Figure 5.

Knockdown of CARMA3 attenuates LPA induced TSLP and CCL20 expression in NHBE cells. (A) A549 cells were transfected with lentiviral vectors expressing shRNA sequences specific to CARMA3 or a control vector. CARMA3 RNA knockdown was assessed by measuring the level of CARMA3 expression by QCPR. Levels were normalized to A549 cells transfected with a control vector and expressed as relative expression compared with control levels. Values are the mean of three samples ± SEM. Western blot of cell lysates from A549 cells transfected with WT CARMA3 and constructs containing sequences for shRNA against CARMA3. Blots were probed with an antibody to Myc. (B) CARMA3 RNA levels measured by QPCR and normalized to GAPDH in NHBE cells after transfection with a construct (shRNA48) containing an shRNA specific to CARMA3 or a control vector. Values are the mean of three samples ± SEM. This experiment was repeated two times. (C) TSLP and CCL20 RNA levels measured by QPCR and normalized to GAPDH in transfected NHBE cells at baseline and after incubation for 6 hours with 10 μM LPA. There are at least six samples in each group pooled from two experiments. *P < 0.05 by unpaired t test. (D) TSLP and CCL20 protein levels in culture supernatants measured by ELISA in transfected NHBE cells at baseline and after incubation for 6 hours with 10 μM LPA. There are at least three samples in each group pooled from two experiments. *P < 0.05 by unpaired t test.