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. Author manuscript; available in PMC: 2017 Jun 15.
Published in final edited form as: Neuron. 2016 Jun 2;90(6):1174–1188. doi: 10.1016/j.neuron.2016.05.005

Figure 5. LncND knock-down leads to reduced cell proliferation and increased neuronal differentiation of SHSY5Y cells.

Figure 5

(A) H9 cells were differentiated into neurons using dual SMAD inhibitors. RNA from cells was isolated at different time points. Left: Expression of LncND, NOTCH-1 and NOTCH-2 was analyzed by RT-qPCR at different time points during differentiation of H9 cells. Right: miRNA expression during neuronal differentiation of H9 cells was measured using Taqman probes and the values were normalized to U6. (B) Representative differentially expressed genes involved in Notch signaling as identified using Cufflinks 2.1.1 after LncND knock-down (n=3, q-value <= 0.05). (C) Significant correlation and overlap between genes differentially expressed or unchanged after LncND knock-down and miR-143-3p overexpression (p<0.0001, Chi-square test). The numbers in the box represent the number of genes overlapping in each category. (D) Gene ontology (GO) term analysis of overlapping down-regulated genes between si-LncND and miR-143-3p overexpression (left), downregulated genes after LncND knock-down (middle) and miR-143-3p overexpression (right). (E) Immunostaining of SHSY5Y cells treated with control siRNA or siRNAs for LncND and NOTCH-1. Cells were stained for Tau (Green), MAP2 (Red) or Hoechst (Blue) for axons, dendrites and nucleus, respectively, and analyzed by fluorescent microscopy. Scale: 100 μm. (F) Bright-field image of SHSY5Y cells treated with either control siRNA, si-LncND, si-NOTCH-1, control miRNA mimic or miR-143-3p mimic. Knock-down of LncND and NOTCH-1 shows neurite-like processes after 72 h of transfection. Scale: 100 μm. (G) BrdU proliferation assay. SHSY5Y cells were treated with 100 nM of either control siRNA or siRNA for LncND or NOTCH-1 for 72 h. Cells stained with BrdU and 7-AAD were analyzed by flow cytometry. Knock-down of LncND and NOTCH-1 significantly reduced the proliferation of SHSY5Y cells. All the experiments were performed in triplicates. Error bars represent standard deviation. p-values indicated were calculated by Student’s t-test (unpaired). ***: p<0.001, **: p<0.01.