Journal: The FASEB Journal

Article Title: Occludin, caveolin‐1, and Alix form a multi‐protein complex and regulate HIV‐1 infection of brain pericytes

doi: 10.1096/fj.202001562R

Figure Lengend Snippet: HIV‐1 infection attenuates cav‐1‐mediated regulation of ocln expression. Pericytes were transfected with 1 µg cav‐1 siRNA per 10 6 cells and either mock‐infected or HIV‐1‐infected with 60 ng/mL HIV‐1 p24 for 48 h. The expression of cav‐1, ocln, and Alix was evaluated by immunoblotting (A), quantified, and compared among groups. GAPDH was used as a loading control. Cav‐1 silencing (B) resulted in upregulation of occludin levels in mock‐infected pericytes and this effect was attenuated in HIV‐1‐infected pericytes (C). Cav‐1 silencing did not affect Alix levels (D). Graphs indicate the mean ± SD from three independent experiments. **** P < .0001, *** P = .0002, ** P = .003, n = 4‐9 per group

Article Snippet: Cav‐1, ocln, and Alix silencing was performance using the following small interfering RNA (siRNA): anti‐occludin 27‐mer siRNA (h): Cat# SR321170, anti‐caveolin‐1 27‐mer siRNA (h): Cat# SR319567 (OriGene, Rockville, MD, USA), and anti‐Alix siRNA (h): Cat# sc‐60149 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Expressing, Transfection, Western Blot, Control

Journal: The FASEB Journal

Article Title: Occludin, caveolin‐1, and Alix form a multi‐protein complex and regulate HIV‐1 infection of brain pericytes

doi: 10.1096/fj.202001562R

Figure Lengend Snippet: HIV‐1 infection obliterates Alix‐mediated regulation of ocln expression. Pericytes were transfected with 1 µg Alix siRNA per 10 6 cells and either mock‐infected or HIV‐1‐infected with 60 ng/mL HIV‐1 p24 for 48 h. The expression of Alix, ocln, and cav‐1 was evaluated by immunoblotting (A) and compared among groups. GAPDH was used as a loading control. Alix silencing (B) resulted in downregulation of ocln in mock‐infected pericytes and this effect was obliterated in HIV‐1‐infected pericytes (C). Alix silencing did not affect cav‐1 levels (D). Graphs indicate the mean ± SD from three independent experiments. **** P < .0001, ** P = .003, * P < .0449, n = 4‐9 per group

Article Snippet: Cav‐1, ocln, and Alix silencing was performance using the following small interfering RNA (siRNA): anti‐occludin 27‐mer siRNA (h): Cat# SR321170, anti‐caveolin‐1 27‐mer siRNA (h): Cat# SR319567 (OriGene, Rockville, MD, USA), and anti‐Alix siRNA (h): Cat# sc‐60149 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Expressing, Transfection, Western Blot, Control

Journal: The FASEB Journal

Article Title: Occludin, caveolin‐1, and Alix form a multi‐protein complex and regulate HIV‐1 infection of brain pericytes

doi: 10.1096/fj.202001562R

Figure Lengend Snippet: Modifications of the cav‐1‐ocln‐Alix complex regulates HIV‐1 infection. A, Pericytes were transfected with cav‐1 siRNA (1 µg), ocln expression vector (2 µg), or/and Alix siRNA (1 µg) per 10 6 cells. When treatment with all three agents was combined, they were used at half the concentrations. Transfected cultures were then either mock‐infected or HIV‐1‐infected as in Figure and p24 was analyzed in cell culture media by ELISA. B, p24 levels in the media from pericyte cultures exposed to conditioned media from (A) for 48 h. C, Confocal microscopy images of HIV‐1 pNL4‐3‐GFP‐infected pericytes after ocln overexpression, cav‐1 silencing, and Alix silencing. Nuclei were stained with DAPI (blue). GFP fluorescence (green) reflects HIV‐1 transcription. D, Quantification of the mean fluorescence intensity (MFI) of GFP from (C). Data represents mean ± SEM from two independent experiments. **** P < .0001, *** P = .0002, ** P = .003, * P < .0449, n = 4 per group. Scale bars, 20 µm

Article Snippet: Cav‐1, ocln, and Alix silencing was performance using the following small interfering RNA (siRNA): anti‐occludin 27‐mer siRNA (h): Cat# SR321170, anti‐caveolin‐1 27‐mer siRNA (h): Cat# SR319567 (OriGene, Rockville, MD, USA), and anti‐Alix siRNA (h): Cat# sc‐60149 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Over Expression, Staining, Fluorescence

Journal: The FASEB Journal

Article Title: Occludin, caveolin‐1, and Alix form a multi‐protein complex and regulate HIV‐1 infection of brain pericytes

doi: 10.1096/fj.202001562R

Figure Lengend Snippet: HIV‐1 infection and/or modifications of the cav‐1/ocln/Alix complex affect anti‐ and pro‐inflammatory cytokine profile in pericytes. Pericytes were transfected with cav‐1 siRNA (Cav‐1‐), ocln expression vector (Ocln+), or Alix siRNA (Alix‐) as in Figure and either mock‐infected or HIV‐1‐infected for 48 or 72 h. Cytokine levels were analyzed in cell culture media by Bio‐Plex Pro Human Cytokine assay kit. INF‐γ levels decreased to non‐detectable (#) concentrations after HIV‐1 infection in control, cav‐1‐, and Alix‐ pericyte cultures. The data are mean ± SEM from two independent experiments. **** P < .0001, *** P = .0002, ** P = .003, * P < .0449, n = 4 per group

Article Snippet: Cav‐1, ocln, and Alix silencing was performance using the following small interfering RNA (siRNA): anti‐occludin 27‐mer siRNA (h): Cat# SR321170, anti‐caveolin‐1 27‐mer siRNA (h): Cat# SR319567 (OriGene, Rockville, MD, USA), and anti‐Alix siRNA (h): Cat# sc‐60149 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Cell Culture, Cytokine Assay, Control

Journal: Molecular medicine reports

Article Title: Dicaine represses apoptosis-linked gene 2-interacting protein X expression to induce airway epithelial barrier dysfunction.

doi: 10.3892/mmr.2015.3433

Figure Lengend Snippet: Figure 2. Assessment of Rpc cell monolayer barrier function. Rpc cell monolayers were treated with dicaine in Transwells with at different concentrations for 24 h. (A) TER of Rpc monolayers. (B) Levels of OVA in the basal chambers of transwells. a, Alix‑knockdown Rpc monolayers; b, rpc monolayers treated with control shRNA; c, rpcs with Alix overexpression; d, rpcs treated with control plasmids. *P<0.01, compared with group 0. (C) Alix gene. (D) Alix over expression. The data represent three separate experiments. Rpc cells, RPMI2650 human airway epithelial cell line; Alix, apoptosis‑linked gene 2‑interacting protein X; TER, transepithelial electric resistance; OVA, ovalbumin; shRNA, small hairpin RNA.

Article Snippet: Antibodies (H‐270, polyclonal; 1:300) and an Alix small hairpin RNA (shRNA) kit were purchased from Santa Cruz Biotechnology Inc. (Shanghai, China).

Techniques: Control, shRNA, Over Expression