Effect of BGP-15 treatment on heat-induced HSF1 acetylation in HEK293T cells and on <i>in vitro</i> SIRT1 activity.

<p><b>A</b>) HEK293T cells transiently co-transfected with mouse HSF1-FLAG and p300 were heat shocked for different lengths of time at 42°C or for 60 min at 42°C followed by 60 min recovery (R). After immunoprecipitation by FLAG, samples were probed for acetylated lysin by western blotting [n = 3, error bars represent standard error of the mean (SEM)]; <b>B</b>) Samples were or were not treated with 10 µM BGP-15 for 60 min before and during 60 min heat shock or 60 min recovery following heat shock (R) Acetylated HSF1 was determined as above. [n = 4, p<0.05, error bars represent standard error of the mean (SEM)]; <b>C</b>) In vitro activity of SIRT1 using activators, inhibitors and BGP-15 [n = 3, error bars represent standard error of the mean (SEM)].</p

Effect of BGP-15 on DChol desorption from lipid monolayers to MBCD.

<p><b>A</b>) MBCD-mediated removal of DChol from lipid monolayers at constant lateral surface pressure. When indicated 10 µM BGP-15 was injected into the subphase underneath SM/DChol monolayer 5 min before MBCD was administered. Surface area was measured before (A₀) and at the indicated time points after (A_t) MBCD injection. <b>B</b>) Effect of BGP-15 concentration on MBCD-mediated DChol desorption from SM/DChol monolayers. The monolayers were equilibrated with BGP-15 for 5 min before MBCD was injected into the subphase.</p

<i>Hsp25</i> mRNA expression in B16-F10 cells modified by Rac1 inhibitor and BGP-15 administration.

<p>B16-F10 cells were pretreated or not by Rac1 inhibitor NSC23766 for 2 hours before 1 h 41.5°C heat shock with or without 10 µM BGP-15. RNA was isolated and the expression of <i>hsp25</i> was tested by RT-PCR. [n = 3, error bars represent standard error of the mean (SEM)].</p

The effect of BGP-15 on the temperature-induced change in cluster fraction of mGFP-GPI.

<p>CHO cells stably expressing mGFP-GPI were subjected to 39.5°C with or without 10 µM BGP-15. TOCCSL experiments were performed and at indicated times the cluster fraction (two or more fluorophores per domain) was calculated [n = 2 error bars represent standard error of the mean (SEM)].</p

The effect of BGP-15 on cholesterol-rich membrane domains and the heat shock inducibility of <i>hsp25</i> in B16-F10 cells with different cell density.

<p><b>A</b>) Fluorescence intensity of fPEG-Chol in B16-F10 cells seeded with low (LCN, 1.5×10⁶/10 cm plate) or high cell number (HCN, 6×10⁶/10 cm plate with or without 10 µM BGP-15). Student's t-test was used for statistical analyses, and p = 0.05 was set as a significance threshold. <b>B</b>) The size distribution profile of membrane microdomains labeled by fPEG-Chol in HCN (with or without 10 µM BGP-15) and LCN cells and imaged by TIRF microscopy. <b>C</b>) HCN (with or without 10 µM BGP-15) and LCN cells were heated at 42°C for 1 hour and the expression of <i>hsp25</i> was tested by RT-PCR.</p