Supplementary information
Synchronized ATP oscillations play a critical role in
prechondrogenic condensation during chondrogenesis
Hyuck Joon Kwon, Yoshihiro Ohmiya, Ken-ichi Honma, Sato Honma, Takeharu Nagai,
Kenta Saito, Kazunori Yasuda
This PDF file includes:
Supplementary Figures 1-4
Supplementary Video legends 1-3
Supplementary methods
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Supplementary Figure 1. Phase contrast images of a time-course of condensation
behaviours during chondrogenesis of ATDC5 cells. ATDC5 cells were cultured with
replacement of insulin medium every other day for the indicated time and then were
recorded with a phase contrast microscopy. Scale bars, 100μm.
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Day 0 Day 2 Day 4
Day 6
Day 8 Day 10 Day 12 Day 14
Supplementary Figure 2. Effect of 2-DG, nifedipine (Nif), 2-APB, ionomycin (Iono),
and thapsigargin (Thaps) pharmacological inhibitors on cellular proliferation. Control
(DMSO). Cellular proliferation was measured by using the highly water soluble
tetrazolium salt, WST-8, 48 h after treatment of pharmacological inhibitors. Error bars,
s.d.m. (n = 4, Dunnett's test, **p < 0.01 vs DMSO)
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control 2-DG Nif 2-APB Thaps Iono
Relat
ive ce
llular
proli
ferat
ion le
vel
**
*
**
*
Supplementary Figure 3. Period length of PACTIN-PxRe oscillations as a function of
temperature. Data are presented as the mean ± SDM. (n = 4 at each temperature)
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02468
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33 34 35 36 37 38 39 40 41Temperature
Perio
d(hou
rs)
(℃)
Supplementary Figure 4. Effect of temperature on cellular proliferation. Cellular
proliferation was measured by using the highly water soluble tetrazolium salt, WST-8,
48 h after culturing at each temperature. Error bars, s.d.m. (n = 4)
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00.20.40.60.8
11.21.4
34℃ 37℃ 40℃
Relat
ive pr
olifer
ation
rate
Supplementary Video legends
Supplementary Video 1. The movie shows a time-lapse observation of YFP/CFP
emission ratio of ATDC5 cells expressing FRET-based ATP sensor from 48 to 86 h
after chondrogenic induction. FRET imaging shows that intracellular ATP levels
oscillate during chondrogenesis. 48 h after chondrogenic induction (time = 0 h). Time,
hr:min:sec.
Supplementary Video 2. The movie shows a time-lapse observation of PACTIN-PxRe
intensity in individual ATDC5 cells from 48 to 146 h after chondrogenic induction.
Bioluminescence imaging shows that about 72 h after chondrogenic induction,
intracellular ATP levels in individual cells start to oscillate collectively by intercellular
synchronization. Time, hr:min.
Supplementary Video 3. The movie shows a time-lapse observation at a low
magnification of PACTIN-PxRe intensity in ATDC5 cells from 36 to 64.5 h after
chondrogenic induction. Bioluminescence imaging shows that synchronized ATP
oscillations propagate as intercellular waves during chondrogenesis. Time, hr:min.
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Supplementary method
Cell proliferation assay
Cell proliferation was measured by using the highly water soluble tetrazolium salt,
WST-8. The ATDC5 cells were seeded at 5000 cells/well on 96-well plates and then
were cultured in each condition for 48 hours. After adding 10 µl of the WST-8 solution
to each well, absorbance was measured at 450 nm using a microplate reader
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