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

Figure EV5. Control of PARylation by TSG101 is ESCRT complex‐independent.

Figure EV5

  • A
    U2‐OS cells were transfected with nontargeting (mock) or TSG101‐targeting siRNAs. Indirect immunofluorescence visualizes TSG101 (red) and PARP1 (green). DAPI staining shows the nuclei in blue. Scale bar is 10 μm. The image is representative of three biologically independent experiments.
  • B
    Z‐scores of the mean values of the depicted ESCRT complex members and accessory proteins (ESCRT‐0: orange, ESCRT‐I: green, ESCRT‐II: brown, ESCRT‐III: purple, and ESCRT‐IV: red) from Dataset EV1. Subsequently analyzed members CHMP4A, PTPN23, TSG101, and UBAP1 are highlighted with red circles.
  • C
    U2‐OS cells were transfected with nontargeting or UBAP1‐targeting siRNAs. Whole‐cell extracts were immunoblotted with antibodies against the indicated proteins. Vinculin was used as a loading control.
  • D
    Total RNA was extracted from cells analyzed in B and converted to cDNA. The relative mRNA expression of UBAP1 was normalized to two housekeeping genes, RPL13A, and TBP2. Data are from six biologically independent experiments. Conditions were compared with an unpaired t‐test with Welch's correction (****P < 0.0001). Error bars represent mean ± SD.
  • E
    U2‐OS cells were transfected with nontargeting siRNAs (mock) or with siRNAs directed against CHMP4A, PTPN23, or TSG101 and irradiated (20 Gy) or not, as indicated. PAR levels and expression of indicated proteins were monitored by western blotting with the respective agents and antibodies. LDH‐A served as a loading control.