Fig 1.
Effects of the psychostimulants cocaine, METH and morphine on HDAC class I proteins in human primary astrocytes.
Cells were exposed to cocaine (1 μM), METH (10 μM), and morphine (5 μM) either alone or in combination with piracetam (10 μM) for 24 h. The protein expression levels of different classes of HDACs (1–3) in astrocytes were determined by western blotting analysis using GAPDH as a loading control. Western blot showing (A) HDAC1, (B) HDAC2 and (C) HDAC3. The densitometric analysis results in D, E and F represent the protein levels (fold-change control) of HDAC1, HDAC2 and HDAC3, respectively. Two-way ANOVA analysis performed to compare the groups between cocaine Vs cocaine + piracetam, METH Vs METH + piracetam and morphine Vs morphine + piracetam. The data are expressed as the mean ± standard error mean of three independent experiments. N = 3. ****P<0.0001, ***P<0.001, **P<0.01, NS—nonsignificant.
Fig 2.
Effects of the psychostimulants cocaine, METH and morphine on HDAC class II proteins in human primary astrocytes.
Cells were exposed to cocaine (1 μM), METH (10 μM), and morphine (5 μM) either alone or in combination with piracetam (10 μM) for 24 h. The protein expression levels of different classes of HDACs (4, 5, 6 and 7) in astrocytes were determined by western blotting analysis using GAPDH as a loading control. Western blot showing (A) HDAC4, (B) HDAC5, (C) HDAC6 and (D) HDAC7. Two-way ANOVA analysis performed to compare the groups between cocaine Vs cocaine + piracetam, METH Vs METH + piracetam and morphine Vs morphine + piracetam. The densitometric analysis results in E, F, G and H represent the protein levels (fold-change control) of HDAC4, HDAC5, HDAC6 and HDAC7, respectively. The data are expressed as the mean ± standard error mean of three independent experiments. N = 3. ****P<0.0001, ***P<0.001, **P<0.01, NS—nonsignificant.
Fig 3.
Effects of the psychostimulants cocaine, METH and morphine on HAT proteins in human primary astrocytes.
Cells were exposed to cocaine (1 μM), METH (10 μM), and morphine (5 μM) either alone or in combination with piracetam (10 μM) for 24 h. The protein expression levels of PCAF, p300 and GCN5 in astrocytes were determined by western blotting analysis using GAPDH as a loading control. Two-way ANOVA analysis performed to compare the groups between cocaine Vs cocaine + piracetam, METH Vs METH + piracetam and morphine Vs morphine + piracetam. Western blot showing (A) PCAF, (B) p300 and (C) GCN5. The densitometric analysis results in D, E and F represent the protein levels (fold-change control) of PCAF, p300 and GCN5, respectively. The data are expressed as the mean ± standard error mean of three independent experiments. N = 3. ****P<0.0001, ***P<0.001, **P<0.01, NS—nonsignificant.
Fig 4.
Effects of the psychostimulants cocaine, METH and morphine on H3 histone proteins in human primary astrocytes.
Cells were exposed to cocaine (1 μM), METH (10 μM), and morphine (5 μM) either alone or in combination with piracetam (10 μM) for 24 h. The protein expression levels of H3K9AC, H3K14AC, H3K18AC, H3K27AC, and H3K56AC in astrocytes were determined by western blotting analysis using total H3 as a loading control. Two-way ANOVA analysis performed to compare the groups between cocaine Vs cocaine + piracetam, METH Vs METH + piracetam and morphine Vs morphine + piracetam. Western blot showing (A) H3K9AC, (B) H3K14AC, (C) H3K18AC, (D) H3K27AC, and (E) H3K56AC. The densitometric analysis results in G, H, I, J and K represent the protein levels (fold-change control) of H3K9AC, H3K14AC, H3K18AC, H3K27AC, and H3K56AC, respectively. The data are expressed as the mean ± standard error mean of three independent experiments. N = 3. ****P<0.0001, ***P<0.001, **P<0.01, NS—nonsignificant.
Fig 5.
Effects of the psychostimulants cocaine, METH and morphine on HDAC and HAT gene expression in human primary astrocytes.
Human primary astrocytes were exposed to cocaine (1 μM), METH (10 μM) and morphine (5 μM) either alone or in combination with piracetam (10 μM) for 24 h. Controls were maintained in drug-free medium (without drug exposure). (A) HDAC1, (B) HDAC3, (C) HDAC4, (D) HDAC6 and (E) HDAC7 (F) GCN5 (G) PCAF mRNA expression levels in astrocytes determined by qRT-PCR analysis using the housekeeping gene β-actin as a loading control. Two-way ANOVA analysis performed to compare the groups between cocaine Vs cocaine + piracetam, METH Vs METH + piracetam and morphine Vs morphine + piracetam. The data are expressed as the mean ± standard error mean of the transcript accumulation index (TAI) of three independent experiments. N = 3. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, NS—nonsignificant.
Fig 6.
Effects of the psychostimulants cocaine, METH and morphine on the expression and translocation of HDAC1 and GCN5 in human primary astrocytes.
Human primary astrocytes were exposed to cocaine (1 μM), METH (10 μM) and morphine (5 μM) either alone or in combination with piracetam (10 μM) for 24 h. Controls were maintained in drug-free medium (without drug exposure). Representative immunostaining images of (A) HDAC1 expression (red) and nuclear staining with DAPI (blue) obtained by confocal microscopy (magnification 100x, scale bar 100 μm). (C) GCN5 expression (red) and nuclear staining with DAPI (blue) obtained by confocal microscopy (magnification 100x). The data are expressed as the mean ± standard error mean of the corrected total cell fluorescence (CTCF) of three independent experiments. Representative graphs depicting immunostaining analysis results showing CTCF values for HDAC1 and GCN5 for all groups in B and D, respectively. N = 3. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, NS—nonsignificant.