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. 2022 Sep 20;41(21):e110372. doi: 10.15252/embj.2021110372

Figure 2. TSG101 is essential for the DNA damage‐induced NF‐κB pathway.

Figure 2

  • A
    IR‐induced NF‐ĸB pathway activation was analyzed via luciferase activity of HEK‐Luc cells, transfected with the indicated siRNAs. The indicated cells were irradiated (20 Gy, 8 h before analysis). A late time point was used for efficient luciferase expression. Luciferase activity was normalized to the viable cell number (TOX fluorescence). The data represent three biological and five technical replicates. Error bars represent mean ± SD. For knockdown efficiency, see Fig EV2A. The conditions were compared with an ordinary one‐way ANOVA (****P < 0.0001).
  • B
    U2‐OS cells were transfected with the indicated siRNAs. The indicated cells were treated with etoposide (50 μM, 90 min. Before analysis). Whole‐cell extracts were immunoblotted with the indicated antibodies. The result is representative of three biological replicates.
  • C
    U2‐OS cells were transfected with the indicated siRNAs and exposed to irradiation (20 Gy, 90 min. Before analysis). The expression of indicated genes was analyzed using RT–qPCR. The mRNA expression was normalized to the expression of the housekeeping genes ACTA1, RPL13A, and TBP2. Data represent three biologically independent experiments +/− SEM. The conditions were compared with an ordinary one‐way ANOVA (*P < 0.05; **P < 0.01, ***P < 0.001).
  • D
    U2‐OS cells were transfected with nontargeting (mock) or TSG101‐targeting siRNAs and irradiated (20 Gy) for 90 min. Before analysis. Lysates were analyzed by EMSA and results of fold changes in densitometric measurements of the three independent experiments were compared with an ordinary one‐way ANOVA (*P < 0.05) in the right panel. Error bars represent mean ± SD.