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1.
Figure 12

Figure 12. Effect of cocaine on induction of ALCAM expression in mouse models of systemic inflammation and T-cell deficiency. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

Effect of cocaine on the ALCAM expression in isolated brain microvessels from C57 BL/6N mice in the presence of systemic inflammation (LPS) and in T-cell deficient mice as assessed by Western blot (A) and immunostaining (B). ALCAM: red; Cav-1: green; nuclei: blue. n=3 per group. *p<0.05 vs saline group; ##p<0.01 vs saline treated C57 BL/6N control group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
2.
Figure 3

Figure 3. Cocaine-mediated induction of ALCAM expression and its translocation to lipid rafts. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Flow cytometric analysis indicating cocaine-mediated changes in ALCAM, ICAM & VCAM in HBMECs. (B) ALCAM expression (green) in cocaine-treated HBMECs. Nuclei stained with DAPI (blue). Scale bar: 20μm. (C) ALCAM localization in cholesterol-rich lipid raft fractions (4-5) of HBMECs. Presence of cholesterol confirms the lipid raft nature of fractions 4-5. (D) Cocaine promoted increased translocation of ACLAM into lipid raft fractions. All the experiments were repeated at least four separate times.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
3.
Figure 5

Figure 5. Cocaine-mediated induction of ALCAM involves PDGF-βR activation. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Cocaine mediated time-dependent phosphorylation of PDGF-βR. (B) Inhibition of cocaine-mediated phosphorylation of PDGF-βR by the tyrosine kinase inhibitor STI-571 (1μM). (C) Inhibition of cocaine-mediated phosphorylation of PDGF-βR by BD1047 (20μM) in HBMECs. (D) Flow analysis demonstrating abrogation of cocaine-mediated induction of ALCAM in HBMECs pretreated with STI571. (E) Knock down of PDGF-βR expression in HBMECs transfected with PDGF-βR siRNA but not Non siRNA. (F) PDGF-βR siRNA, but not non-siRNA, inhibited cocaine-mediated induction of ALCAM expression. All the data are presented as mean±SD of four independent experiments. *p<0.05 vs control group; ##p<0.01 vs cocaine-treated HBMECs in non-specific siRNA group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
4.
Figure 9

Figure 9. Involvement of σ-1R, Src kinase and PDGF-βR in the regulation of MAPKs and PI3K/Akt cell signaling pathways. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

Pretreatment of HBMECs with either BD1047 (20μM) /STI571 (1μM) (A), σ-1R siRNA (B), PDGF-βR siRNA or (C) c-Src inhibitor PP2 (10μM) (D), resulted in inhibition of cocaine-mediated phosphorylation of signaling pathways. Representative immunoblots and the densitometric analyses of p-ERK/ERK, p-JNK/JNK, p-p38/p38 and p-Akt/Akt from 4 separate experiments are presented. All the data are indicated as mean±SD of four independent experiments. *p<0.05; **p<0.01vs control group; #p<0.05; ##p<0.01 vs cocaine-treated group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
5.
Figure 4

Figure 4. Engagement of σ-1R is critical for cocaine-mediated induction of ALCAM expression. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Detection of σ-1R RNA by RT-PCR analysis in HBMECs. (B) Flow analysis of ALCAM expression in cocaine-exposed HBMECS pretreated with/without σ-1R antagonist BD1047 (20 μM). (C) Whole cell lysates from HBMECs transfected with either σ-1R or nonsense (Non) siRNAs subject to Western blot analysis. (D) σ-1R siRNA, but not non-siRNA inhibited cocaine-mediated induction of ALCAM expression. All the data are presented as mean±SD of four independent experiments. *p<0.05 vs control group; ##p<0.01 vs cocaine-treated HBMECs in non-specific siRNA group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
6.
Figure 2

Figure 2. Cocaine-mediated induction of ACLAM enhances monocyte adhesion and transmigration in vivo. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Cocaine induced increased expression of ALCAM in isolated microvesssels compared with those from saline-injected animals (upper panels). ALCAM: red; Cav-1: green; nuclei: blue. n=6 per group. (B) Representative fluorescence micrographs demonstrating cocaine-mediated adhesion of PKH26-labeled monocytes in the perivascular cuff (see arrowheads) and the parenchymal (see arrows) areas of the mice brain. (C) Cocaine-mediated increased monoctye transmigration was ameliorated in mice pre-treated with ALCAM neutralizing antibody. *p<0.05; **p<0.01 vs saline group; #p<0.05 vs cocaine group counted from the perivascular cuff and parenchyma.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
7.
Figure 8

Figure 8. Cocaine-mediated induction of ALCAM expression involves MAPKs and PI3K/Akt cell signaling pathways. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Western blot analysis demonstrating time-dependent activation of ERK, JNK and p38 by cocaine. (B) Inhibition of the ERK and JNK pathway by MEK1/2 (U0126, 20μM), JNK (SP600125, 20μM) and p38 (SB203580, 20μM) inhibitor resulted in amelioration of cocaine-mediated induction of ALCAM. (C) Western-blot analysis demonstrating time-dependent activation of Akt in cocaine-treated HBMECs. (D) Pharmacological inhibition of the PI3K pathway by LY294002, (20μM) resulted in amelioration of cocaine-mediated induction of ALCAM. (E) Transduction of HBMECs with the DN-Akt, but not WT-Akt resulted in abrogation of cocaine-mediated induction of ALCAM. All the data are presented as mean±SD of four independent experiments. *p<0.05; **p<0.01vs control group; ##p<0.01vs cocaine-treated group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
8.
Figure 7

Figure 7. Cocaine-mediated induction of ALCAM involves activation of c-Src tyrosine kinase. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Cocaine-mediated time-dependent phosphorylation of c-Src in HBMECs. (B) Pre-treatment of HBMECs with Src kinase inhibitor PP2 (10μM), but not PP3 (1μM), abrogated cocaine-induced phosphorylation of c-Src. (C) Western blot analysis demonstrating inhibition of cocaine-mediated phosphorylation of PDGF-βR in HBMECs pretreated with the Src inhibitor-PP2, but not PP3. (D) Western blot analysis demonstrating inability of the STI-571 (1μM) to inhibit cocaine-mediated phosphorylation of c-Src in HBMECs. (E) Quantification of flow cytometric analysis demonstrating abrogation of cocaine-mediated induction of ALCAM in HBMECs pretreated with the Src kinase inhibitor PP2 (10 μM), but not with the Src kinase ortholog PP3 (1μM). All the data are presented as mean±SD of four independent experiments. *p<0.05 vs control group; #p<0.05, ##p<0.01 vs cocaine-treated group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
9.
Figure 11

Figure 11. Cocaine-mediated induction of ACLAM enhances monocyte adhesion and transmigration in vitro. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

Exposure of HBMECs to varying concentrations of cocaine resulted in induction of HIV-infected (A) and uninfected (B) monoctye adhesion. (C) Cocaine-mediated induction of monoctye adhesion was ameliorated in HBMECs pretreated with the ALCAM-blocking antibody (1μg/ml). (D) Cocaine-mediated increase in monoctye adhesion was ameliorated in HBMECs pretreated with BD1047 (20μM), STI571 (1μM), PP2 (10μM) but not PP3 (1μM). (E) Cocaine-mediated increase in monoctye adhesion was ameliorated in HBMECs pretreated with MEK1/2 (U0126; 20μM), JNK (SP600125; 20μM), p38 (SB203580; 20μM), PI3K (LY294002; 20μM) or IκB inhibitor (SC514; 5 μM). All the data are presented as mean±SD of four individual experiments. ***p<0.001vs control group; ###p<0.001 vs cocaine-treated group. (F) Cocaine-mediated increase in monoctye transmigration was ameliorated in HBMECs pretreated with the ALCAM-1-blocking antibody (1μg/ml). All the data are presented as mean±SD of four individual experiments. *p<0.05;**p<0.01;***p<0.001; #p<0.05; ###p<0.001 vs cocaine-treated group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
10.
Figure 6

Figure 6. Interaction of σ-1R and PDGF-βR. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Pull-down assay with GST fusion protein to identify the binding of σ-1R and PDGF-βR. (B) FRET analysis of the interaction between PDGF-βR and σ-1 R. Negative control of CHO cells expressing GFP and σ-1R-RFP. Top-left: GFP; top-right: σ-1R-RFP; bottom-left: merged image; bottom-right: normalized FRET with a scale and references of fluorescent intensities (Left panel). FRET in CHO cells expressing PDGF-βR-GFP and σ-1 R-RFP. Top-left: PDGF-βR-GFP; top-right: σ-1R-RFP; bottom-left: merged image; bottom-right: normalized FRET. The scale (scale bar=10 μm) and references are the same to A (Right panel). (C) Cocaine affects the interaction between σ-1R and PDGF-βR using co-immunoprecipitation assay. (D) HBMECs were treated with cocaine and double-stained using antibodies specific for σ-1R (red) or PDGF-βR (green). Nuclei stained with DAPI (Blue). Representative pictures from three typical experiments. Scale bars: 20 μm. (E) Z-stack reconstruction was used for analysis of σ-1R and PDGF-βR co-localization (yellow) in HBMECs.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
11.
Figure 10

Figure 10. Cocaine-mediated induction of ALCAM expression involves NF-κB activation. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Western blot analysis demonstrating cocaine-mediated increase in the phosphorylation of the p65 subunit of NF-κB in the nuclear fraction of HBMECs, with a concomitant decrease in the cytosolic fraction. Flow cytometric analysis indicating inhibition of cocaine-mediated induction of ALCAM in HBMECs: (B) pretreated with the IκBα inhibitor, SC514 (5 μM) and (C) over-expressing the mutant, but not the full-length p65/RelA NF-κB construct. Nuclear extracts from HBMECs exposed to cocaine in the presence or absence of σ-1R siRNA (D), or PDGF-βR siRNA (E) were examined for cocaine-mediated translocation of NF-kB by Western blot analysis. Representative immunoblots and the densitometric analysis of p-P65 NF-kB/histone from four separate experiments. (F) Schematic illustration of NF-κB binding consensus sequence on the ALCAM promoter region. (G) ChIP assay demonstrating cocaine-mediated binding of p65NF-κB to the ALCAM promoter. The image is representative of three independent experiments. All the data are indicated as mean±SD of four independent experiments. *p<0.05; **p<0.01; vs control group; #p<0.05 vs cocaine-treated group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.
12.
Figure 1

Figure 1. Cocaine-mediated induction of ALCAM expression in human brains. From: Cocaine hijacks sigma-1 receptor to initiate induction of ALCAM: Implication for increased monocyte adhesion and migration in the central nervous system.

(A) Increased accumulation of CD68 positive cells around the microvessel that stained positive for ALCAM in drug-abusing HIV+ individuals (lower panel) compared with HIV- or HIV+ with no drug abuse history. Confocal microscopy of paraffin-embedded sections of the basal ganglia from HIV- (upper panel), HIV+/no drug abuse history (middle panel) or HIV+/drug abusers stained with antibodies specific for CD68 (red) and ALCAM (green). Scale bar=20 μm. Representative figures were shown from four experiments with 4 HIV-, 3 HIV+/no drug abuse and 4 HIV+/drug abusers. (B) ALCAM expression was upregulated in the CNS vessels of HIV (+) individuals with drug (cocaine) abuse history (lower panel). Confocal microscopy of paraffin-embedded sections of the basal ganglia from HIV(-) (upper panel), HIV(+)/no drug abuse (middle panel) or HIV(+)/drug abusers stained with antibodies specific for Cav-1 (red), ALCAM (green), nuclei are stained with DAPI (blue). Scale bar=20μm. Semiquantitative analysis of the signal intensity from 3-μm z-stack reconstructions along the line markers in the images in left panels. (C) Peak fluorescence intensity averaged from two distinct measurements on each vessel. Each symbol represents one vessel image; small horizontal lines indicate the mean. **p<0.01 vs HIV(-) group; ##p<0.01 vs HIV (+)/no drug abuse group counted from 12 vessels per group.

Honghong Yao, et al. J Neurosci. ;31(16):5942-5955.

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