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

Figure EV6. Efficient apoptosis protection and DNA repair following DNA double strand break generation requires TSG101.

Figure EV6

  • A
    U2‐OS cells were lentivirally transduced with two independent guide RNAs targeting the TSG101 locus. Due to the lethal side effects of TSG101 deletion on long‐term proliferation, bulk cells were used instead of the clonally expanded cells. DNA damage was induced by irradiation (20 Gy, 3 or 9 h before analysis) in wild‐type or TSG101 CRISPR knockout bulk cells. Total mRNA was extracted and expression of NUAK, PTX3, and PUMA was analyzed with RT–qPCR. The mRNA expression was normalized to the housekeeping genes ACTA1, RPL13A, and TBP2. Data are from three biologically independent experiments. The conditions were compared with an ordinary one‐way ANOVA (***P < 0.001; ****P < 0.0001). Error bars represent mean ± SEM.
  • B
    The efficiency of TSG101 CRISPR in the cells used in A is shown with immunoblotting. Whole‐cell extracts were immunoblotted with antibodies against TSG101. Vinculin was used as a loading control.
  • C
    Irradiation‐induced cleaved caspase‐3 activation in nontargeting gRNA or Tsg101‐targeting gRNA transduced MEF cells was measured using a colorimetric assay. For knockout efficiencies see Fig EV6E. Bulk cells from Tsg101 guide‐1 were used in this assay. Results were obtained from four biologically independent experiments. The conditions were compared with an unpaired t‐test (****P < 0.0001). Error bars represent mean ± SD.
  • D
    Percentage of γH2AX positive staining from Fig 6D is shown. Results were obtained from blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. The conditions were compared with an unpaired t‐test (****P < 0.0001). Error bars represent mean ± SD.
  • E
    Wild‐type MEF cells from Fig 6J (control or Tsg101 guide RNA transduced cells) were analyzed for Tsg101 CRSIPR/Cas9 knockout efficiency. Whole‐cell extracts were obtained from these cells and immunoblotted with the indicated antibodies. Vinculin was used as a loading control.
  • F
    U2‐OS cells were transfected with nontargeting (mock) or two different TSG101‐targeting siRNAs. Nonirradiated cells (samples 1–3) were compared with senescent cells 7 days postirradiation (samples 4–6). Total mRNA was extracted and expression of CXCL8, IL‐6, and TSG101 was analyzed by RT–qPCR. The mRNA expression of these genes was normalized to the three housekeeping genes ACTA1, RPL13A, and TBP2. Data are from three biologically independent experiments. Conditions were compared with an ordinary one‐way ANOVA (ns, P > 0.05; *P < 0.05; **P < 0.01, ***P < 0.001). The conditions were compared with an unpaired t‐test (****P < 0.0001). Error bars represent mean ± SEM.
  • G
    Representative brightfield images of TSG101‐targeting or nontargeting siRNA‐transfected BRCA1 wild‐type and mutant MDA‐MB 231 and 436 breast cancer cells are shown.