Figure 6.

Effect of VEGF-B on TRPV4 expression and functionality under oxidative stress. (A–C) QMMuC-1 cells were treated with 4-HNE (10 µM) with or without recombinant VEGF-B (100 ng/mL) for 24 hours. The expression of TRPV4 was examined by immunofluorescence staining (A, B) and Western Blot (C). (A) Representative image of TRPV4 (green) and phalloidin (red) in untreated control, 4-HNE treated and 4-HNE + recombinant VEGF-B (100 ng/mL) treated QMMuC-1 cells. Arrow in control indicates cluster aggregation of TRPV4. Arrows in 4-HNE–treated group indicates contracted cell body. (B) Quantitative analysis of different types of TRPV4-expressing QMMuC-1 cells (A, spread cell with evenly distributed TRPV4; B, spread cell with aggregated or uneven TRPV4 expression; C, contracted cell with aggregated TRPV4 expression). ^****P < 0.001; n = 4 wells per experiment. Two-way ANOVA followed by Bonferroni post hoc test. (C) Western Blot of TRPV4 expression on 4-HNE–treated QMMuC-1 cells with/without recombinant VEGF-B, n = 6–7. (D and E) Effect of TRPV4 and VEGF-B on intracellular Ca²⁺ concentration in 4-HNE–treated QMMuC-1 cells and its representative plots (D). Intracellular Ca²⁺ levels were measured using the FlexStation. The y-axis was the 340 nm/380 nm fluorescence ratio (ΔR340 nm/380 nm). (D and E) TRPV4 agonist GSK101 (100 nM) alone did not elicit a measurable calcium response in QMMuC-1 cells. A 24 hours 4-HNE (10 µM) treatment significantly increased the GSK101-evoked response, which was fully blocked by the TRPV4 antagonist HC06 (10 µM) and by recombinant VEGF-B (100 ng/mL). Mean ± SD, n = 4–8 ^****P < 0.001; one-way ANOVA followed by Newmann-Keuls post hoc test. All experiments were performed at least twice.