Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 4

Research Article

Cell Line-Dependent Variability of Coordinate Expression of p75NTR and CRABP1 and Modulation of Effects of Fenretinide on Neuroblastoma Cells

Figure 4

p75NTR, CRABP1, and response to fenretinide in SK-N-AS neuroblastoma cells. (a) Western blot of lysates from SK-N-AS cells in their native state (Wildtype), stably transfected with empty vector (Vector), a scrambled construct (Scr), or shRNA for p75NTR (clones NC-1 and NC-2). Knockdown of p75NTR is more efficient in NC-1 cells than in NC-2 cells. Blotting for β-actin serves as a loading control. The graph below the blot depicts the mean optical density and SEM for 3 blots performed. Open bars, p75NTR; solid bars, CRABP1 (b) Alamar blue assay of SK-N-AS cells treated as in (a) after treatment with fenretinide ( for each point; results for NC-1 (×, solid line; concentration required for growth inhibition by 50% [GI50] = 15) differ from those for Wildtype (gray triangle; GI50 = 7.5), Vector (□, solid line; GI50 = 5), and Scr (□, dashed line; GI50 = 6) with and from those for NC-2 (×, dashed line; GI50 = 10) with ; Student’s -test). Note that while NC-1 cells are more resistant to fenretinide than empty vector- and scrambled construct-transfected cells, NC-2 cells are not. Western blot (c) and Alamar blue assay (d) of SK-N-AS cells transfected with an expression construct for CRABP1 (CRABP1 (black triangle)) or an empty vector (Vector (□)) or examined in their native state (Wildtype (gray triangle)). Results for Wildtype differ from those for Vector (; ), indicating that transfection with an empty construct changes the fenretinide sensitivity of the cells; CRABP1 cells differ from Vector cells (; ) at 4 and 8 μM fenretinide. The top band detected with anti-CRABP1 antibody in the CRABP1 lane is from Flag-CRABP1, the expression of which is induced. The graph below the blot depicts the mean optical density and SEM for 3 blots performed. Expression of p75NTR does not change significantly with induction of altered total expression of CRABP1 (Flag-CRABP1 + CRABP1). α-Tubulin is used as a loading control for Western blotting. Open bars, p75NTR; solid bars, CRABP1.