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. 2024 Feb 9;43(6):904–930. doi: 10.1038/s44318-024-00044-1

Figure 4. Ubiquitin-dependent recruitment of NEMO to mitochondria is essential for NF-κB activation after MOMP.

Figure 4

(A) Timelapse of U2OS cells expressing GFP-NEMO. Cells were treated with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh. Mitochondria and nuclei are visualised using PkMito DeepRed and Hoechst, respectively. Cells were treated for 1 h with images taken every 10 min. Image is representative for three independent experiments. Scale bar is 20 μm. (B) U2OS cells expressing GFP-NEMO treated with treated with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh were quantified for mitochondrial localised GFP-NEMO puncta over time. Puncta are calculated using ImageJ/Fiji “trainable Weka segmentation plug-in”. The graph is representative of three biological repeats and shows the mean +/− s.e.m. (error bars) of five fields of view taken over time. (C) SVEC4-10 cells treated for 3 h with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh. Mitochondria were isolated using Dounce homogeniser and cellular fractions were probed with relevant antibodies. Mitochondrial localised NEMO was quantified normalising to mitochondrial content defined by HSP60 signal. (D) U2OS EMPTYCRISPR and BAX/BAKCRISPR cells expressing GFP-NEMO were treated for 3 h with 10 μΜ ABT-737, 2 μΜ S63845 and 20 μΜ Q-VD-OPh. Cells were immunostained for mitochondrial COXIV. Scale bar is 20 μm. Images are maximum projections of Z-stacks and are representative for three independent experiments. (E) Quantification of (D) showing the percentage of cells with mitochondrial localised GFP-NEMO puncta. (F) U2OS cells expressing GFP-NEMO, GFP-NEMOD311N or GFP-NEMOΔZF were treated for 3 h with 10 μΜ ABT-737, 2 μΜ S63845 and 20 μΜ Q-VD-OPh. Cells were immunostained for mitochondrial TOM20 and DAPI. Scale bar is 20 μm and images are representative for three independent experiments. (G) Quantification of (F) showing the percentage of cells with mitochondrial translocation of GFP-NEMO. (H) Parental SVEC4-10 cells and SVEC4-10 cells expressing GFP-NEMO, GFP-NEMOD311N or GFP-NEMOΔZF were treated for 1 h with 10 μΜ ABT-737, 10 μΜ S63845 and 30 μΜ Q-VD-OPh. Cells were immunostained for p65 and DAPI. Scale bar is 50 μm and images are representative for three independent experiments. (I) Quantification of (H) showing the GFP+ cells with nuclear translocation of p65. Data information: (E, G, I) graphs display mean values ± s.e.m. (error bars) of n  =  3 independent experiments. Statistics are performed using two-way ANOVA with Tukey correction. **P < 0.01, ***P < 0.001. ****P < 0.0001. Source data are available online for this figure.