PMC

Distribution of all differentially regulated total and phospho-proteins in 2-DE in the form of functional categories

ELISA analysis of PrP^c levels in parental and SH-SY5Y cells stably transfected with pCIneoPRNP vector following DMSO/STS treatment. PrP^c levels were analyzed following treatment of parental (expressing endogenous PrP^c; designated ctrl) and PrP^c-overexpressing (designated PrP) SH-SY5Y cells with either DMSO or 1 μM STS for 2 h. Note markedly, 7-and 5-fold, higher PrP^c expression in pCIneoPRNP transfected versus parental cells following DMSO and STS treatment, respectively. PrP^c concentration was measured in 50 μg of proteins. Values represent the mean±S.D. of four independent experiments (two-sided unpaired Student's t-test ***P⩽0.001)

2-DE phospho-proteome of empty vector- and PrP^c-overexpressing SH-SY5Y in the presence of DMSO/STS. Linear 17 cm IPG strips (pH 5-10) were used and loaded with 130 μg of proteins. Labelling on the gel represents the location of 11 protein spots detected as differentially regulated between distinct transfection/treatment groups (spot no. 394 remained unidentified). Upper panels show representative gels of control cells, expressing endogenous levels of PrP^c, whereas lower panels show representative gels of PrP^c-overexpressing SH-SY5Y cells. The panels on the left side: DMSO (vehicle control); the panels on the right side: STS treatment. The protein identity of spots is listed in

Silver-stained 2-DE gels of empty vector- and PrP^c-overexpressing SH-SY5Y in the presence of DMSO/STS. Linear 17 cm IPG strips (pH 3-10) were used and loaded with 130 μg of proteins. Labelling on the gel represents the location of 14 protein spots detected as differentially regulated between distinct transfection/treatment groups. Upper panels represent 2-DE gel pattern of SH-SY5Y cells stably transfected with an empty vector (designated ctrl), whereas lower panels represent the 2-DE pattern of SH-SY5Y cells stably overexpressing PrP^c (designated PrP). The panels on the left side depict 2-DE pattern following DMSO treatment, whereas panels on the right side depict 2-DE pattern following STS treatment. The protein identity of spots is listed in

Co-localization of profilin-1 and PrP^c in SH-SY5Y cells following DMSO/STS treatment. Co-immunostaining of profilin-1 and PrP^c shows a marked altered localization of profilin-1 in both empty vector (designated ctrl) and PrP^c-overexpressing (designated PrP) SH-SY5Y cells subjected to STS as compared with DMSO treatment (a). Quantification of images taken in different regions of SH-SY5Y cells fixed after STS treatment showed a significant re-distribution of profilin-1 in PrP-positive cells. Pearson's co-localization correlation coefficient rp (−1⩽ rp⩽1) and graph was generated by ImageJ (Bethesda, MD, USA) (WCIF plugin) software. Densitometric analysis was performed from four independent experiments and the level of significance was calculated using one-way ANOVA Friedman test: *P⩽0.05; **P⩽0.01; ***P⩽0.001 (b–d)

Profilin-1, transgelin-2 and p53 are differentially regulated between empty vector- and PrP^c-overexpressing SH-SY5Y cells following DMSO/STS treatment. Western blot analysis shows a marked upregulation of profilin-1 in both empty vector (designated ctrl) and PrP^c-overexpressing (designated PrP) SH-SY5Y cells subjected to STS as compared with DMSO treatment. An overexpression of PrP^c significantly decreases profilin-1 expression, regardless of DMSO/STS treatment. Transgelin-2 expression was detected only in cells overexpressing PrP^c subjected to STS treatment, whereas no signal was detected in PrP^c-overexpressing cells subjected to DMSO. Likewise, no signal was obtained in control cells subjected either to DMSO or STS. p53 displays a similar expression pattern as profilin-1 with significantly higher expression in control as compared with PrP^c-overexpressing cells under both experimental conditions. β-actin expression below is given as a control for an equal protein load. The displayed Western blots are representatives of three independent experiments (a). Expression level of each protein displayed by western blot was quantified by densitometric analysis and is shown as a diagram (b–d). Data were normalized against ß-actin and are given as a ratio of each protein/β-actin±S.D., *P⩽0.05; **P⩽0.01; ***P⩽0.001

Silencing of profilin-1 expression in SH-SY5Y cells using siRNA. Western blot displays profilin-1 expression after transfection with non-targeting mock control (designated Mock-ctrl) and following transfection with profilin-1 specific siRNA (siRNA-PFN-1). Cells expressing endogenous levels of PrP^c (designated ctrl) and PrP^c-overexpressing cells (designated PrP) were both subjected to 2 h treatment with 1 μM STS or DMSO. Markedly higher profilin-1 expression is present in control versus PrP cells both treated with mock control. No profilin-1 expression after transfection with siRNA-PFN-1 was visible after immunoblotting. Beta-actin expression is given as a control for an equal protein load (a). Cell viability was measured by MTS assay 24 h post-transfection and 2 h after 1 μM STS or DMSO treatment. Note, increase in cell viability of PrP cells after silencing profilin-1 expression under STS treatment versus control cells subjected to the same conditions. PrP cells treated with siRNA-PFN-1 exhibit a significant increase in viability as compared with PrP cells treated with mock control. (b) Caspase-3 activity was reduced in PrP cells depleted of profilin-1 and treated with STS as compared with control cells under same experimental conditions. In addition, slight but significant decrease in caspase-3 activity was observed in PrP-siRNA-PFN-1 treated cells as compared with PrP^c-overexpressing cells not depleted of profilin-1 (PrP), both exposed to STS (c). Values represent the mean±S.D. of four independent experiments. Level of significance was calculated using Student's t-test: *P⩽0.05; **P⩽0.01; ***P⩽0.001

Cell viability (a) and caspase-3 activity (b) in empty vector transfected and PrP^c-overexpressing SH-SY5Y cells subjected to apoptotic agent STS. SH-SY5Y cells expressing either endogenous PrP^c (designated ctrl/black bars) or overexpressing PrP^c (designated PrP/grey bars) were subjected to DMSO (vehicle control) or 1 μM STS treatment for 2 h. As a negative control both, ctrl and PrP SH-SY5Y cells were incubated in the cell medium only, without addition of DMSO or STS. During performance of caspase assay a potent caspase-3 activity inhibitor (Z-VAD-FMK) was used as a positive control. Note, a significantly lower cell viability in PrP as compared with control cells under both, treatment-free (cell medium) and DMSO treatment conditions with no obvious changes in caspase-3 activity (ns). Conversely, after STS treatment a survival rate of PrP^c-overexpressing cells was significantly higher as compared with cells expressing endogenous levels of PrP^c. At the same time STS treatment resulted in caspase-3 activity in control, but not in PrP cells. Cell viability and caspase-3 activity were expressed as a percent of relative absorbance to untreated control cells expressing endogenous level of PrP^c. Data are means±S.D. of duplicate measurements from four independent experiments. Level of significance was calculated using Student's t-test: *P⩽0.05; ***P⩽0.001