JCS004184 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Movie 1 -

  Movie 1. Movie of calcium images corresponding to Fig. 5. The images have been processed by compressing the range of calcium to 8 bits and then converting the look-up table to display relative Ca²⁺ signal as false color (see Materials and methods). Fluo3 imaging in human osteoclasts showing increased motility and Ca²⁺ after preincubation with a cGMP agonist. Ten frames at 4- minute intervals, with frame 1 taken 30 minutes after addition of 100 μM 8-pCPT-cGMP. Note that many of the cells have elevated Ca²⁺ by fluo3 (arrows) and these cells are also moving. The white cell at the bottom of the frame is dead. The false color image shows Ca²⁺ signal as white>orange>red>lavender>blue, with no signal being black. Fluo3 measurements cannot be calibrated accurately, but ambient Ca²⁺ (∼1 mM) is white (note dead cell) and the high Ca²⁺ (red) in living cells correspond to the low μM range in other studies (Radding et al., 1999) and in dual-wavelength studies here (see Fig. 3). Frame size is 290×225 μM.

• Movie 2 -

  Movie 2. Movie of calcium images corresponding to Fig. 5. The images have been processed by compressing the range of calcium to 8 bits and then converting the look-up table to display relative Ca²⁺ signal as false color (see Materials and methods). Fluo3 imaging in human osteoclasts that show low motility and Ca²⁺ after preincubation with a cGMP antagonist. Ten frames at 4-minute intervals. After a 30-minute preincubation with 50 μM 8-pCPT-cGMP, only one cell with high Ca²⁺ is seen (arrow), this cell is moving. The moving cell is an atypical, fusiform cell. It may represent a macrophage derivative rather than an osteoclast (see text). False color Ca²⁺ scale shows Ca²⁺ signal as white>orange>red>lavender>blue, with no signal being black. Frame size is 290×225 μM.

• Movie 3 -

  Movie 3. Movie of calcium images corresponding to Fig. 5. The images have been processed by compressing the range of calcium to 8 bits and then converting the look-up table to display relative Ca²⁺ signal as false color (see Materials and methods). Ratio imaging using Oregon Green 488 BAPTA and Fura Red AM showing increased motility and Ca²⁺ after preincubation with the NO donor SNP. Six frames at 10- to 12-minute intervals. Thirty minutes after exposure to 100 μM SNP, many cells have elevated Ca²⁺ (left frame) and are moving as shown by subtraction of an image at 32 minutes (right frame). Peak Ca²⁺ in the motile cells (red), determined as described (Yap et al., 2000), averaged 5-10 μM, whereas blue-background cells gave Ca²⁺ activities of approximately 100 nM. False color Ca²⁺ scale shows Ca²⁺ signal as white>orange>red>lavender>blue, with no signal being black.. Frame size is 580×450 μM.