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

Figure EV1. Characterization of NEAT1 mutants with deletions in the NEAT1_2 3′ terminal regions.

Figure EV1

  1. (left) SRM images of the paraspeckles in HAP1 WT cells detected by NEAT1_5′ (green) and NEAT1_3′ (magenta) FISH probes with MG132 treatment (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends to the core and shell or the shell in WT cells (n = 167).
  2. Graph showing the proportion of paraspeckles with localization of the NEAT1 5′ ends to the core and shell or the shell in WT, Δ3′, Δ16.6–20.2 kb, and Δ20.2–22.6 kb cells treated with MG132 (5 μM for 6 h; WT: n = 115, Δ3′: n = 21, Δ16.6–20.2 kb: n = 161, and Δ20.2–22.6 kb: n = 89).
  3. (left) Detection of NEAT1_2 by single‐molecule FISH (smFISH; magenta) in HAP1 WT, Δ3′, Δ16.6–20.2 kb, and Δ20.2–22.6 kb cells treated with MG132 (5 μM for 6 h). Nuclei were stained with DAPI. Scale bar, 10 nm. (right) Quantitation of area and sum intensity per paraspeckle in each cell line (WT: n = 407, Δ3′: n = 314, Δ16.6–20.2 kb: n = 302, and Δ20.2–22.6 kb: n = 609). (***P = 0.0001, ****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).
  4. (left) EM observations of the paraspeckles in MG132‐treated (5 μM for 17 h) HAP1 Δ3′ cells using NEAT1_5′ probe. Scale bar, 100 nm. (middle) Graph showing the proportion of the localization of NEAT1_5′ probes (838 gold particles) within the paraspeckles in Δ3′ cells. (right) Graph showing the proportion of localization of NEAT1_5′ probes in each paraspeckle in Δ3′ cells (n = 21). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top).
  5. (left) EM observation of the paraspeckles in MG132‐treated (5 μM for 17 h) HAP1 WT cells using NEAT1_5′ probe. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (601 gold particles) within the paraspeckles in WT cells. (right) Graph showing the proportion of localization of NEAT1_5′ probes in each paraspeckle in WT cells (n = 16). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top).
  6. (left) EM observation of the paraspeckles in MG132‐treated (5 μM for 17 h) HAP1 WT cells using the NEAT1_D2 probe. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_D2 probes (461 gold particles) within the paraspeckles in WT cells. (right) Graph showing the proportion of localization of NEAT1_D2 probes in each paraspeckle in WT cells (n = 24). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and 10–90 percentiles (whisker bottom to top).
  7. Quantitation of the relative expression levels of NEAT1_1 and NEAT1_2 by RT–qPCR in HAP1 WT, Δ16.6–20.2 kb, and Δ20.2–22.6 kb cells treated with MG132 (5 μM for 6 h). Data are represented as mean ± SD (n = 3).
  8. SRM images of the paraspeckles in MG132‐treated (5 μM for 6 h) WT cells detected by NEAT1_5′ (green) and NEAT1_19k (magenta) FISH probes. Scale bar, 500 nm.
  9. Graph showing the proportion of paraspeckles with localization of the NEAT1_19k probes to the core and shell or the core in WT cells treated with MG132 (5 μM for 6 h; n = 103).

Source data are available online for this figure.