Fig 1: Inhibition of FOXO1 abolished enhanced sensitivity to venetoclax of AGK knockdown. A-B. The mRNA and protein expression of BCL-2 were measured in SU-DHL4 shRNA AGK#1 cells upon treatment of AS1842856 for 24 h. The band intensities were quantified from three independent experiments. C. Cell viability of SU-DHL4 shRNA AGK#1 cells with or without AS1842856 treatment was determined. The data showed mean ± SEM from three independent experiments with duplicates. D-E. NCG mice were implanted with shRNA control cells or shRNA AGK#1 cells treated with venetoclax or venetoclax plus AS1842856. At day 10 after i.p administration, mice were sacrificed. Tumor volume change curves were recorded (D) and tumor weights (E) were presented (n ≥ 5, each group). F-G. The levels of BCL-2 were measured (E) and quantified (F) in the tumor tissues isolated from shRNA AGK groups treated with venetoclax alone and co-treated with venetoclax and AS1842856 (n = 4, each group). H-I. Immunohistochemistry staining of BCL-2 in the four groups as shown, and the percentages of positive areas of BCL-2 were quantified. J-K. H&E staining and cleaved-caspase3 and TUNEL staining of the four groups as shown, and the percentages of positive areas of cleaved-caspase3 and TUNEL were quantified. Scale bar, 100 μm. Data were expressed as mean ± SEM and analyzed for statistical significance by One-way ANOVA (A, C, E, I, K) or t-test (G). (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).
Fig 2: AGK expression is inversely associate with the sensitivity to venetoclax in DLBCL cells. A. AGK protein expression was determined using immunoblotting in SU-DHL2, OCI-LY1, TMD8, SU-DHL4, SU-DHL6, SU-DHL10, and SU-DHL16 DLBCL cell lines. B. The band intensities were quantified from three independent experiments. C-D. The IC50 of seven DLBCL cell lines to venetoclax was calculated with log (inhibitor) vs normalized response. E. The AGK expression and IC50 of venetoclax in seven DLBCL cell lines were presented by dual Y axis graph. The AGK/Actin ration in SU-DHL10 cells was set as 1. Data were expressed as mean ± SEM. The statistical significance was determined by One-way ANOVA (B, D).
Fig 3: The expression of AGK is inversely associated with BCL-2 expression in DLBCL patients. A. H&E staining, BCL-2, AGK and FOXO1 staining of BCL-2low and BCL-2high DLBCL tissue samples. n = 33, scale bar, 100 μm or 25 μm. Black arrow: cytoplasmic FOXO1; Red arrow: nuclear FOXO1. B. Quantitation of the percentages of AGK positive cell in BCL-2low (n = 17) and BCL-2high (n = 16) DLBCL paraffin sections. C. Quantitation of the ratios of nuclear and cytoplasmic FOXO1 in BCL-2low (n = 15) and BCL-2high (n = 14) DLBCL paraffin sections. D. Spearman correlation analysis of the ratios of nuclear versus cytoplasmic FOXO1 with the expression of AGK. E. Schematic figure of the regulation of AGK on venetoclax sensitivity. In DLBCL cells, AGK phosphorylates and inactivates PTEN, leading to enhanced phosphorylation of AKT and FOXO1 and reduced FOXO1 nuclear translocation and its target gene BCL-2 expression. AGK knockdown could reverse the effect of AGK-PTEN-FOXO1-BCL-2 axis, upregulate the expression of BCL-2 and increase the sensibility to venetoclax-induced apoptosis. Ibrutinib inhibits the expression of AGK in DLBCL cells. Data were expressed as mean ± SEM and analyzed for statistical significance by t test (B, D). (**P < 0.01; ***P < 0.001; ns, not significant).
Fig 4: AGK inhibits BCL-2 expression by phosphorylation PTEN and subsequent AKT-FOXO1 signaling. A. The levels of AGK and BCL-2 were determined in SU-DHL2, OCI-LY1, TMD8, SU-DHL4, SU-DHL6, SU-DHL10, and SU-DHL16 DLBCL cell lines. B. Spearman correlation analysis of AGK and BCL-2 expression in DLBCL cell lines. C. AGK and BCL-2 expression was measured with immunoblotting and BCL-2 expression was quantified in shRNA control and shRNA AGK SU-DHL4 cells from three independent experiments. D. AGK and BCL-2 expression was measured in control and AGK overexpression SU-DHL4 cells, and BCL-2 expression was quantified from three independent experiments. E. The amounts of p-PTEN and PTEN in SU-DHL2, OCI-LY1, TMD8, SU-DHL4, SU-DHL6, SU-DHL10, and SU-DHL16 DLBCL cell lines were determined by immunoblotting. F. Spearman correlation analysis of AGK and PTEN phosphorylation levels in DLBCL cell lines. G-H. Phosphorylation levels of PTEN, AKT and FOXO1 were measured and quantified in control and AGK knockdown SU-DHL4 cells from three independent experiments. I-J The levels of FOXO1 and p-FOXO1 were measured in cytoplasmic and nuclear fractions (I) and the band intensities were quantified (J). K. The binding of FOXO1 to BCL-2 promoter in SU-DHL4 was determined by chromatin immunoprecipitation analysis. Data were expressed as mean ± SEM from three independent experiments (A-J) or two independent experiments with triplicates (K), and analyzed for statistical significance by t-test. (*P < 0.05; **P < 0.01; ***P < 0.001).
Fig 5: AGK inhibits venetoclax-induced apoptosis in DLBCL cells. A. SU-DHL4 cells were stably transduced with shRNA control lentiviral particles or shRNA AGK lentiviral particles. AGK protein expression was determined by immunoblotting and the band intensities were quantified from three independent experiments. B. AGK knockdown SU-DHL4 and control cell were treated with 0-10 μM venetoclax for 72 h, and cell viability was assessed by CCK-8 assays. C. SU-DHL4 cells were transduced with control retrovirus or AGK retrovirus and selected with 5 μg/mL puromycin for 14 days to generate stable cell lines. AGK protein expression was measured by immunoblotting and quantified from three independent experiments. D. Cell viability of control SU-DHL4 cells and AGK overexpression SU-DHL4 cell that were treated with venetoclax for 72 h. E-F. Apoptosis of control and AGK-knockdown SU-DHL4 cells was detected using Annexin Ⅴ/PI staining, and the percentage of apoptotic cells was quantified. G. TMD8 cells were stably transduced with shRNA control lentiviral particles or shRNA AGK lentiviral particles. AGK protein expression was determined by immunoblotting and the band intensities were quantified from three independent experiments. H. AGK knockdown TMD8 and control cell were treated with 0-10 μM venetoclax for 72 h, and cell viability was assessed by CCK-8 assays. Data were expressed as mean ± SEM from three independent experiments (A, C, F, G) or three independent experiments with duplicates (B, D, H), and analyzed for statistical significance by t-test (A, C, G) or Two-way ANOVA (B, D, F, H) (*P < 0.05; **P < 0.01; ***P < 0.001).
Supplier Page from Abcam for Anti-AGK antibody