Rai1, Richa Pathak1, Neeraj Kumari1, Dhananjay Kumar Sah1, Sanjay Pandey1, Namita Kalra1, Ravi Soni1, B S Dwarakanath2 and Anant Narayan Bhatt1
Correspondence: Anant Narayan Bhatt Institute of Nuclear Medicine and Allied Sciences Brig S K Mazumdar Road Delhi 110 054 INDIA Telephone: (+91) 11 2390-5138 Fax: (+91) 11 2391-9509 E-mail:
[email protected],
[email protected]
Supplementary figure 1
Metabolic viability (MTT) 2.0 Control Rad.2Gy Rad.3Gy Rad.5Gy Rad.7Gy
OD
A549
1.5
1.0
8
(i)
Control Rad.2Gy Rad.3Gy Rad.5Gy Rad.7Gy
6
Nt/N0
(A)
Cell number (Nt/N0)
4
2
0.5
0
0.0 0
24
0
48
24
1.0
7
(i)
Control Rad.2Gy Rad.3Gy Rad.5Gy Rad.7Gy
6 5
Nt/N0
Control Rad.2Gy Rad.3Gy Rad.5Gy Rad.7Gy
OD
MDA-MB-231
(B) 1.5
48
Time (h)
Time (h)
0.5
4
(ii)
3 2 1 0
0.0 0
24
0
48
24
Control Rad.2Gy Rad.3Gy Rad.5Gy Rad.7Gy
OD
2.0
1.5
6
(i)
Control Rad.2Gy Rad.3Gy Rad.5Gy Rad.7Gy
5
Nt/N0
(C)
48
Time (h)
Time (h)
HeLa
(ii)
4 3
(ii)
2
1.0
1
0.5
0
0
24
Time (h)
48
0
24
48
Time (h)
Radiation dose dependent analysis of metabolic viability (MTT) and cell number: Metabolic viability does not correlate with viable cell number in radiation induced growth inhibition: Radiation dose response measured by MTT assay in A549 (A i), MDA-MB231(B i) and HeLa (C i) cells and compared with cell number in same cell lines A549 (A ii), MDAMB-231(B ii) and HeLa (C ii), under similar experimental conditions at various radiation dose. Data are presented as mean ± SD from four independent experiments. *p < 0.05 vs. respective control cells.
Supplementary figure 2
(139bp)
R-48h
C-48h
R-24h
R-48h
C-24 h
MDA-MB-231 C-48h
R-24h
C-24 h
HeLa
R-48h R-48h
C-48h C-48h
R-24h
R-24h
C-24 h
C-24 h
R-48h
R-48h
C-48h
C-48h
R-24h
(39bp)
Nuc. Pol
(39bp)
h C-24 R-24h
C-24 h
Leu-tRNA
(139bp)
Figure 3 (B) is cropped and taken from these original images of the supplementary figure 1: Full length DNA gel blot images are presented here for Leu-tRNA (139 bp) and Nuclear pol γ (39 bp) in HeLa and MDAMB-231 cells at 24 and 48 hours post irradiation with respective control.
R-48h
R-24h
R-8h
R-24h R-48h M
R-24h R-48h
R-48h
R-24h
R-8h
R-48h
R-24h
R-8h
Control
R-8h
R-8h
Control
Control
PGC-1α
Control
β Actin
HeLa
Control
R-48h
R-24h
R-8h
Control
TFAM
Supplementary figure 3
MDA-MB-231
Supplementary figure 3 continue:
R-48h R-48h
R-24h R-24h
R-8h R-8h
Control Control
R-48h R-48h
R-24h
MDA-MB-231
R-24h
R-8h R-8h
Control Control
β Actin
SDH-A
HeLa
Figure 3 (C) is cropped and taken from these original images of the supplementary figure 3: Full length western blot images are presented here for PGC-1α, TFAM, SDH and its respective β Actin in HeLa and MDAMB-231 cells. Cropped images marked at 8, 24 and 48 hours with respective control and analyzed by densitometry.
