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. 2021 Oct 20;12:697071. doi: 10.3389/fimmu.2021.697071

Figure 3.

Figure 3

rhHMGB1-mediated early barrier dysfunction is largely dependent on ROCK1 signaling. (A, B) ECs were transfected with ROCK1/2 siRNA. Western blot was used to assess the protein expression of ROCK1/2 in HPMECs. (C) Cell viability was assessed by the CCK-8 assay after transfection with different concentration of ROCK1/2 siRNA for 24 h or transfection with 10 nM ROCK1/2 siRNA for the indicated times. There was no evidence of cytotoxicity found in ROCK1/2 siRNA transfected cells. (D) Examination of FITC-dextran flux of HPMECs. ROCK1 knockdown ameliorated rhHMGB1-induced early permeability increases (at 60 min). (E) Effects of ROCK1 siRNA on the expression of VE-cadherin and ZO-1 induced by rhHMGB1 at 24 h. (F) ECs were transfected with ROCK1 siRNA and then stimulated with rhHMGB1 for 60 min. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was detected by fluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (G) ROCK1 knockdown attenuated the ROCK1 expression induced by rhHMGB1 at 60 min. (H) ROCK1 knockdown downregulated the rhHMGB1-induced pMLC expression in cells at 60 min. Mean ± SD of 3 independent trials was shown. *p < 0.05 vs. corresponding control group. # p < 0.05 vs. rhHMGB1 60-min group. NC, negative control.