Figure 1.
Comparative analysis of MCF-7 cell viability, cell number and mitochondrial activity.
Cells were treated with 5 mM IPTG, 10 nM ICI 182780, 5 µM FTY720, or serum deprived (serum free) 48 h post-seeding. Using the Trypan Blue exclusion method cells were harvested and viable cells counted using a haemocytometer at days indicated. MTS and SYBR-DNA assays were performed, as detailed in materials and methods, at days indicated. The treatment results are shown as a percentage of the uninduced vehicle control (±SE) correlating with viable cell number (Trypan blue counts), colorimetric measurement (MTS), and fluorescent intensity (SYBR assay). Each experiment was performed in triplicate at least 3 times with similar results.
Figure 2.
Flow cytometric analysis of cell cycle phases post ICI 182780 and IPTG treatment.
Cells were treated with 10 nM ICI 182780 or 5 mM IPTG (p14ARF-induction) 48 h post seeding. At 48 h post-treatment cells were harvested, stained with propidium iodide solution as described in materials and methods and analysed for cell cycle distribution by flow cytometry using Modfit software. A. Fluorescence histograms showing cell cycle distribution of control, IPTG and ICI 182780 treated MCF-7 cells (representative experiment). B. Representative column graph showing the percentage of cells (± SD) in each cell cycle. This experiment was performed three times with three different cell lines showed similar results.
Figure 3.
EdU incorporation post ICI 182780 and IPTG (p14ARF) treatment.
Cells were seeded on cover slips and treated with 5 mM IPTG or 10 nM ICI 182780 24 h post-seeding. EdU was added to the medium on days 1 and 4 and cells were incubated for a further 20 h. EdU incorporation was visualized by staining with Alexafluor 488 (green). The nucleus was stained with Hoechst 33342 (blue) and images were taken on a Nikon fluorescence microscope (magnification ×200). Column graph shows % cells staining for EdU compared to Hoechst 33342 stained nuclei (± SE). Experiments were performed in triplicate (duplicate biological experiments). A minimum of 500 cells was counted for each treatment.
Figure 4.
ICI 182780 and p14ARF increase mitochondria activity.
Cells were treated with 10 nM ICI 182780 (A) or 5 mM IPTG (B), 48 h post seeding. At 72 h post treatment cells were counted (see inset) and equal number of cells plated in 96 well plates. Mitochondrial activity was measured using the MTS assay. Treatment results were presented as percentage of control (± SE) in column graphs. Each experiment was performed in duplicate at least 3 times.
Figure 5.
Induction of p14ARF increases mitochondrial biomass and maintains membrane potential.
A. Cells were treated with 5 mM IPTG 48 h post-seeding. At day 3 post-IPTG treatment, live cells were incubated with Mitotracker (red), CellTracker (green) and Hoechst 33342 (blue) and imaged using an inverted fluorescent microscope (magnification ×400). Cells treated with IPTG noticeably increased in size. B. Images processed by high content imaging (magnification ×200) and mitochondria (per cell) counted using BD Attovision™ software. C. Cells were treated with 5 mM IPTG 48 h post-seeding. On day 3 post-IPTG-treatment cells were stained with TMRE for 15 min (+), or left unstained (−) and run through a flow cytometer (IPTG = black, control = white). TMRE-IPTG-treated cells showed increased fluorescence intensity compared to the TMRE-control cells, which is indicative of an increase in ΔΨmt in IPTG-treated cells. D. The median FL2 relative fluorescence units (RFU) of control and IPTG treated cells (day 3) were determined by flow cytometry. The column graph shows the median RFU of TMRE-stained cells minus unstained cells (±SE). This experiment was performed at least twice in triplicate.