Figure 4.

PNP Inhibitors and dG Synergistically Induce Cell Death in Cells Lacking SAMHD1

(A–C) BMDMs were treated with the indicated doses of dG and forodesine. Viability was tested as in Figure 1A after 24 or 48 h.

(D) BMDMs treated for 24 h with dG and forodesine were fixed and stained with crystal violet. After washing, cell-associated dye was solubilized and quantified by absorbance at 570 nm. For each genotype, values from untreated control cells were set to 100%.

(E and F) BMDMs were treated for 8 h with dG and forodesine. Levels of PARP and cleaved PARP (E) or cleaved CASPASE 3 and SAMHD1 (F) in total cell extracts were determined by western blot. β-Actin served as a loading control. cld, cleaved.

(G–I) Jurkat cells were reconstituted with SAMHD1 as described in Figures 3J and 3K. Uninfected cells (NI) served as control.

(G and H) Cells were treated for 18 h with 10 μM dG and 1 μM forodesine. Cells were then stained with Annexin V and 7AAD and analyzed by flow cytometry. Representative FACS plots are shown in (G) and Annexin V⁺ 7AAD⁻ cells are quantified in (H).

(I) SAMDH1 levels in total cell extracts were determined by western blot. β-Actin served as a loading control.

(J) HeLa cells were infected with VLPs containing Vpx (VLP_vpx) or not (VLP_ctrl). After 6 h, cells were treated with 20 μM dG and 2 μM forodesine, and brightfield images were acquired after an additional 48 h. Scale bar represents 300 μm.

(K) BMDMs were treated with the indicated doses of dG and forodesine. Viability was tested as in Figure 1A after 24 h. Means from three biological replicates are shown ± SEM.

(L) Samhd1^−/− BMDMs were treated with the indicated doses of dG in the presence or absence of 1 μM forodesine. Cell viability was determined by CellTiter-Glo assay after 24 h. Data were normalized by setting the values for the lowest and highest dG concentrations to 100 and 0, respectively. Means from three biological replicates are shown ± SEM. Half maximal inhibitory concentration (IC₅₀ ) values were calculated from the non-linear regression curves shown on the graph.

(M and N) BMDMs were treated with the indicated doses of dG and homo-DFPP-DG (M) or 6C-DFPP-DG (N). Viability was tested as in Figure 1A after 24 h.

Data are representative of three independent experiments. In (A)–(D), (M), (N), and (H), dots represent BMDMs from individual mice and technical replicates, respectively. Mean ± SD is shown. The p values determined with two-way ANOVA are indicated. ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001