Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet: dG Treatment Kills Samhd1 −/− Cells by Apoptosis (A) BMDMs were treated with 0.5 mM of each dN for 24 h and stained with Annexin V and 7AAD. AnnexinV + 7AAD − and AnnexinV + 7AAD + cells were quantified by flow cytometry. Data from triplicate measurements are shown with mean ± SD. p values determined with two-way ANOVA are indicated. (B) Caspase activity was assessed in BMDMs 6 h after treatment with the indicated doses of dG, or Staurosporine as control, using the Caspase 3/7 Glo assay. For each genotype, values from untreated control cells were set to 100. Data from triplicate measurements are shown with mean ± SD. The p value determined with an unpaired t test is indicated. (C and D) Live-cell imaging of Samhd1 −/− BMDMs treated with 0.5 mM dG. Alexa 488-labeled Annexin V and propidium iodide (PI) were added to the culture medium to visualize early apoptotic cells and cells that lost membrane integrity, respectively. (C) Representative images of a Samhd1 −/− cell treated with dG. Numbers show the time after dG exposure (h:min). (D) Enumeration of AnnexinV + PI + cells after 24 h of treatment with or without 0.5 mM dG. Six images per condition were analyzed, and means ± SEM are shown. The p value determined with an unpaired t test is indicated. (E and F) BMDMs were treated with 0.5 mM dG or 1 μg/mL cycloheximide (CHX, added to WT cells in F) for 8 hours. (E) Levels of the indicated proteins in total cell extracts were determined by western blot. (F) Cells were fractionated into cytosol and a pellet containing organelles. Levels of the indicated proteins were determined by western blot. β-Actin served as a loading control. (G) WT and Samhd1 −/− BMDMs were co-cultured at the indicated ratios. Cell viability was determined as in Figure 1 A 24 h after treatment with 0.5 mM dG. Data from triplicate measurements are shown with mean ± SD. (A)–(G) are representative of at least three independent experiments. ns, p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S1 .

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Staining, Flow Cytometry, Activity Assay, Control, Glo Assay, Live Cell Imaging, Labeling, Membrane, Western Blot, Cell Culture

Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet: dG Induces the Death of Cancer Cell Lines (A) HeLa cells were infected with VLPs containing Vpx (VLP vpx ) or not (VLP ctrl ). After 24 h, cells were treated with 0.5 mM of each dN for an additional 24 h. Cell viability was assessed as in Figure 1 A. (B and C) HeLa (B) and MDA-MB231 (C) cells were left uninfected (NI) or were infected with VLPs containing Vpx (VLP vpx ) or not (VLP ctrl ). After 24 h, cells were treated with 0.5 mM dG, and brightfield images were acquired after an additional 10–12 h. Scale bars represent 300 μm. (D) MDA-MB231 cells were treated as in (C), and confluency was monitored after dG addition using a live-cell imaging system in the incubator (Incucyte). The mean of 9 measurements ± SD is shown. (E–G) Wild-type and Samhd1 −/− B16F10 cells were treated with dG as indicated for 20 h. (E and F) Cells were then stained with Annexin V and 7AAD and analyzed by flow cytometry. Representative fluorescence-activated cell sorting (FACS) plots are shown in (E), and Annexin V + 7AAD − and Annexin V + 7AAD + cells were quantified in (F). (G) Confluency was determined as in (D). (H) Jurkat cells were treated for 20 h with dG as indicated or with 25 μM etoposide. Cell viability was determined as in Figure 1 A. (I) Jurkat cells were treated with dG as indicated for 20 h and then seeded in semi-solid medium containing dG. After 13 days, cell colonies were counted, and the number colonies per field of view are shown. (J and K) Jurkat cells were reconstituted with hemagglutinin (HA)-tagged wild-type or K11A mutant SAMHD1 using a lentivector. Uninfected cells (NI) served as control. (J) Cells were then treated with dG for 48 h. Cell viability was determined as in Figure 1 A. (K) SAMHD1 levels in total cell extracts were determined by western blot. β-Actin served as a loading control. (A), (D)–(H), and (J)–(K) are representative of three independent experiments and (B) and (C) of two experiments. In (A), (F)–(H), and (J), dots represent technical triplicates and means ± SD are shown. In (I), data from two independent experiments were pooled, and dots represent the mean of technical duplicates per experiment. The p values determined with two-way ANOVA are indicated. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also and .

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Infection, Live Cell Imaging, Staining, Flow Cytometry, Fluorescence, FACS, Mutagenesis, Control, Western Blot

Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet: 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 1 A 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 J 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 1 A 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 (IC 50 ) 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 1 A 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

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Staining, Control, Western Blot, Flow Cytometry, Infection, Glo Assay, Concentration Assay

Journal: Cell Reports

Article Title: SAMHD1 Limits the Efficacy of Forodesine in Leukemia by Protecting Cells against the Cytotoxicity of dGTP

doi: 10.1016/j.celrep.2020.107640

Figure Lengend Snippet:

Article Snippet: FITC annexinV / 7AAD detection kit , Biolegend , Cat# 640922.

Techniques: Virus, Recombinant, Staining, Blocking Assay, Cell Viability Assay, Plasmid Preparation, Software

Journal: iScience

Article Title: A series of xanthenes inhibiting Rad6 function and Rad6-Rad18 interaction in the PCNA ubiquitination cascade

doi: 10.1016/j.isci.2022.104053

Figure Lengend Snippet:

Article Snippet: AlphaScreen FLAG (M2) detection kit , PerkinElmer , Cat#6760613.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Modification, Amplified Luminescent Proximity Homogenous Assay, Plasmid Preparation, Software