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1.
Figure 6

Figure 6. From: Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

ATP oscillations have a critical role for prechondrogenic condensation during chondrogenesis in micromass culture of mMSC. (a) Bioluminescence monitoring of PACTIN-PxRe activity in mMSCs after changing the maintenance medium, the chondrogenic medium, or chondrogenic medium implemented with 2-DG. (b) Condensation behaviours of mMSCs were examined with phase contrast images after 2 days of replacement with the maintenance medium, the chondrogenic medium, or chondrogenic medium implemented with 2-DG. Scale bars, 100 μm. (c) Gene expression analysis of type II collagen (COL2A1), aggrecan, and Sox9 in the micromass culture of mMSCs after 2 days of replacement with the maintenance medium, the chondrogenic medium (Chondro), or chondrogenic medium implemented with 2-DG (Chondro + 2-DG). Data show mean±S.D. (n=4). Dunnett's test, **P<0.01 by ANOVA compared with maintenance medium

H J Kwon, et al. Cell Death Dis. 2012 March;3(3):e278.
2.
Figure 4

Figure 4. From: Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

ATP oscillations are driven by Ca2+ oscillations, leading to oscillatory secretion in chondrogenesis of ATDC5 cells. (a) Effects of nifedipine, 2-APB, ionomycin, and thapsigargin treatment on insulin-induced PACTIN-PxRe oscillations. Time course analysis from (b) the raw data and (c) the detrended data taken with simultaneous monitoring of PACTIN-PxRe intensity (red line) and PACTIN-AqBl intensity (blue line) during chondrogenesis. (d) Time course data taken with simultaneous monitoring of PxRe intensity (red line) and secreted CLuc intensity (blue line) during perfusion with insulin-implemented medium. (e) Effect of 2-DG treatment on the oscillatory secretion of CLuc during perfusion with insulin-implemented medium. (f) Immunofluorescent staining of N-cadherin (green) at the peak and the trough of PxRe oscillations. Nuclei are stained blue by Hoechst 33342. Scale bars, 100 μm

H J Kwon, et al. Cell Death Dis. 2012 March;3(3):e278.
3.
Figure 7

Figure 7. From: Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

The degree of cellular condensation increases with the frequency of ATP oscillations. (a) Bioluminescence monitoring of PACTIN-PxRe activities after chondrogenic induction (time=0 h) at 34°C, 37°C, and 40°C. (b) Condensation behaviours observed with phase contrast images at 34°C, 37°C, and 40°C. Scale bars, 500 μm. (c) Temperature effect on gene expression of type II collagen (COL2A1), aggrecan, and Sox9 at 34°C (white bars), 37°C (grey bars), and 40°C (black bars), respectively. Data show mean±S.D. (n=4). Tukey-Kramer test. **P<0.01 by ANOVA compared with 34°C. ++P < 0.01 by ANOVA compared with 37°C

H J Kwon, et al. Cell Death Dis. 2012 March;3(3):e278.
4.
Figure 2

Figure 2. From: Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

ATP oscillations are synchronized via gap junctions among ATDC5 cells in chondrogenesis. (a and b) Bioluminescence imaging at the single-cell level shows that PACTIN-PxRe intensities in individual cells start to oscillate collectively by intercellular synchronization during chondrogenesis. (a) Time course data of PACTIN-PxRe intensities of individual cells indicated by red squares after chondrogenic induction. (b) Sequential images of PACTIN-PxRe intensities in representative cells at 1-h intervals from 97 to 144 h after chondrogenic induction. (c and d) Bioluminescence imaging at a low magnification shows that synchronized oscillations of PACTIN-PxRe intensities propagate as intercellular waves during chondrogenesis. (c) Sequential images of PACTIN-PxRe intensities at 30 min intervals from 53.5 to 57.5 h after chondrogenic induction. (d) Detrended data of time course of PACTIN-PxRe intensities in individual areas indicated by coloured squares from 48 to 60 h after chondrogenic induction. (e) Effect of carbenoxolone treatment on PACTIN-PxRe oscillations during chondrogenesis

H J Kwon, et al. Cell Death Dis. 2012 March;3(3):e278.
5.
Figure 5

Figure 5. From: Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis of ATDC5 cells. (a) Bioluminescence monitoring of PACTIN-PxRe intensity in the culture; the insulin-supplemented medium was changed every other day for 2 weeks. (b and c) Cellular condensation and chondrogenic differentiation were examined with (b) phase contrast images and (c) staining images with FITC-labeled peanut agglutinin of ATDC5 cells 1 week after culturing in medium implemented with insulin plus 2-DG, nifedipine, 2-APB, ionomycin, and thapsigargin, respectively. Scale bars, 100 μm. (d) Effect of 2-DG, nifedipine (Nif), 2-APB, ionomycin (Iono), and thapsigargin (Thaps) on gene expression of type II collagen (COL2A1), aggrecan, and Sox9 1 week after chondrogenic induction. Data show mean±S.D. (n=4). Dunnett's test, **P<0.01 by ANOVA compared with Control (insulin−)

H J Kwon, et al. Cell Death Dis. 2012 March;3(3):e278.
6.
Figure 1

Figure 1. From: Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

ATP oscillations are generated in chondrogenesis of ATDC5 cells. (a) Bioluminescence monitoring of PACTIN-PxRe activity in ATDC5 cells after changing the maintenance medium (black line) or the insulin-implemented medium (red line). (b) Protein expression levels of PxRe and β-actin at a trough and a peak of PACTIN-PxRe oscillations. Control: untransfected cells. (c) Relative value of ATP concentration at peak points and trough points of PACTIN-PxRe oscillations. Data represents the combined results from two independent experiments performed with six samples. (n=6, paired t test, **P<0.01). (d and e) Monitoring of cytoplasmic ATP level using a FRET-based ATP sensor. (d) Sequential images of the YFP/CFP emission ratio from 69 to 80 h after chondrogenic induction and (e) the time course of the YFP/CFP emission ratio inside the red circle from 66 to 84 h after chondrogenic induction

H J Kwon, et al. Cell Death Dis. 2012 March;3(3):e278.
7.
Figure 3

Figure 3. From: Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

ATP oscillations depend on both glycolysis and mitochondrial respiration in chondrogenesis of ATDC5 cells. (a) Treatment effect of 2-DG or oligomycin on PACTIN-PxRe oscillations. (b) Metabolite analysis shows the mean log ratio of concentration of metabolites involved in glycolysis and the TCA cycle in the peaks relative to the troughs of PACTIN-PxRe oscillations. Data represent the combined results from three independent experiments performed with two samples. (n=3, paired t test, *P<0.05). 1,3BPG, 1,3-bisphosphoglycerate; 1,3BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate. (c) Simultaneous monitoring of PACTIN-PxRe activity (red line) and PACTIN-ReBl activity (blue line) from 60 to 72 h after chondrogenic induction. The data were detrended by subtracting the 6 h running average from the raw data

H J Kwon, et al. Cell Death Dis. 2012 March;3(3):e278.

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