Figure 1.
Construction of the Nox biosensor p47-roGFP.
The coding sequences of human p47phox and roGFP2, carrying a 30-amino-acid linker, were PCR amplified from pcDNA3.1-p47phox and pLPCX-Grx1-roGFP2, respectively as described in Materials and Methods. The forward and reverse primers for the two reactions were designed to incorporate HindIII, SpeI and NotI restriction sites. Both PCR products were purified and ligated to form a linear p47phox-linker-roGFP expression cassette, which was cloned into the pcDNA3.1 vector using HindIII and NotI restriction sites.
Figure 2.
p47-roGFP allows for live-cell imaging of redox changes during RAW264.7 cell activation.
(A) Representative confocal image of RAW264.7 cells transfected with p47-roGFP shows cytosolic distribution of p47-roGFP in control cells (−LPS). Upon LPS-mediated activation (20 ng/ml, 45 min) p47-roGFP localized with the membrane dye FM4-64®. (B) Representative time course of LPS-induced p47-roGFP oxidation. Biosensor oxidization occurred dynamically upon LPS-induced activation (black squares), while in the presence of the Nox peptide inhibitor gp91ds (5 µm, 60 min) minimal oxidation occurred (red circles). After 45 min of LPS, cells were treated with 1 mM H2O2 to maximally oxidize followed by addition of 10 mM DTT to maximally reduce the biosensors. (C) Average (±SEM) of 9 cells for each condition from (B) during the last 3 minutes of LPS. ** p<0.01 (Tukey statistical analysis for p value).
Figure 3.
LPS-induced stimulation increases the rate of DCF fluorescence in macrophage cells from 8–10 wk old mice.
(A) Representative data (ΔF/F0) from macrophages of WT (C57Bl/6J, black line), p47phox−/− (red line) and Nox2−/y (blue line) mice showing the temporal change in DCF fluorescence upon LPS-treatment (20 ng/ml). F0 is the basal fluorescence taken over the first 100 seconds prior to LPS. (B) LPS-induced stimulation (boxed areas of A) significantly increased the normalized rate of DCF fluorescence (black bar) compared to pre-stimulated values (white bar). The increased intensity of DCF fluorescence was significantly higher in WT macrophages compared to macrophages from p47phox−/− and Nox2−/y mice. Error bars represent s.e. from the mean. Data are analyzed from nanimals = 6 per strain, 6 replicates per animal. * p<0.05 (Tukey statistical analysis for p value).
Table 1.
Temporal response of DCF and p47-roGFP to LPS.
Figure 4.
p47-roGFP biosensor shows specificity to the Nox complex in primary spleen macrophages.
(A) Macrophage cells from wild-type mice (C57Bl/6j) expressing transiently transfected p47-roGFP showed homogeneous cytosolic distribution of p47-roGFP in the absence of LPS-mediated activation (−LPS). Upon LPS-mediated activation p47-roGFP localized with the membrane dye FM4-64® (+LPS). (B) In the absence of LPS (−LPS), p47-roGFP is distributed homogeneously throughout the cytosol of macrophages isolated from Nox2 deficient (Nox2−/y) mice. Upon LPS stimulation (+LPS), p47-roGFP translocated to the cell membrane of Nox2−/y macrophages, localizing with FM4-64®. (C) LPS stimulation of primary spleen macrophages resulted in rapid oxidation of p47-roGFP in wild-type macrophages (black squares). Cells incubated with gp91ds (5 µM, 60 min.) demonstrated a significant reduction in the rate and extent of p47-roGFP oxidation (red squares). In Nox2−/y macrophages (blue squares) LPS did not induce oxidation of p47-roGFP. There was no oxidation of p47-roGFP in the absence of LPS (Time CTRL, green squares). Error bars represent s.e. from the mean. Representative images (A & B) are shown from at least 9 cells. Data (C) are representative of nanimals = 6 per strain, 3 cells per animal. ** p<0.01, * p<0.05 (Tukey statistical analysis for p value).
Figure 5.
Localization of p47-roGFP in skeletal muscle.
(A) Immunostaining of enzymatically dissociated single FDB myofibers for endogenous p47phox and the ryanodine receptor shows that p47phox co-localizes with the ryanodine receptor at the triad. (B) Fluorescent live cell image of an FDB electroporated with p47-roGFP and counter stained with the membrane and t-tubule dye FM4-64® shows that p47-roGFP is localized at the t-tubule. The line plots represent the longitudinal spatial profile of fluorescence averaged over the transverse direction within the boxed regions.
Figure 6.
Electrical stimulation induces oxidation of DCF in myofibers from WT but not p47−/− or Nox2−/y mice.
Tetanic electrical stimulation increased the normalized rate of DCF fluorescence (red bar) compared to pre-stimulated values (black bar). In addition, the rate of change of DCF fluorescence was significantly lower in FDB fibers from p47phox−/− and Nox2−/y mice compared to WT FDB fibers. There was no difference between non-stimulated and stimulated condition for either p47−/− or Nox2−/y. Data are analyzed from nanimals = 6 per strain, 12 replicates per animal. ** p<0.01 (Tukey statistical analysis for p value).
Figure 7.
Physiological perturbation facilitates Nox2-dependant ROS formation in skeletal muscle.
(A) Oxidation (H2O2, 1mM) and reduction (DTT, 10mM) of fibers expressing p47-roGFP with exogenous agents show reversibility of p47-roGFP. (B) Passive stretch to 2.4 µm (20% increase in sarcomere length) increased the oxidation state of p47-roGFP in wild-type (WT) myofibers (black squares) but not in myofibers from Nox2−/y mice (black circle). (C) Repetitive contractile activity resulted in rapid oxidation of p47-roGFP sensor in WT fibers, which plateaued around 15 min. (black square). Oxidation of p47-roGFP was significantly attenuated in WT fibers treated with the Nox peptide inhibitor gp91ds (5 µm, 60 min (open circles). FDBs from Nox2−/y mice also showed very minimal oxidation (black circle) of p47-roGFP. (D) Average data from experiments in (C) during the first 2 minutes (No stim.) and during the last 3 minutes of stimulation. (E) The oxidation of p47-roGFP was reversible after cessation of contraction. Data are from nanimals = 6 per strain, 3 cells per animal. * p <0.05, ** p <0.01 (Tukey statistical analysis for p value).
Figure 8.
p47-roGFP measures Nox dependent ROS produced in the extracellular space.
Catalase (4 µM) in the extracellular space prevented the oxidation of p47-roGFP (A) but did not alter the oxidation of Grx1-roGFP2 (B) during electrical stimulation of FDB myofibers.
Figure 9.
Nox-mediated ROS formation is rescued by p47-roGFP in p47phox−/− cells.
(A) Macrophage cells from p47phox−/− mice transiently transfected with p47phox- showed homogeneous cytosolic distribution of p47-roGFP in the absence of LPS-mediated activation (left). Cells incubated with LPS (20 ng/ml, 45 min, right) showed p47-roGFP localization with the membrane dye FM4-64® around the periphery of the cell. (B) LPS stimulation of primary macrophages resulted in rapid oxidation of p47-roGFP in wild-type macrophages (WT, black squares). p47phox−/− cells showed a very similar level of p47-roGFP oxidation, but with a slower rate (blue squares). In the absence of LPS-stimulation, wild-type cells showed almost no change in oxidation of p47-roGFP sensor (green square). (C) Average data from (B) before addition of LPS (−LPS) and during the last 3 minutes of LPS stimulation (+LPS). (D) Tetanic electrical stimulation of single FDBs from wild-type and p47phox−/− mice expressing p47-roGFP resulted in similar levels of p47-roGFP oxidation compared to WT. Representative images (A) are shown from at least 9 cells. Data (B–C) are representative of nanimals = 8 per strain, 3 cells per animal and for (E) nanimals = 4 per strain, 3 cells per animal. ** p<0.01 (Tukey statistical analysis for p value).