Cell surface translocation and secretion of ANXA1 during myogenic differentiation in C2C12 cells.

<p>Cell surface (a) and extracellular (b) ANXA1 from C2C12 cells in GM (0 differentiation day) and after exposure for the indicated times (3, 5, and 7 differentiation days) to DM was analyzed by Western blot with anti-ANXA1 (a, b) antibody. Total cell protein extracts were analyzed by Western blot with anti-ANXA1 (c) and with anti-MyoD (d), anti-Myogenin (e), and anti-MyHC (f) antibodies to assess myogenic differentiation rate. The protein bands were normalized on tubulin levels (g). The data are representative of 5 experiments with similar results.</p

FPR detection and effects of Ac2-26, fMLP and CsH on the FPR-induced rise in intracellular Ca²⁺. (A)

<p>Qualitative PCR products for full-length Fpr-rs1 and Fpr-rs2 genes with only cDNA isolated from C2C12 cells. Product electrophoresis was performed on 1% agarose gel stained with ethidium bromide. Lane 1: negative control. Lane 2∶1 kb DNA ladder. Lane 3: primer pairs 1 for Fpr-rs1 amplicon, 240 bp. Lane 4: primer pairs 1 for Fpr-rs2 amplicon, 297 bp. Lane 5: primer pairs 2 for Fpr-rs1 amplicon, 240 bp. Lane 6: primer pairs 2 for Fpr-rs2 amplicon, 297 bp. <b>(B)</b> C2C12 were treated as described in Materials and Methods. The histogram shows the fluorescence ratio calculated as F340/F380 nm in the presence or in the absence of extracellular Ca²⁺.Control represents unstimulated cells. Data are means ± SEM (n = 3). *** <0.001, ** <0.01 vs corresponding controls; §§§ <0.001 vs Ac 2-26 or fMLP.</p

Cell surface translocation and secretion of ANXA1 after Ac2-26 treatment in Wound-healing migration assay of C2C12 cells.

<p>(<b>A</b>) Results for control, fMLP, Ac2-26, CsH, fMLP + CsH and Ac2-26+ CsH are reported as means of three experiments, measuring individual cell migrations at different times. Bars represent standard errors. (<b>B</b>) Cytosolic, cell surface and extracellular ANXA1 from C2C12 cells after exposure for the indicated time (16 h) to fMLP, Ac2-26, CsH, Ac2-26+ CsH and fMLP + CsH were analyzed by Western blot with anti-ANXA1 and anti-tubulin antibodies. The data are representative of 5 experiments with similar results. *** <0.001 vs control; §§§ <0.001 vs Ac 2-26 or fMLP.</p

Proteins identified as possible Ac2-26 partners by chemical proteomics.

<p>Proteins identified as possible Ac2-26 partners by chemical proteomics.</p

Effects of geraniin and 17-AAG on cell cycle progression.

<p>Percentage of cell cycle stages was analyzed by flow cytometry, (<b>A</b>) PI-stained viable Jurkat cells treated with DMSO, 0.7 µM geraniin or 10 µM 17-AAG for 24 h. (<b>B</b>) PI-stained viable HeLa cells treated with DMSO, 5 µM geraniin or 0.2 µM 17-AAG for 24 h, (<b>C</b>) The percentage of hypodiploid cells as treated in <b>A</b> and <b>B</b>. Results are expressed as means ± SD of three experiments performed in duplicate (***<i>P</i><0.001).</p

Thermodynamic constants measured by SPR for the interaction between tested compounds and immobilized HSP90α.

<p>Thermodynamic constants measured by SPR for the interaction between tested compounds and immobilized HSP90α.</p

Structure of the 54 tested compounds.

<p>Geraniin structure (54) is evidenced.</p

Inhibition of Hsp90α activity.

<p>ATPase activity of the chaperone was evaluated in the presence of different concentrations of geraniin, 17-AAG and HA (<b>A</b>). Data are reported as the residual ATPase activity (%) compared to that observed for an untreated sample. Data are the mean of three independent experiments performed in triplicate and were analyzed by t test (HA vs testing compounds): The error bar represents the standard deviation of nine measurements, while * indicates significance at P<0.01. Aggregation kinetics of citrate synthase (CS) at 43°C determined by light scattering <b>(B</b>)<b>.</b> The spontaneous aggregation of CS at 43°C (♦) and the aggregation of CS at 43°C in the presence of 0.075 µM Hsp90 α and 0.3 µM ATP (▴), 0.075 µM Hsp90 α, 0.3 µM ATP and 0.3 µM geraniin (▪), or 0.075 µM Hsp90α, 0.3 µM ATP and 0.3 µM HA (•) are shown. Kinetics traces reported are the averages of three separated measurements; the error bar represents the standard deviation of three measurements.</p

Effect of geraniin on Hsp90 client protein levels.

<p>Equal amounts (30 µg) of whole-cell lysates were separated on SDS-PAGE and client proteins were visualized by western blot analysis using specific antibodies. Actin was used as loading control. Total cellular proteins were extracted 24 h after treatment with geraniin (2.5 µM, 5 µM and 10 µM) in Jurkat cells (<b>A</b>) or geraniin (1 µM, 0.7 µM and 0.5 µM) in HeLa cells (<b>B</b>), ctrl = control cells treated with DMSO). The blots shown are representative of three different experiments with similar results.</p

SPR results.

<p>Sensorgrams obtained by injecting 25(▪), 50 nM (♦), 250 nM (▴) and 1 µM (•) of geraniin (<b>A</b>), compound <b>1</b> (<b>B</b>), 17-AAG (<b>C</b>), and HA (<b>D</b>) on immobilized Hsp90α.</p