SUPPLEMENTAL MATERIAL
Article title: Modulation of ROS Levels in Fibroblasts by Altering Mitochondria Regulates the Process of Wound
Healing
Journal name: Archives of Dermatological Research
Author names: Jaroslav Janda, Valentine Nfonsam, Fernanda Calienes, James E. Sligh and Jana Jandova
Corresponding author: Jana Jandova, PhD
University of Arizona, Department of Surgery
1501 N Campbell Avenue
Tucson, AZ 85724
Phone: (520) 626 1674
E-mail: [email protected]
FIGURE
Supplemental Figure 1: Differences in proliferation, ROS levels and abilities to close the wound in three
transmitochondrial cybridd cells. A) mtBALB cybrids showing the highest and mt501-1CAP-R the lowest
proliferation rates after 24, 48 and 72 hours. B) The levels of intracellular (upper panel) and mitochondrial ROS
(lower panel) are significantly higher in mtBALB cybrids and significantly lower in mt501-1CAP-R comepred to
mtB6 cybrids (*P<.05, **P<.0005, ***P<.0001). C) mt501-1CAP-R cybrid cells generating the lowest levels of ROS
were able to close the wound more effectively than the mtB6 and mtBALB cybrid generating higher levels of ROS.
METHODS
Proliferation assay
Individual cell lines were plated in the 6-well plates at the concentration of 1x105 cells per well. The cells were
grown for 24 h, 48h and 72h.After each time period the cells were collected and viable cells were counted with a
hemocytometer every other day by the trypan blue exclusion method.
Measurement of ROS
Intracellular: ROS production was detected by using the fluorescent probe DCFH 2-DA (Molecular Probes,
Carlsbad, CA). Cybrid cells were seeded at 150,000 in 6-well plates and allowed to grow for 48h. Then the cells
were trypsinized and resuspended in PBS buffer containing 15µM DCFH2-DA. After 30 minutes of incubation in
dark at 37 °C, the fluorescent intensity was analyzed by flow cytometry using appropriate wave length (excitation
and emission wavelengths of 490 and 525 nm, respectively).
Mitrochondrial: Mitochondrial superoxide was detected using the MitoSOX™ Red dye (Thermo Fisher Scientific,
Grand Island, NY). Cybrid cells were seeded at 150,000 in 6-well plates and allowed to grow for 48h, then the
cells were stained with 3µM of MitoSOX™ Red dye dissolved in Hanks' Balanced Salt Solution (Invitrogen
Corporation, Carlsbad, CA) for 30 min in the dark at 37 °C. After incubation with a dye, cells were washed with
PBS, trypsinized and resuspended in PBS. MitoSOX™ Red fluorescence was analyzed by flow cytometry with
488nm excitation laser using appropriate filters to detect fluorescence emission shift from green (529nm) to red
(590nm).
Scratch assay
Cybrid cell lines were plated to near confluency in 6-well plates overnight. The next day scratches were made in the
cells with a sterile 200 µL pipet tip. Plates were marked on the underside as a landmark for taking pictures. Pictures
were immediately taken with a 10× objective on a Nikon eclipse TE2000-S microscope with Metamorph software.
Pictures were taken 24 hours later using the marked underside as a landmark for alignment.