Protein and mRNA markers of ER stress related apoptosis. Cells were treated with BSAconjugated palmitate, oleate, elaidate or vaccenate (800 µM) or with palmitate (800 µM) and oleate, elaidate or vaccenate at a 4:1 or 2:1 molar ratio (200 or 400 µM) at 70-80% confluence for 8 h. (a) cDNA was prepared and CHOP expression was detected by qPCR. GAPDH was used as a constitutive reference gene. Relative expression levels were determined as CHOP/GAPDH ratios. Data are shown as mean values ± S.D.; n = 6; statistically significant differences: * p < 0.05 vs. BSA treated control; ! p < 0.05 vs. palmitate treated samples; # p < 0.05 vs. palmitate and oleate treated cells in case of combinational treatments at the corresponding ratio. (b) Cleaved caspase-3 was detected in cell lysates by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a constitutive reference protein. The image shows a typical picture of two independent experiments with two parallels.

Contexts in source publication

Context 1

... an acyl-CoA surplus in FA treated cells may cause alterations in the cellular ceramide content. The sharp contrast, which was seen between the modulation of TG and DG levels, was also observed in relation with TGs and ceramides (Table 4, Figure 7 and Figure S9-columns). The total ceramide content of the cells reached the highest levels (765.3 ± 197.4 ng/mg protein at 4 h and 1 993.9 ± 100.5 ng/mg protein after 8 h) upon palmitate supplementation. ...

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... values represent a more than 4-fold and 13-fold increase compared to the controls (171.1 ± 12.3 and 149.3 ± 52.4 ng/mg protein, respectively). The accumulating ceramides contained saturated acyl groups, the concentration of palmitoyl-sphingosine (16:0) and stearoyl-sphingosine (18:0) increased parallelly while oleoyl-sphingosine remained near the limit of detection (Figure 7 and Figure S9, Table S8). Table 4. Overall ceramide content of hepatoma cells in lipotoxicity. ...

Context 3

... a single unsaturated FA was added to the cells, palmitoyl-and stearoylsphingosine were consistently unchanged, yet the alteration of unsaturated acyl-sphingosine (ceramide 18:1) depended greatly on the cis or trans nature of the FA. While no significant elevation was found in oleate treated cells, vaccenate induced a 25-fold (4 h) and 18-fold (8 h) elevation and elaidate caused an even higher, 39-fold (4 h) and 25-fold (8 h), increase compared to the control (Figure 7 and Figure S9, Table S8). ...

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... the moderation of the total ceramide buildup was of nearly the same extent, and TFAs were only slightly less effective than oleate, a remarkable difference was again seen in the production of unsaturated acyl-sphingosine (ceramide 18:1), i.e., these ceramides only appeared in substantial amounts when TFAs were present. Intriguingly, their production seemed to be enhanced by palmitate because the half or quarter dose of a TFA co-administered with palmitate yielded the same or even more of these unusual ceramides as a large dose of the same TFA alone (Figure 7 and Figure S9-line, Table S8). ...

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... CHOP induction was assessed in our experiments at mRNA level by employing qPCR. A large increase was caused by palmitate while no change could be seen after unsaturated FA administration, either cis or trans (Figure 9a). Oleate helped the cells evade palmitate-induced CHOP induction in combination treatments, particularly at a 2:1 ratio. ...

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... helped the cells evade palmitate-induced CHOP induction in combination treatments, particularly at a 2:1 ratio. The two TFAs were also protective but to a significantly smaller extent than oleate (Figure 9a). To have a closer look into the activation of apoptosis, we examined the level of cleaved Caspase-3, the major effector caspase governing the process of apoptosis. ...

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... have a closer look into the activation of apoptosis, we examined the level of cleaved Caspase-3, the major effector caspase governing the process of apoptosis. In accordance with the observed stress signals, palmitate treatment resulted in the most pronounced activation of Caspase-3, while all unsaturated FAs were basically ineffective when applied on their own (Figure 9b). In combination treatments, oleate prevented the cleavage of Caspase-3 at the highest degree in a dose-dependent manner, and the TFAs exerted a similar, albeit milder, effect (Figure 9b). ...

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... accordance with the observed stress signals, palmitate treatment resulted in the most pronounced activation of Caspase-3, while all unsaturated FAs were basically ineffective when applied on their own (Figure 9b). In combination treatments, oleate prevented the cleavage of Caspase-3 at the highest degree in a dose-dependent manner, and the TFAs exerted a similar, albeit milder, effect (Figure 9b). Protein and mRNA markers of ER stress related apoptosis. ...