Table 1.
List of primers used for qPCR.
Figure 1.
mRNA expression levels of genes involved in cholesterol metabolism after TSA treatment.
SH-SY5Y neuroblastoma cells were treated with 250 nM TSA for 16 h. mRNA transcript levels of HMGCS, HMGCR, MVK, LDLR, SREBP1c and 2, APOE, NPC1, ABCA1 and CYP46A1 were analyzed by real-time RT-PCR. Values were normalized to the internal standard β-actin and expressed as fold change relative to untreated cells. Values represent means ± SEM from at least three individual experiments (* p<0.05, ** p<0.01, *** p<0.001).
Figure 2.
HDAC inhibition by TSA decreases SREBP2 proteolytic cleavage.
A) Western blot analysis of SREBP2 protein levels in SH-SY5Y cells after treatment with 250 nM TSA for the indicated time points. The levels of GAPDH are shown as a loading control. B) Quantification of the relative levels of SREBP2 after TSA treatment. Results are expressed as fold change relative to vehicle-treated cells and represent means ± SEM from at least three individual experiments. C) NT2-N post-mitotic neurons were treated with 250 nM TSA or vehicle for 16 h. mRNA transcript levels of HMGCR were analyzed by real-time RT-PCR. Values were normalized to the internal standard β-actin and expressed as fold change relative to vehicle-treated cells. Values represent means ± SEM from at least three individual experiments (* p<0.05, ** p<0.01, *** p<0.001).
Figure 3.
TSA treatment significantly decreases total cholesterol content in neuroblastoma cells.
A) Quantification of total cholesterol was performed in SH-SY5Y neuroblastoma cells treated with vehicle, 250 nM TSA or 500 nM TSA for indicated time-points. Values were normalized to total protein content and expressed as ng of cholesterol per µg of total protein. Data represent means ± SEM from at least three individual experiments (* p<0.05, ** p<0.01). B) βIII-tubulin immunostaining of SH-SY5Y cells treated with vehicle or 250 nM TSA for 48 h, or with 250 nM TSA for 48 h and additionally 48 h without TSA (washout). The results shown are representative of those obtained in at least three independent experiments (scale bar = 40 µm).
Figure 4.
TSA removal completely reverts the transcriptional changes observed after HDAC inhibition.
SH-SY5Y neuroblastoma cells were treated with vehicle or 250 nM TSA for 16 h, or with 250 nM TSA for 16 h and additionally 24 h without TSA (washout). mRNA transcript levels of HMGCR, MVK, LDLR, SREBP2 and ABCA1 were analyzed by real-time RT-PCR. Values were normalized to the internal standard β-actin and expressed as fold change relative to vehicle-treated cells. Values represent means ± SEM from at least three individual experiments (* p<0.05, *** p<0.001).
Figure 5.
TSA reverts the increase in total cholesterol levels observed after U18666A treatment.
A) Filipin III immunofluorescence staining of SH-SY5Y cells treated for the indicated time-points with 1 µg/ml U18666A or vehicle (scale bar = 40 µm). B) SH-SY5Y cells were treated with 3 µg/ml U18666A for 24 h and with or without 250 nM TSA for 48 h, and total cholesterol levels were determined. Values were normalized to total protein content and expressed as ng of cholesterol per µg of total protein. Data represent means ± SEM from at least three individual experiments (** p<0.01, *** p<0.001).
Figure 6.
TSA treatment partially reverts the effect of U18666A on the expression of genes involved in cholesterol synthesis, uptake and efflux.
SH-SY5Y neuroblastoma cells were pre-treated with 3 µg/ml U18666A for 6 h and with or without 250 nM TSA for 16 h. mRNA transcript levels of HMGCR, MVK, LDLR and ABCA1 were analyzed by qPCR. Values were normalized to the internal standard β-actin and are expressed as fold change relative to untreated cells. Data represent means ± SEM from at least three individual experiments (* p<0.05, ** p<0.01, *** p<0.001).
Figure 7.
TSA treatment partially reverts the U18666A-induced phenotype by promoting cholesterol redistribution in neuroblastoma cells.
A) Filipin III, lysosome-associated membrane protein 2 (LAMP-2) and acetyl-histone 4 (AcH4) immunofluorescence staining of SH-SY5Y cells pre-treated with 1 µg/ml U18666A for 24 h and with or without 250 nM TSA for 16 h. After the 24 h treatment with U18666A, the medium was removed and replaced with new medium without U18666A, and with or without TSA. Colocalization images of filipin III and LAMP-2 immunostaining are also presented. The results shown are representative of those obtained in at least three independent experiments. (scale bar = 40 µm). Quantification of filipin III fluorescence (B) and filipin III and LAMP-2 colocalization (C) was performed, the values were normalized to total cell number assessed by AcH4 immunostaining and are expressed as fold change relative to U18666A treated cells. Data represent means ± SEM of at least three independent experiments (* p<0.05).