Journal: American Journal of Translational Research

Article Title: LncRNA TUG1 regulates autophagy-mediated endothelial-mesenchymal transition of liver sinusoidal endothelial cells by sponging miR-142-3p

doi:

Figure Lengend Snippet: Starvation activated autophagy in LSECs. A. WST-1 assay revealed that starvation significantly inhibited LSEC proliferation. B. Flow cytometry showed an increased proportion of LSECs in the G0/G1 phase when cultured under starvation. C. Quantitative RT-PCR showed the expression of autophagy-related genes (LC3B, ATG5, ATG7, Beclin1, and P62) in LSECs under starvation. D. The protein expression of LC3B and P62 in LSECs under starvation and starvation plus CQ conditions was assessed by western blot analysis. E, F. An increase in autophagic flux in LSECs under starvation was validated by punctate mCherry-GFP-LC3 fusion protein expression using confocal microscopy and double-membraned cytoplasmic vesicles (black arrow) using TEM. CQ, 1.5 μM; **, P < 0.01; *, P < 0.05.

Article Snippet: Lentiviral vectors expressing shRNA against ATG5 and corresponding negative lentiviral vector control were purchased from Genechem (Shanghai, China).

Techniques: WST-1 Assay, Flow Cytometry, Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Confocal Microscopy

Journal: American Journal of Translational Research

Article Title: LncRNA TUG1 regulates autophagy-mediated endothelial-mesenchymal transition of liver sinusoidal endothelial cells by sponging miR-142-3p

doi:

Figure Lengend Snippet: LPS promoted EndMT of LSECs by increasing autophagy. A. As indicated by ectopic expression of LC3B, ATG5, and P62, LPS treatment promoted starvation-induced autophagy in a dose-dependent manner. B, C. Dose- and time-dependent expressions of autophagy (LC3B, ATG5, and p62) and EndMT (α-SMA, VE-cadherin, and CD31) proteins in LSECs under starvation treated with LPS was examined by western blot analysis. D. The increased expression of LC3B and P62 after LPS treatment was further elevated by CQ. E. Transwell assay revealed that ATG5 knockdown suppressed the migration ability of LSECs, which was partially reversed by LPS treatment. F. Knockdown of ATG5 decreased autophagy and EndMT of LSECs as shown by the decreased expressions of LC3B and α-SMA, and increased expressions of P62, CD31, and VE-cadherin. These effects were partially reversed LPS treatment. CQ, 1.5 μM; **, P < 0.01; *, P < 0.05.

Article Snippet: Lentiviral vectors expressing shRNA against ATG5 and corresponding negative lentiviral vector control were purchased from Genechem (Shanghai, China).

Techniques: Expressing, Western Blot, Transwell Assay, Knockdown, Migration

Journal: American Journal of Translational Research

Article Title: LncRNA TUG1 regulates autophagy-mediated endothelial-mesenchymal transition of liver sinusoidal endothelial cells by sponging miR-142-3p

doi:

Figure Lengend Snippet: TUG1 promoted LPS-mediated autophagy and EndMT of LSECs. A. Poly(A) RNA of LSECs was detected by deep sequencing. B. Expression levels of lncRNAs comparable with that of VEGFA. C. RT-PCR analysis of five highly expressed lncRNAs (MALAT1, NEAT1, MEG3, SNHG5, and TUG1) in LSECs. TUG1 was most markedly elevated in LPS-treated LSECs and was upregulated in a dose-dependent manner. D. Transwell assay revealed that TUG1 knockdown suppressed the migration ability of LSECs, which was partially reversed by LPS treatment. E, F. TUG1 knockdown suppressed autophagy and EndMT of LSECs as evidenced by the changes in LC3B, ATG5, P62, VE-cadherin, CD31, and α-SMA in LSECs. LPS, 1 μg/ml. **, P < 0.01; *, P < 0.05.

Article Snippet: Lentiviral vectors expressing shRNA against ATG5 and corresponding negative lentiviral vector control were purchased from Genechem (Shanghai, China).

Techniques: Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Transwell Assay, Knockdown, Migration

Journal: American Journal of Translational Research

Article Title: LncRNA TUG1 regulates autophagy-mediated endothelial-mesenchymal transition of liver sinusoidal endothelial cells by sponging miR-142-3p

doi:

Figure Lengend Snippet: TUG1 modulated ATG5 through cross-talk with miRNA-142-3p. A. RNA FISH assay of TUG1 in LSECs. B. RIP with an antibody against Ago2 in LSECs showed that TUG1, ATG5, and miRNA-142-3p were more preferentially enriched in the anti-Ago2 group than in the anti-normal IgG group. C. Dual-luciferase reporter assay revealed that miR-142-3p-overexpressing plasmid decreased the luciferase activity in the WT vectors of TUG1 and ATG5 compared with that in the MUT vectors. D. Expression of miR-142-3p in LSECs after TUG1 knockdown or LPS-induced overexpression was confirmed by RT-PCR. E, F. Migration ability and changes in LC3B, ATG5, P62, α-SMA, VE-cadherin, and CD31 expression after transfection of miRNA-142-3p mimics in LSECs treated with LPS were analyzed by Transwell assay, RT-PCR, and western blot analysis. LPS, 1 μg/ml. **, P < 0.01; *, P < 0.05.

Article Snippet: Lentiviral vectors expressing shRNA against ATG5 and corresponding negative lentiviral vector control were purchased from Genechem (Shanghai, China).

Techniques: Luciferase, Reporter Assay, Plasmid Preparation, Activity Assay, Expressing, Knockdown, Over Expression, Reverse Transcription Polymerase Chain Reaction, Migration, Transfection, Transwell Assay, Western Blot