Fig. 4: Overexpression of hnRNPM up-regulates NEAT1 expression and induces neurotoxicity.

a NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the cell lysates were subjected to immunoblotting (IB) and dot blotting analysis using indicated antibodies. Intensities of immunodetected signals of endogenous hnRNPM were densitometrically examined with an ImageJ software. b, c NSC-34 cells were infected with adenovirus encoding FLAG-PR100 at MOIs of 0–800. To keep the constant total MOIs of adenoviruses, appropriate MOIs of LacZ-encoding adenovirus were added for each infection. At 48 h after the infection, the quantitative real-time PCR analysis of hnRNPM was performed (b). The cell lysates were subjected to dot blotting analysis using indicated antibodies (c). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. d, e NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed (d). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies (e). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test. f, g NSC-34 cells were transfected with 0.2 μg/well of the pCMV-GFP or -mouse NEAT1_2 on 6-well plate. After the transfection, NSC-34 cells were infected with adenovirus encoding LacZ or hnRNPM at an MOI of 400. At 48 h after the infection, the quantitative real-time PCR analysis of NEAT1 was performed (f). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies (g). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Dunnett’s multi comparisons test. h, i NSC-34 cells were infected with adenovirus encoding LacZ, hnRNPM, MATR3, or hnRNPQ at an MOI of 400. At 48 h after the infection, the cell viability was detected by WST-8 assay (h). The cell lysates were subjected to immunoblotting (IB) using indicated antibodies (i). The data are presented as means ± SD (N = 3). Statistical analysis was performed by one-way ANOVA followed by the Tukey’s multi comparisons test