Figure 1.

Induction of neurite outgrowth by overexpression of PKCε. (A) SK-N-BE(2) neuroblastoma cells were transfected with expression vectors encoding full-length (FL) and RD from PKCδ, ε, η, and θ, fused to EGFP. Empty EGFP vector (−) was used as control. Cells were fixed 16 h after transfection and transfected cells with neurites longer than the length of two cell bodies were counted. Data (mean ± SEM, n = 2) are presented as percentage of transfected cells with long neurites. (B) Amount of EGFP fusion proteins in individual cells was analyzed with laser scanning cytometry. For each experiment, 50–150 cells were analyzed and the average of these cells was used as the observation value from that experiment. Data (mean ± SEM) are presented as arbitrary fluorescence intensity units from two separate experiments. (C) Cells expressing full-length PKCε or the PKCε RD fused to EGFP with fluorescence intensity of 350,000–550,000 U were scored for the presence of neurites. Twenty cells were scored in each experiment and data are presented as percentage of cells with neurites (mean ± SEM, five separate experiments) and arbitrary fluorescence intensity units (mean ± SEM, 100 different cells). SK-N-BE(2) cells were grown with or without 10% serum (D) or in serum-free medium with or without 16 nM TPA (E) for 16 h after transfection with vectors encoding EGFP (−), full-length PKCε (FL), or the PKCε RD (RD). Transfected cells were scored for neurites longer than two cell bodies and data, mean ± SEM, n = 3 (D) or 5 (E), are presented as percentage of transfected cells with neurites.