Figure 2.
SOX11 impairs tetrameric configuration of SAMHD1 and confers ara-C sensitivity. (A) Representative western blot of native gel electrophoresis performed using disuccinimidyl glutarate (DSG)-crosslinked in JVM-2vector and JVM-2iSOX11 96 hours after treatment with 0.1 μM doxycycline. The tetrameric form of SAMHD1 is detected at a size of ∼250 kDa, dimer at ∼150 kDa, monomer at 71 kDa, and GAPDH at 37 kDa. (B) Curve shows tetramer/monomer ratio in crosslinked in JVM-2vector and JVM-2iSOX11 with different concentrations of DSG (related to panel A). Data are represented as mean ± SEM of 3 independent biological repeats. P value (2-tailed, 2-way ANOVA) is indicated on the curve. (C) Dose response curve for ara-C in Granta-519, JeKo-1, and JVM-2 treated for 72 hours by CellTiter MTS assay. The values on the y-axis represent the relative viability values, which were calculated by normalizing 100% values to respective untreated controls. Data are represented as mean ± SEM of 3 independent experiments. (D) Western blot showing the depleting efficiency of SAMHD1 by Vpx in the 3 cell lines compared to nontargeting dX. One representative western blot out of 3 is shown. Cells were treated with dX or Vpx for 3 hours before ara-C treatment and cultured thereafter for 72 hours before harvesting. SAMHD1 was detected at 71 kDa and GAPDH at 37 kDa. (E-G) Dose response curves for ara-C determined in JVM-2 (E), JeKo-1 (F) and Granta-519 (G) with Vpx or dX for 3 hours before ara-C treatment. Viability was measured by CellTiter MTS assay after 72 hours of ara-C treatment. The values on the y-axis represent the relative viability values which were calculated by normalizing absorbance value at each dose of ara-C for each condition to respective untreated controls. Data are represented as mean ± SEM of 3 independent biological replicates. P (2-tailed) was calculated using the 2-way ANOVA. ∗∗∗∗P < .0001. (H) Western blot showing the efficiency of SOX11 silencing in Granta-519 and JeKo-1 by siRNA for 48 hours. One representative western blot out of 2 is shown. SOX11 was detected at 74 kDa and GAPDH at 37 kDa. (I-J) Dose response curve for 72 hours of ara-C treatment in nontargeting control siRNA- and SOX11 siRNA-transfected Granta-519 (I) and JeKo-1 (J). ara-C treatment was applied 8 hours after transfection. Viability was measured using Celltiter MTS assay after 72 hours of ara-C treatment. Data are represented as mean ± SEM of 2 independent biological replicates. P (2-tailed) was calculated using 2-way ANOVA. ∗∗P < .01; ∗∗∗∗P < .0001.

SOX11 impairs tetrameric configuration of SAMHD1 and confers ara-C sensitivity. (A) Representative western blot of native gel electrophoresis performed using disuccinimidyl glutarate (DSG)-crosslinked in JVM-2vector and JVM-2iSOX11 96 hours after treatment with 0.1 μM doxycycline. The tetrameric form of SAMHD1 is detected at a size of ∼250 kDa, dimer at ∼150 kDa, monomer at 71 kDa, and GAPDH at 37 kDa. (B) Curve shows tetramer/monomer ratio in crosslinked in JVM-2vector and JVM-2iSOX11 with different concentrations of DSG (related to panel A). Data are represented as mean ± SEM of 3 independent biological repeats. P value (2-tailed, 2-way ANOVA) is indicated on the curve. (C) Dose response curve for ara-C in Granta-519, JeKo-1, and JVM-2 treated for 72 hours by CellTiter MTS assay. The values on the y-axis represent the relative viability values, which were calculated by normalizing 100% values to respective untreated controls. Data are represented as mean ± SEM of 3 independent experiments. (D) Western blot showing the depleting efficiency of SAMHD1 by Vpx in the 3 cell lines compared to nontargeting dX. One representative western blot out of 3 is shown. Cells were treated with dX or Vpx for 3 hours before ara-C treatment and cultured thereafter for 72 hours before harvesting. SAMHD1 was detected at 71 kDa and GAPDH at 37 kDa. (E-G) Dose response curves for ara-C determined in JVM-2 (E), JeKo-1 (F) and Granta-519 (G) with Vpx or dX for 3 hours before ara-C treatment. Viability was measured by CellTiter MTS assay after 72 hours of ara-C treatment. The values on the y-axis represent the relative viability values which were calculated by normalizing absorbance value at each dose of ara-C for each condition to respective untreated controls. Data are represented as mean ± SEM of 3 independent biological replicates. P (2-tailed) was calculated using the 2-way ANOVA. ∗∗∗∗P < .0001. (H) Western blot showing the efficiency of SOX11 silencing in Granta-519 and JeKo-1 by siRNA for 48 hours. One representative western blot out of 2 is shown. SOX11 was detected at 74 kDa and GAPDH at 37 kDa. (I-J) Dose response curve for 72 hours of ara-C treatment in nontargeting control siRNA- and SOX11 siRNA-transfected Granta-519 (I) and JeKo-1 (J). ara-C treatment was applied 8 hours after transfection. Viability was measured using Celltiter MTS assay after 72 hours of ara-C treatment. Data are represented as mean ± SEM of 2 independent biological replicates. P (2-tailed) was calculated using 2-way ANOVA. ∗∗P < .01; ∗∗∗∗P < .0001.

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