Journal: Cancer Research Communications

Article Title: Schlafen 12 Modulation and Targeting in Acute Myeloid Leukemia

doi: 10.1158/2767-9764.CRC-25-0283

Figure Lengend Snippet: Effects of velcrin treatment on SLFN12 protein levels, PDE3A/B binding, and induction of cell death. A, HEL cells were treated with vehicle control (DMSO) or with the indicated doses of BAY 2666605 for 4 or 8 hours. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. GAPDH is shown as a loading control. B, SLFN12 mRNA expression of HEL cells after BAY 2666605 treatment at the indicated concentrations for 4 hours (left) or 8 hours (right). SLFN12 expression was assessed by qRT-PCR normalized to GAPDH and DMSO was used as a vehicle control (indicated by the dotted line). Data represent means ± SEM of three independent experiments, each conducted in triplicate. At each concentration, statistical significance was determined using a one-sample Student t test compared with 100%; ns, not significant. C, mRNA expression of respective genes in HEL cells transfected with a non-targeting siRNA control (indicated by the dotted line) and siRNA targeting PDE3A (left) or PDE3B (right). Data are expressed as the percentage of respective mRNA expression over non-targeting siRNA control (%) and represent means ± SEM of three independent experiments, each conducted in triplicate. Statistical significance was determined using a one-sample Student t test compared with 100%; ns, not significant, **, P ≤ 0.01. D, Immunoblot analysis of HEL cells. HEL cells were transfected with respective siRNA for 24 hours (see panel C ), followed by 8 hours of low (1 μmol/L) or high (10 μmol/L) doses of BAY 2666605, using DMSO as a vehicle control. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies ( E ). HEL (left) or U937 (right) cells were treated with vehicle control (DMSO) or with low (1 μmol/L) or high (10 μmol/L) concentrations of BAY 2666605 for 24 hours. Cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. F and G, Upon treatment with increasing doses of BAY 2666605 for 24 hours, HEL ( F ) and U937 ( G ) cells were stained with annexin V/propidium iodide and subjected to flow cytometry. The percentage of apoptotic cells for HEL ( F ) and U937 ( G ) cells treated with increasing BAY 2666605 concentrations is shown as mean ± SEM of three independent experiments. Statistical significance was determined using a one-way ANOVA, followed by a Dunnett multiple comparisons test to adjust for multiple comparisons for each dose vs. control; **, P ≤ 0.01; ****, P ≤ 0.0001. H and I, Co-immunoprecipitation analysis of SLFN12 interaction with ( H ) PDE3A in HEL cells or ( I ) PDE3B in U937 cells. HEL and U937 cells were treated with low (1 μmol/L) or high (10 μmol/L) concentrations of BAY 2666605 for 24 hours. DMSO was used as vehicle control. Cell lysates were used for immunoprecipitation with anti-PDE3A and anti-PDE3B antibodies or control rabbit immunoglobulin, as indicated. H, After probing for PDE3A, the membrane was stripped and reprobed with antibody for PDE3B. BAY, BAY 2666605; Cl casp-3, cleaved caspase-3; IP, immunoprecipitation; RIgG, rabbit immunoglobulin; WCL, whole-cell lysate.

Article Snippet: To immunoprecipitate PDE3A or PDE3B, cell lysates were incubated overnight at 4°C with either anti-PDE3A rabbit polyclonal antibody (1:100), anti-PDE3B rabbit polyclonal antibody (1:100), or rabbit IgG polyclonal antibody isotype control (Proteintech, cat. #30000-0-AP, RRID: AB_2819035) with rotation.

Techniques: Binding Assay, Control, SDS Page, Expressing, Quantitative RT-PCR, Concentration Assay, Transfection, Western Blot, Staining, Flow Cytometry, Immunoprecipitation, Membrane