Panel A shows four histograms representing FACS analysis of p53-deficient MEFs. The x-axis represents cell count, and the y-axis represents DNA content. The histograms are labeled Asy, plus Noc, plus Release [0 hours, no drug], plus Release [2 hours, no drug], and plus Release [2 hours, Chx], showing the distribution of cells in G1, S, and G2/M phases. Panel B is a vertical bar graph showing qRT-PCR analysis of normalized Malat1 RNA levels in MEFs. The x-axis labels conditions (Asy, Release [0 hours], Release [2 hours]) with and without Chx treatment, and the y-axis represents RNA levels. Panel C is a vertical bar graph showing qRT-PCR analysis of Neat1_2 RNA levels in MEFs. The x-axis labels conditions (Asy, Release [0 hours], Release [2 hours]) with and without Chx treatment, and the y-axis represents RNA levels. Panel D is a vertical bar graph showing qRT-PCR analysis of NEAT1_2 RNA levels in RPE cells. The x-axis labels conditions (Asy, Release [0 hours], Release [2 hours]) with and without Chx treatment, and the y-axis represents RNA levels.
Supporting data for translation inhibition studies, related to Fig. 3. (A) FACS analysis of p53-deficient MEFs treated as described in Fig. 3 A. Data confirm enrichment of G2/M cells in samples treated for 4 h with 50 ng/ml Noc and entry into G1 of cells released from Noc in the absence or presence of 50 μg/ml Chx for 2 h. (B) qRT-PCR analysis of normalized Malat1 RNA levels in samples, described in Fig. 3 B, using a primer set specific to region D, excludes production of truncated Malat1. (C and D) qRT-PCR analysis of normalized Neat1_2 (C) or NEAT1_2 (D) RNA levels in murine and human cells, described in Fig. 3, B and C, respectively, shows that Neat1_2 and NEAT1_2 are not subject to the same translation-dependent regulatory mechanism as Malat1/MALAT1. In this figure, bar graphs show individual values and mean ± SD of n ≥ 3 technical (B) and biological (C and D) replicates. Unpaired t test (B) and paired t test (C and D), *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant.