Figure 2.

Tspan18-knockdown endothelial cells have impaired Ca²⁺ mobilization. (A-D) Human umbilical vein endothelial cells (HUVEC) were transfected with a negative control siRNA (CON) or with one of two independent siRNA targeting Tspan18 (T18 KD). After 48 hours, HUVEC were loaded with the Ca²⁺-sensitive dye Fluo-4 NW and Ca²⁺ measurements taken using a FlexStation fluorescence reader during addition (arrow) of (A) 1 U/mL thrombin, (B) 20 μM histamine, or (C) 10 μM ionomycin. Representative Ca²⁺ traces are shown for Tspan18-knockdown HUVEC (left), with quantitation of maximum intracellular Ca²⁺ concentrations (right). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent the Standard Error of Mean (SEM) from three independent experiments. *P<0.05; ***P<0.001. (D) siRNA-transfected HUVEC from (A) to (C) were harvested, mRNA extracted, cDNA produced and Tspan18 mRNA levels were assessed by quantitative real-time polymerase chain reaction (qPCR). Data were normalized to 18S and actin as internal controls and adjusted such that the non-siRNA-trans-fected mock value was 1 in each experiment. Data were then normalized by logarithmic transformation, and analyzed by one-way ANOVA and Tukey’s multiple comparison test. Error bars represent the Standard Error of the Mean from three independent experiments. ***P<0.001. (E) HUVEC were subjected to Tspan18 siRNA knockdown as described for (A-D), stimulated with 1 U/mL thrombin or 20 μM histamine for 5 minutes, then whole cell lysates were analyzed by western blotting with phospho-ERK1/2 and total ERK1/2 antibodies. (Left) Representative blots. (Right) Quantitation of three independent experiments. Error bars represent SEM. Knockdown efficiency was similar to that shown in (D) (data not shown). secs: seconds; RFU: relative fluorescence unit.