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. 2021 Apr 22;40(12):e107270. doi: 10.15252/embj.2020107270

Figure EV2. Characterization of NEAT1 mutants with deletions in the NEAT1_2 5′ terminal regions.

Figure EV2

  1. Quantitation of the relative expression levels of NEAT1_1 and NEAT1_2 by RT–qPCR in HAP1 WT, Δ5′, and Δ5′/ΔPAS cells treated with MG132 (5 μM for 6 h). Data are represented as mean ± SD (n = 3).
  2. (upper) Detection of NEAT1_2 by smFISH (magenta) in WT, Δ0–0.8 kb, Δ5′, and Δ5′/ΔPAS kb cells treated with MG132 (5 μM for 6 h). Nuclei were stained with DAPI. Scale bar, 10 nm. (lower) Quantitation of area and sum intensity per paraspeckle in each cell line (WT: n = 839, Δ0–0.8 kb: n = 845, Δ5′: n = 535, and Δ5′/ΔPAS: n = 652). (*P = 0.0420, ****P < 0.0001, compared with WT: Kruskal–Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top).
  3. Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends to the core and shell or the shell in WT, Δ0–0.8 kb, and Δ5′ cells treated with MG132 (5 μM for 6 h) by SRM analyses (WT: n = 167, Δ0–0.8 kb: n = 77, Δ5′: n = 48).
  4. (upper panels) EM observations of the paraspeckles in MG132‐treated (5 μM for 17 h) WT and Δ5′ cells using the NEAT1_D2 probe. Scale bar, 100 nm. (bottom, left) Graph showing the proportion of localization (three layers: outer, middle, and inner layers [black circles indicate the boundaries]) of NEAT1_D2 probes (WT: 307 gold particles, Δ5′: 167 gold particles) within the paraspeckles in HAP1 Δ5′ cells. (bottom, right) Graph showing the proportion of localization of NEAT1_D2 probes in each paraspeckle in WT (n = 22) and Δ5′ cells (n = 15). (***P = 0.0001, ****P < 0.0001, compared with WT: Mann–Whitney test (two‐tailed)). Each box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top).
  5. Quantitation of the relative expression levels of NEAT1_1 and NEAT1_2 by RT–qPCR in HAP1 WT and Δ0–2.8 kb cells. Data are represented as mean ± SD (n = 3).
  6. Detection of the paraspeckles by smFISH in WT and Δ0–2.8 kb cells. Scale bar, 10 nm.
  7. (left) The paraspeckles in Δ5′/ΔPAS cells treated with MG132 treatment (5 μM for 6 h) detected with SRM by NEAT1_2k (green) and 3′ (magenta) FISH probes. Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in Δ5′/ΔPAS cells (n = 31).

Source data are available online for this figure.