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. 2023 Aug 9;65:102838. doi: 10.1016/j.redox.2023.102838

Fig. 3.

Fig. 3

The hydrogen peroxide producing NADPH oxidase 4 (NOX4) promotes the active trafficking of cytoglobin to the nucleus in human vascular smooth muscle cells. (a) Representative confocal immunofluorescence images of sub-cultured human vascular smooth muscle cells following 0-, and 24-h stimulation with 5% serum containing growth media (Blue – DAPI, red – CYGB). Cells were then fixed and analyzed by indirect immunofluorescence using an antibody against cytoglobin; scale bars, 25 μm. (b) Time course of cytoglobin nuclear import following the addition of 5% serum containing growth media. The solid line represents the non-linear regression obtained from a single exponential model yielding a half-life 1.2 h; n = 4. (c) Quantitative analysis of the ratio of nuclear to cytosolic cytoglobin (CYGB) pixel intensity after pre-treatment with ivermectin (nuclear import inhibitor, left panel) or Leptomycin B (right panel, LMB – nuclear export inhibitor). A ratio greater than 1 is indicative of nuclear accumulation. (d) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) results showing the relative expression for NADPH oxidase (NOX) isoforms in sub-cultured human vascular smooth muscle cells. (e) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) results showing the relative expression of NADPH oxidase 4 (NOX4). Human smooth muscle cells were electroporated with scrambled SiRNA (SCR) or siRNA mix targeting NOX4. Each individual symbol represents an independent experiment. (f) Left panel, representative indirect immunofluorescence pictures of human vascular smooth muscle cells stained for NOX4 following silencing of NOX4 as described in panel (e); bar scale, 50 μm. Right panel, quantitation of experiments shown in left panel. (g) Left panel, representative indirect immunofluorescence pictures of human vascular smooth muscle cells stained for cytoglobin following silencing of NOX4 as described in panel (e); bar scale 20 μm. Center panel, quantitative pixel analysis of the ratio of nuclear to cytosolic cytoglobin (CYGB) pixel intensity following silencing of NOX4. Right panel, quantitative pixel analysis of total cytoglobin (CYGB) pixel intensity following silencing of NOX4. (h) Right panel, representative indirect immunofluorescence pictures of serum-starved (control) human vascular smooth muscle cells stained for cytoglobin following treatment with 200 μM hydrogen peroxide (+H2O2) for 1 h. Right panel, time course of cytoglobin nuclear import following the addition 200 μM hydrogen peroxide as described for left panel. The line represents the non-linear regression derived from a single exponential model yielding a half-life 18 min; n = 4; p values were determined by Student's t-test or one way ANOVA, followed by Tukey's post hoc test. Red bars represent the mean for each condition and error bars represent SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)