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

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1.2

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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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