|
Figure 2.
(a) Quantification of TRPV4 transcript by qRT-PCR in control
human lymphoblast lines (n = 4), human dorsal spinal cord (n =
3), ventral spinal cord (n = 3) and tracheal cartilage (n = 3)
using primers specific for the following exon regions: exons
3–4, exons 5–6, exons 7–8, exon 5 and exon 7. Values are
normalized to the first tracheal cartilage sample. Data is
averaged; error bars, s.e.m. (b) DRG neurons were transfected
with wild-type and mutant forms of TRPV4 (green). At 16 h, some
cells expressing mutant forms of TRPV4 show evidence of early
cellular toxicity with a collapsed cytoplasm. The nuclear DAPI
stain is blue. Scale bar, 40 μm. (c) Quantification of
propidium iodide uptake in DRG neurons expressing wild-type and
mutant TRPV4 reveals a marked increase of cell toxicity in
mutant expressing cells at 48 h. *P < 0.0001. (d) HEK293 cells
expressing R269C and R269H mutants show an increase in the
number of dead cells (red channel is EthD-1 stain) at 48 h; this
increase is prevented by the TRP channel blocker ruthenium red
(RR). Green channel is calcein-AM stain for live cells. Scale
bar, 200 μm. (e) Quantification of cell death in HEK293 cells
indicates a time-dependent increase in cell death that is
blocked by the TRP channel blocker ruthenium red (RR). *P <
0.01, **P < 0.001. Data in c and e are averaged from three
independent experiments; error bars, s.e.m.
|
