Fig. 1. Western blot analysis of HSP90, HSP70, HSP27 and HSC70 expression levels in ARPE-19 cells. Whole cell extracts (20 µg) of untreated cells, heat shocked (H/S) cells (1 h + 42 °C, recover 5 h in normal cell culture incubator) and the cells that were exposed to geldanamycin (0.1 µM or 5 µM GA) or radicicol (0.1 µM or 5 µM RA) for 6, 24 and 48 h. C represents untreated control. HSC70 was used as a loading control. The columns represent densitometric analyses of HSP90, HSP70 or HSP27 expression levels normalized to HSC70. Values for assays are expressed as percent of control value.

Fig. 2. Protein carbonyl levels in ARPE-19 cells. Cells were either unexposed or exposed to geldanamycin (0.1 µM or 5 µM GA) or radicicol (0.1 µM or 5 µM RA) for 6, 24 and 48 h. The differences between control samples and exposed samples were tested by two-way ANOVAs. In Fig. 2A (samples taken from cells, n = 3 per time point and exposure) there were statistically significant differences between samples (F = 31.63; df = 4,30; P < 0.0005) and statistically significant interactions between time and samples (F = 4.33; df = 8,30; P = 0.001). The differences between controls and exposed samples at certain time points were evaluated by one-way ANOVA. At the 6 h time point, there were no statistically significant differences between controls (C) and exposed samples (Bonferroni's multiple-comparison correction was applied). At the 24 h time point, there were statistically significant differences between controls (C) and samples that were exposed to 0.1 µM or 5 µM radicicol (RA 0.1 µM, RA 5 µM). P-values were 0.048 and 0.030 respectively (Bonferroni's multiple-comparison correction was applied). At the 48 h time point, there were statistically significant differences between controls (C) and samples that were exposed to 5 µM geldanamycin (GA 5 µM), 0.1 µM or 5 µM radicicol (RA 0.1 µM, RA 5 µM). P-values were 0.045, 0.018 and 0.046 respectively (Bonferroni's multiple-comparison correction was applied). In Fig. 2B (samples taken from medium) no statistically significant differences were found.

Fig. 3. Oxygen radical absorbance capacity (ORAC) levels in ARPE-19 cells. Cells were either unexposed or exposed to geldanamycin (0.1 µM or 5 µM GA) or radicicol (0.1 µM or 5 µM RA) for 6, 24 and 48 h. The differences between control samples and exposed samples were tested by two-way ANOVA. In Fig. 3A (samples taken from cells, n = 3 per time point and exposure) no statistically significant differences were found. In Fig. 3B (samples taken from medium, n = 3 per time point and exposure) there were statistically significant differences between samples (F = 6.82; df = 4,30; P = 0.001) but no statistically significant interactions between time and samples (F = 1.69; df = 8,30; P = 0.141). The Dunnett's t-test indicated that samples exposed to 5 µM (GA 5 µM) geldanamycin or 5 µM radicicol (RA 5 µM) differed from control samples. P-values were 0.005 and 0.002 respectively.

Fig. 4. (A) Western blot analysis of superoxide dismutase (SOD), hydroxynonenal (HNE) adducts and β-actin levels in ARPE-19 cells. Whole cell extracts (20 µg) of untreated cells and cells that were exposed to geldanamycin (0.1 µM or 5 µM GA) or radicicol (0.1 µM or 5 µM RA) for 6, 24 and 48 h. C refers to untreated control. β-actin was used as a loading control. The columns represent densitometric analyses of SOD or HNE-adducts levels normalized to β-actin. Values for assays are expressed as percent of control value. (B) SOD activity from RPE cells either unexposed or exposed to geldanamycin (0.1 µM or 5 µM GA) or radicicol (0.1 µM or 5 µM RA) for 6, 24 and 48 h. Variations are represented by standard deviation (n = 5). Mann–Whitney U-test showed statistically significant differences (P < 0.05, P < 0.01).

Fig. 5. MTT-assay of ARPE-19 cells. The columns represent the cellular viability of exposed cells compared to untreated cells. Values for assays are expressed as percent of control value. Variations are represented by standard deviation (n = 6). Mann–Whitney U-test showed statistically significant differences (P < 0.05, P < 0.01). Cells were either unexposed or exposed to geldanamycin (0.1 µM or 5 µM GA) or radicicol (0.1 µM or 5 µM RA) for 24 and 48 h.

Fig. 6. Calcein-AM test of ARPE-19 cells. The cells were exposed to geldanamycin (0.1 µM, 1 µM or 5 µM GA) or radicicol (0.1 µM, 1 µM or 5 µM RA) for 15 min before adding calcein-AM for 20 min. Cellular fluorescence was measured in cold buffer immediately after the experiment. Mann–Whitney U-test indicated statistically significant efflux inhibition when cells were exposed to 1 µM or 5 µM (P < 0.05) radicicol; n = 4. Values are means ± S.D.