Figure 11.

Enhanced expression of HSP70 in 1-heptanol-treated BMMCs and the thermal stability of the plasma membrane in the presence of 1-heptanol and inhibitors affecting HSP70 function or expression. (A) RT-PCR quantification of HSP70 mRNA in non-activated (n = 6) BMMCs pretreated for 15 min with 2.5 mM 1-heptanol or vehicle (Control). (B) HSP70 expression levels in BMMCs untreated (−) or treated (+) with 2.5 mM 1-heptanol for 75 min. (C) Quantitative analysis of the HSP70 levels normalized to β-actin expression as in B. Values are means ± SEM calculated from seven biological replicates. (D) RT-PCR quantification of HSP90 mRNA in non-activated (n = 6) BMMCs pretreated for 15 min with 2.5 mM 1-heptanol or vehicle (Control). (E) Expression levels of HSP90 in control BMMCs and BMMCs treated with 2.5 mM 1-heptanol for 75 min. (F) Quantitative analyses of the HSP90 levels normalized to β-actin expression as in E. Values indicate means ± SEM calculated from seven biological replicates. * p < 0.05. (G) Membrane thermal stability analysis of BMMCs pretreated overnight with vehicle (DMSO; Control; n = 6), HSP inhibitor I (n = 3), or VER 155008 (n = 3) in the absence (0 mM) or presence of 2.5 mM or 5 mM 1-heptanol. (H) Membrane thermal stability analysis of RBL-2H3 cells pretreated overnight with vehicle (DMSO; Control; n = 6), HSP inhibitor I (40 μM, n = 3), or VER 155008 (25 μM, n = 3) in the absence (0 mM) or presence of 2.5 mM or 5 mM 1-heptanol. Cells in (G,H) were exposed to the indicated concentrations of 1-heptanol and PI. The cells were plated in 96-well plates for RT-PCR and incubated for 15 min at 37 °C, followed by PI fluorescence measurement at a slowly increasing temperature in the RT-PCR instrument. Values indicate means ± SEM calculated from n, which show the numbers of biological replicates. Intergroup differences between control and heptanol-treated cells were examined by two-way ANOVA; * p < 0.05, **** p < 0.0001.