Fig. 4

Knockdown of nestin in Neuro-2a cells has no impact on RA-induced neurite outgrowth. (A) Neuro-2a cells grown in proliferation medium were transfected with either a nontargeting control (siCTL) or four different nestin-specific siRNAs (siNES#1, #3, #4, and #17). At 48 h after transfection, the cytoskeleton was prepared and processed for western blot analysis using antibodies against nestin and vimentin. Bands of interest were excised from different blots and grouped together. Full-length blots are presented in Supplementary File 3. (B) Quantification of nestin protein levels in control and nestin-depleted cells. Chemiluminescent signals were quantified using a Bio-Rad ChemiDoc imaging system and normalized to the vimentin level in control cells. The data are presented as the means ± SEMs (error bars) of three separate experiments. Each replicate has its own vimentin control. Statistical significance was determined using a two-tailed unpaired Student’s t test. **, p < 0,01; ***, p < 0,001. (C) Neurite length (mm) per cell-body cluster was measured every 4 h in control and nestin siRNA-transfected cells that were exposed to RA for 72 h. Data on graph represent the individual values obtained from five independent experiments at each time point with means connected. Multiple unpaired t tests were used for statistical analysis. ns, not significant compared with the control cells