Figure 10: DHHC21-mediated PLCβ1 palmitoylation contributes to barrier dysfunction in inflammation. | Nature Communications

Figure 10: DHHC21-mediated PLCβ1 palmitoylation contributes to barrier dysfunction in inflammation.

From: Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome

Figure 10

Molecular manipulations of DHHC21 or PLCβ1 via knockdown and site-specific mutagenesis indicate the involvement of DHHC21-PLCβ1 palmitoylation in endothelial barrier response. (a) Verification of PLCβ1 siRNA knockdown by Western blotting. β-actin serves as loading control. Results represent mean±s.e.m. from three independent experiments. *P<0.05 versus control siRNA. (b) Verification of wild-type PLCβ1 or C17S mutant PLCβ1 (V5 tagged) overexpression in Zdhhc21+/+ or Zdhhc21dep/depECs. PLCβ1 C17S mutant was created by site-specific mutagenesis at Cys17, the highly predicted palmitoylation site. Empty pLX304 vector serves as vector control. β-actin serves as loading control. (c) Dynamic recording of TER responses to thrombin (10 U ml−1) in PLCβ1 knockdown ECs. Solid line tracing represents the mean resistance, and shadow represents±s.e.m. Embedded bar graph shows peak TER changes and presented as mean±s.e.m. from three independent experiments. ***P<0.001. (d) The effect of overexpressing wild-type PLCβ1 on TER responses to thrombin (10 U ml−1) in Zdhhc21+/+ ECs. Solid line tracing represents the mean resistance, and shadow represents±s.e.m. Embedded bar graph shows peak TER value changes. Bar graph represents mean±s.e.m. from three independent experiments. ***P<0.001. (e) The effect of overexpressing C17S mutant PLCβ1 (palmitoylation impaired) on TER responses to thrombin (10 U ml−1) in Zdhhc21+/+ ECs. Solid line tracing represents the mean resistance, and shadow represents s.e.m. Embedded bar graph shows peak TER value changes. Bar graph represents mean±s.e.m. from three independent experiments. n.s.=not significant. (f) The effect of overexpressing wild-type PLCβ1 on TER responses to thrombin (10 U ml−1) in Zdhhc21dep/dep ECs. Solid line tracing represents the mean resistance, and shadow represents s.e.m. Embedded bar graph shows peak TER changes. Bar graph represents mean±s.e.m. from three independent experiments. n.s.=not significant.

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