Functional validation of CR-specific lncRNAs In Vitro and Ex Vivo.
(A) Immunostaining of neural cells treated with control (CAG-control) or overexpression lentiviruses for CR-specific lncRNAs Ln-CR2 (CAG-Ln-CR2) and Ln-CR3 (CAG-Ln-CR3) after in vitro culture for 5 days. RELN (red), TUJ1 (cyan), or DAPI (blue) for CR neurons, neurons, and nucleus, respectively. Scale bar, 50 μm. (B-E) Overexpression of CR-specific lncRNAs Ln-CR2 and Ln-CR3 increased CR neuron number, promoted NSC differentiation and generated more neuron-like small cell clones. All the experiments were performed 4 times and were counted 8 randomly selected microscopy fields each time. Data represented mean ± SEM. p values indicated were calculated by Student’s t test (unpaired), *p<0.05. (F) Quantification of process branch depth, branch levels, total dendrites length and soma size of CR neurons after overexpression of CR-specific lncRNAs Ln-CR2 and Ln-CR3 compared to control. All the experiments were performed 3 times and were counted 8 randomly selected microscopy fields each time. Data represented mean ± SEM. p values indicated were calculated by Student’s t test (unpaired), *p<0.05. (G) Comparison of dendritic spine, filopodia and processes of cultured cortical wholemounts overexpressing Ln-CR1 (CAG-Ln-CR1), Ln-CR2 (CAG-Ln-CR2) and Ln-CR3 (CAG-Ln-CR3) to wholemounts expressing CAG-control. 10 days culture. Scale bar, 15 μm. (H) Quantification of CR neuron processes branch depth, branch levels and total dendrites length after overexpression of CR-specific lncRNAs, and compared to control group. All the experiments were performed 3 times and were counted 4 randomly selected microscopy fields each time. Data represent mean ± SEM. p values indicated were calculated by Student’s t test (unpaired), **p<0.01, *p<0.05. (I) Quantification of CR neuron molecular markers as Reln, Ebf2, and Calb2 at 72h after lentiviral transduction in the NSC culture assay comparing the effect of overexpression of CR-specific lncRNAs Ln-CR2 and Ln-CR3 to control. Data represented mean ± SEM (**p<0.01, *p<0.05, n>3, Student’s T test).
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