Data on retinoic acid and reduced serum concentration induced differentiation of Neuro-2a neuroblastoma cells

Abstract

The present data describe the relative neuro-2a cellular differentiation induced by reducing serum concentration (0.1% FBS) in DMEM in the presence/absence of 20 μM retinoic acid (RA). Neurite outgrowth was observed within 24 h in DMEM supplemented with reduced serum and retinoic acid (GpIV). The CFSE based proliferation assay data signified cessation of neuro-2a cellular proliferation in GpIV. An increase in the number of cells arrested at G₀/G₁ phase was also evident in GpIV and DMEM supplemented with 0.1% FBS (GpIII). Moreover, GpIV cells had improved mRNA and protein expression of Rbfox3/NeuN and choline acetyltransferase (ChAT).

Keywords: Neuro-2a; Reduced serum; Retinoic acid.

Fig. 1

Phase contrast microscopy for morphological analysis. Neuro-2a cells were subjected to various culture conditions, namely DMEM + 10% FBS (GpI), DMEM + 10% FBS + 20 μM Retinoic acid (GpII), DMEM + 0.1% FBS (GpIII), DMEM + 0.1% FBS + 20 μM Retinoic acid (GpIV) and then photographed with Nikon-C100 at 20× magnification. Scale bar 5 μM. Data are represented as photomicrographs (A–D) and its analysis of neurite length (E), the branching level (F) and average cell density (cells/475 μm², G) in bar graphs. Values are mean ± S.E. P ≤ 0.05 (α, β, γ), P ≤ 0.01 (αα, ββ, γγ), P ≤ 0.001 (ααα, βββ, γγγ) were considered to be statistically significant. α, Compared to GpI; β, Compared to GpII; γ, Compared to GpIII. Additionally, mean values, S.E., ANOVA, and Tukey׳s multiple comparisons statistics (post hoc) are shown in tabular form below their respective graphs.

Fig. 2

Modulation in cell cycle and proliferation pattern of neuro-2a under varied serum conditions with presence/absence of retinoic acid. Neuro-2a cells were subjected to culture conditions, namely DMEM + 10% FBS (GpI), DMEM + 10% FBS + 20 μM Retinoic acid (GpII), DMEM + O.1% FBS (GpIII), DMEM + 0.1% FBS + 20 μM Retinoic acid (GpIV) for 24 h, processed as per respective protocols and then scanned using FACS Calibur™. Cell cycle data are represented as the histogram (A–D) and CFSE assay data is represented in bar graphs (E, F) showing mean fluorescence intensity values (MFI values) and cell counts (cells/9.6 cm²) respectively. Values are mean ± S.E. P ≤ 0.05 (α, β, γ), P ≤ 0.01 (αα, ββ, γγ), P ≤ 0.001 (ααα, βββ, γγγ) were considered to be statistically significant. α, Compared to GpI; β, Compared to GpII; γ, Compared to GpIII. Additionally, mean data values, S.E., ANOVA statistics and P. Values from Tukey׳s multiple comparisons (post hoc) are represented besides respective graphical representations.

Fig. 3

Protein and mRNA expression data for the neuronal marker of differentiation using various serum concentrations in the presence/absence of retinoic acid. A; Represents western blot of ChAT, Rbfox3, GAPDH. B, C; Relative quantification of ChAT and Rbfox3 w.r.t. GAPDH as the reference control. D, E; Real-time PCR data for ChAT and Rbfox3, analyzed by the 2^-ddCt method using SDHA as the reference gene. Values are mean ± S.E. P ≤ 0.05 (α, β, γ), P ≤ 0.01 (αα, ββ, γγ), P ≤ 0.001 (ααα, βββ, γγγ) were considered to be statistically significant. α, Compared to GpI; β, Compared to GpII; γ, Compared to GpIII. Additionally, mean values, S.E., ANOVA, and Tukey׳s multiple comparisons statistics are shown in tabular form below their respective graphs.