Fig. 2.

TRPV4 knockdown by siRNA significantly reduces the hypotonicity-induced [Ca²⁺]_i response and RVD in rat astrocytes. (A) The TRPV4 antibody recognizes three major bands of ∼90, 100, and 110 kDa. The decrease in TRPV4 protein level, based on densitometric analysis in three different experiments, was most pronounced for siRNA 9 (89% reduction for the 120-kDa band, 61% for the 100-kDa band, and 69% for 90-kDa band) followed by siRNA 7 (89%, 59%, 51%) and siRNA 11 (49%, 54%, 72%). The density of bands of samples treated with control siRNA was set at 100%. The level of actin is not affected by siRNA treatment. (B) Typical [Ca²⁺]_i dynamics recorded in fura-2–loaded primary astroglial cells treated with control (CT) siRNA. (C) Knockdown of TRPV4 by siRNA 9 abolishes the [Ca²⁺]_i response. (D) Quantitative analyses of hypotonicity-induced [Ca²⁺]_i signal in primary astrocytes. Astrocytes treated with CT siRNA (n = 20) are shown by the gray bar and astrocytes treated with TRPV4-specific siRNA (n = 22) by the white bar. P < 0.001 for the comparison between control group and TRPV4 knockdown astrocytes, independent t-test; P < 0.001 for experiment in presence and absence of [Ca²⁺]_o, paired t-test. ***P ≤ 0.001. (E) Calcein-quenching method for measurement of osmotically induced volume changes in primary astroglial cells treated with control siRNA (closed symbols) and TRPV4-specific siRNA 9 (open symbols). The cells were exposed to a hypotonic medium (ΔOsm = 60 mOsm).