TSG101 Associates with PARP1 and is Essential for PARylation and DNA Damage-induced NF-κB Activation (2022)
Figure 6

Figure 6. Loss of TSG101 Sensitizes Cells to Apoptosis and Impairs DNA Repair

A: Survival curve assay. U2-OS cells were transfected with non-targeting or TSG101-targeting siRNAs. Cells were treated with increasing concentrations of etoposide (10, 25 and 50 µM). Relative numbers of viable cells in culture were measured using a fluorogenic, cell-permeant peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC) at 2, 3 or 4 days post-treatment. The data represent five biologically independent experiments. The heatmap displays the relative survival fractions of TSG101-targeted compared to non-targeting siRNA-transfected cells exposed to increasing etoposide concentration at each time point, 2, 3 or 4 days post treatment, respectively.

B: U2-OS cells were transfected with non-targeting (mock) or TSG101-directed siRNAs, kept untreated (UT) or irradiated (20 Gy) and analyzed 1 day later. Total RNA was extracted and the expression of BCL2L1 analyzed by RT-qPCR. BCL2L1 expression was normalized to the housekeeping genes ACTA1, RPL13A and TBP2.in three biologically independent experiments. The conditions were compared with an unpaired t-test (**p<0.01). Error bars represent mean ± SEM.

C: Cells were transfected with non-targeting or TSG101-targeting siRNAs. Immunofluorescence staining was performed with the indicated antibodies (red: cleaved caspase 3; green: γH2AX). Nuclei were stained with DAPI (blue). The image is a representative of 3 biologically independent experiments. Scale bar: 30 µm. See Fig EV6D for percentage of γH2AX positive cells as an evidence of IR-treatment.

D: Percentage of cleaved caspase 3 was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. Conditions were compared with an unpaired t-test (*p < 0.05, **p < 0.01). Error bars represent mean ± SEM.

E: γH2AX staining of TSG101-targeting or non-targeting siRNA transfected U2-OS cells reveals puncta-like foci at 2 or 7 days after irradiation. Scale bar: 30 µm.

F: The number of puncta-like γH2AX foci per cell was determined by blind counting of approximately 100 cells for each condition from 3 biologically independent experiments. Conditions were compared with an unpaired t-test (***p < 0.001, ****p < 0.0001). Error bars represent mean ± SD.

G: MDA-MB-231 and MDA-MB-436 cells were transfected with TSG101-targeting or non-targeting siRNAs. Cells were treated with vehicle (DMSO) or olaparib (10 µM) 96 hours prior to the analysis, as indicated. Results were obtained from five biologically independent experiments. Relative number of viable cells in culture was measured using GF-AFC). See Fig EV6G for brightfield images of cells expressing TSG101-targeting or non-targeting siRNAs. Conditions were compared with an unpaired t-test (ns, ****p < 0.0001). Error bars represent mean ± SD.

H: MDA-MB-436 cells were transfected with TSG101-targeting or non-targeting (mock) siRNAs. Cells were treated with vehicle (DMSO) or olaparib (10 µM) 96 hours prior to the analysis, as indicated. Results were obtained from three biologically independent experiments. Relative number of viable cells in culture was measured using GF-AFC). Conditions were compared with an unpaired t-test (ns, ***p<0.001, ****p < 0.0001). Error bars represent mean ± SD.

I: Analysis of breast cancer cell lines bearing damaging BRCA1/2 mutations in the DepMap database. The CERES score represents the gene effect, which is based on cell depletion assays of the CRISPR (Avana) public 22Q1 dataset. A CERES score lower than (-1) indicates a high likelihood that the gene of interest is essential in the given cell line. Correspondingly, a -1 CERES score was indicated with a discontinued red line. The median dependency of all breast cancer cell types to TSG101 expression was indicated with a discontinued black line.

J: BRCA1 deficient MEF cells were lentivirally transduced with two independent guide RNAs targeting mouse Tsg101 or with empty CRISPR. Cell viability was analyzed with crystal violet staining. CRISPR knock-out of Tsg101 in wild-type MEF cells did not cause cell death. Note that the figure is composed of two separate culture images. The knockout efficiency in wild-type cells was determined by immunoblotting (See Fig EV6E).