Fig. 3
Nestin expression in DLK-depleted Neuro-2a cells. (A) Relative level of nestin mRNA in control and DLK-depleted Neuro-2a cells incubated without or with 20 µM RA for 24 h. The mRNA level of nestin was analyzed by quantitative RT‒PCR, normalized to three housekeeping genes (Psmc4, Pum1, and Txnl4b) and calculated with the ΔΔCT method. The data represent the mean ± SEM of three separate experiments. Statistical significance was determined using a two-tailed unpaired Student’s t test. *, p < 0,05, relative to control cells not exposed to RA. (B) Representative Western blots showing the levels of nestin, DLK and vimentin in control and DLK-depleted Neuro-2a cells incubated without (-) or with (+) 20 µM RA for 24 h. Bands of interest were excised from different blots and grouped together. Full-length blots are presented in Supplementary File 2. (C) Quantification of nestin protein levels in control and DLK-depleted cells. Chemiluminescent signals were quantified using a Bio-Rad ChemiDoc imaging system and normalized to the vimentin level in control cells not exposed to RA. The data are presented as the means ± SEMs (error bars) of two or three separate experiments. Each replicate has its own vimentin control. Statistical significance was determined using a two-tailed unpaired Student’s t test. *, p < 0,05; ****, p < 0,0001, relative to control cells not exposed to RA