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. 2019 Jul 4;38(15):e101341. doi: 10.15252/embj.2018101341

Figure 4. Compartmentalization of DRiPs and polyUb proteins is catalyzed by the self‐assembly of PML into nuclear bodies.

Figure 4

  1. HeLa cells were lipofected with non‐targeting siRNA control or a specific siRNA against PML. 72 h post‐transfection, total proteins were extracted and expression levels of PML were analyzed by immunoblotting. TUBA4A was used as loading control.
  2. HeLa cells were lipofected with non‐targeting siRNA control or a specific siRNA against PML. 72 h post‐transfection, cells were treated with MG132 (10 μM) and OP‐puro (25 μM) for 4 h. Left panel: Automated quantitation of the number of PML‐NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 334–364; P < 10−10. Right panel: automated quantitation of the number of DRiP foci/nucleus. Cell number analyzed: 334–364; P < 10−10. Automated DRiP segmentation is based on azide signal.
  3. DRiPs and polyUb (FK1) protein distribution in cells treated as described in (A). Automated DRiP segmentation is based on azide signal. Quantitation of the number of DRiP foci enriched for polyUb (> 2) is shown; n = 590–2,114; statistical significance via 2‐tailed Student's t‐test; P < 10−10. Scale bars: 10 μm.
  4. Immunoblotting on protein extracts from HeLa cells lipofected with non‐targeting siRNA control or a specific siRNA against Ubc9. A dilution curve of the control sample is shown. TUBA4A was used as loading control.
  5. Quantitation of the number of PML‐NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192–430; P < 10−10. Automated PML‐NB segmentation is based on PML signal.
  6. Cells lipofected for 72 h with non‐targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 (10 μM) and OP‐puro (25 μM) for 4 h. Automated quantitation of the number of PML‐NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 151–225; P < 10−10 (see panel G).
  7. Representative pictures showing that Ubc9‐depleted cells cannot efficiently induce the self‐assembly of PML‐NBs upon treatment with MG132 (10 μM) and OP‐puro (25 μM) for 4 h. Scale bars: 10 μm.