Fig 2: sCD146 promotes BBB permeability in vitro. (A) Analysis of paracellular barrier function by permeability assay. hCMEC/D3 cells were seeded into the upper chambers of a transwell system, and permeability was measured with 0.5 µg/mL HRP after hCMEC/D3 cells were incubated with 5 µg/mL BSA, 0.05-5 µg/mL rhsCD146 for 2 h. *p<0.05; **p<0.01; and ***p<0.001. (B) Immunofluorescence staining of the TJPs (occludin, ZO-1 and JAM-1) and F-actin after hCMEC/D3 cells were treated with 5 µg/mL BSA or rhsCD146 for 4 h. Bar, 10 µm. (C) hCMEC/D3 cells were preincubated with 5 µg/mL BSA, 0.5 µg/mL or 5 µg/mL rhsCD146, TJP expression levels were verified by western blotting. (D) hCMEC/D3 cells were treated with 5 µg/mL BSA or 0.5, 2 or 5 µg/mL rhsCD146 for 12 h, and apoptosis was detected by flow cytometry with Annexin V and 7-AAD. (E) hCMEC/D3 cells were treated with 5 µg/mL BSA, 0.5 rhsCD146 or 5 µg/mL rhsCD146 for 12 h, and cell lysates were used to detect the expression of caspase 9, caspase 3, Bcl-2 and Bax.

Fig 3: JAM-A 3' UTR regulates VCAN expression by sequester microRNAs in hDPCs. (A and B) Quantitative analysis of JAM-A and VCAN expression in lentiviral mediated Dicer knockdown hDPCs with and without JAM-A and VCAN knockdown. (C) The Quantification of JAM-A and VCAN targeted miRNAs between early and late passage hDPCs. (D) Dual-luciferase assay detecting the targeting effect of candidate miRNAs to VCAN and JAM-A 3' UTR. (E) Dual-luciferase assay detecting the targeting effect of candidate miRNAs to site mutated VCAN and JAM-A 3' UTR constructs. (F) Illustration of the mechanism of RNA immunoprecipitation using the MS2 system. The western blot analysis showing the efficacy of MS2 mediated 3' UTR pulldown in hDPCs is showing in the lower panel. (G) The quantification of microRNAs in MS2 mediated JAM-A 3' UTR pulldown of hDPCs. (H) The quantification of microRNAs in MS2 mediated VCAN 3' UTR pulldown of hDPCs. All quantifications were done with three independent repeats, and the expression of GAPDH was used as a PCR internal references. Data are represented as means ± SD. *P < 0.05, **P < 0.01.

Fig 4: JAM-A promotes agglutinative growth and VCAN expression of hDPCs through the 3' UTR region. (A) Quantitative analysis of JAM-A and VCAN expression in JAM-A knockdown hDPCs, the phenotypic agglutinative colonies were lost in the siJAM-A group. NC, transfected with a scrambled siRNA negative control. (B) Western blot analysis revealed the expression of JAM-A and VCAN were decreased in the siJAM-A group compared to NC. (C) CCK-8 assays were conducted to determine the cell proliferation. (D) Cell viability was assessed by FCM in hDPCs with and without JAM-A knockdown. (E) Quantitative analysis of JAM-A and VCAN expression in JAM-A overexpressed hDPCs, the phenotypic agglutinative colonies were shown in the upper panel. The JAM-A CDS represents overexpression of only JAM-A coding region, while JAM-A 3' UTR represents overexpression of JAM-A 3' UTR region only. (F) Western blot analysis showing the protein expression of JAM-A and VCAN in JAM-A overexpressed hDPCs. (G) CCK-8 assays were conducted to determine the cell proliferation of hDPCs with JAM-A CDS or 3' UTR overexpression. (H) Cell viability was assessed by FCM. (I) Quantitative analysis of JAM-A and VCAN expression in hDPCs of different treatments, the phenotypic agglutinative colonies were shown in the left panel. The JAM-A CDS represents overexpression of only the JAM-A coding region, while JAM-A 3' UTR represents overexpression of only the JAM-A 3' UTR region. (J) CCK-8 assays were conducted to determine the cell proliferation of hDPCs with of different treatments. (K) Cell viability analysis of hDPCs with different treatments by FCM. All quantifications were done with three independent repeats, and the expression of GAPDH was used as PCR internal references. CTL means control. Data are represented as means ± SD. *P < 0.05, **P < 0.01. Scale bars show 50 µm.

Fig 5: METH and HIV-Tat synergistically induce oxidative stress that damages the BBB model in vitro. The hCMEC/D3 cells were treated with or without 1.0 mM of NACA for 1 h before 0.5 mM of METH and 100 nM of HIV-Tat for 24 h. (A) ROS fluorescence was observed by an inverted fluorescence microscope (Scale bar: 1 µm), and the fluorescence intensity was analyzed by Image J software; (B–E) the activity of CAT, GSH-PX, and SOD and the MDA level were measured using commercial kits; (F) the cell viability was measured using the CCK8 kit; (G) apoptosis was assessed via TUNEL assay (Scale bar: 50 µm), apoptosis level was determined by the number of TUNEL-positive cells expressed as a percentage of the total cell number (analyzed by Image J software); (H) western blot was performed to determine JAMA, Occludin, and ZO1 expression levels. Compared with the control, **p < 0.01, ***p < 0.001; compared with the METH + HIV-Tat, # p < 0.05, ## p < 0.01, n = 3. C=Control, N = 1.0 mM of NACA, M + T = 0.5 mM of METH+100 nM of HIV-Tat, and N + M + T = 1.0 mM of NACA+0.5 mM of METH+100 nM of HIV-Tat.