Figure 1.
SOX11 binds to SAMHD1 in MCL. (A) Identification of SOX11 protein interactors by mass spectrometry–based proteomics analysis of coimmunoprecipitation of SOX1 in Granta-519 and JeKo-1 cell lines. Three biological replicates were analyzed per cell line, and the mean number of peptide spectrum matches (PSMs) across the 3 replicates per protein are displayed on the axes. Proteins with 0 PSMs indicate absence of interaction in that cell line. See “Methods” for defining SOX11 interacting proteins. (B) PLA performed on 2 primary MCL cells with different SOX11 expression levels (supplemental Table 1) using rabbit polyclonal anti-SOX11 and mouse monoclonal anti-SAMHD1. The DAPI channel represents stained nuclei, whereas the red channel (TRITC) represents SOX11-SAMHD1 colocalization. Original magnification, 60×; scale bar, 50 μm; and the pinhole was set at 1.2. The red fluorescent foci represent the colocalization between SOX11 and SAMHD1. (C) PLA performed on Granta-519, JeKo-1, and Rec1 cells using rabbit polyclonal anti-SOX11 and mouse monoclonal anti-SAMHD1. Original magnification, 60×; and pinhole set at 1.2. (D) Representative western blot showing the efficiency of doxycycline-induced expression SOX11 in JVM-2vector and JVM-2iSOX11 at 72 hours after doxycycline treatment. SOX11 band is detected at 74 kDa and GAPDH at 37 kDa. (E) Representative images of PLA performed on JVM-2vector and JVM-2iSOX11 using rabbit polyclonal anti-SOX11 and mouse monoclonal anti-SAMHD1; original magnification, 60×; scale bar, 10 μm; and pinhole set at 1.2. Cells were treated with doxycycline 0.1 μM for 72 hours. (F) Scatterplot shows number of fluorescent foci per cell. The number of foci per cell were analyzed in total 350 cells of JVM-2vector or JVM-2iSOX11 using CellProfiler software. The data are represented as mean ± standard error of the mean (SEM) of 3 independent biological replicates. P < .0001 was calculated by unpaired, 2-tailed t test with Welch correction. (G) CETSA performed using JVM-2vector and JVM-2iSOX11 72 hours after treatment with 0.1 μM doxycycline. A representative western blot showing band intensities of SAMHD1 (71 kDa) and thermostable superoxide dismutase-1 (SOD-1) (20 kDa). (H) Sigmoidal Boltzmann curve of percentage of remaining SAMHD1 protein on y-axis and log10 temperature on x-axis. Data are represented as mean ± SEM of 3 independent biological replicates. ∗∗∗∗P < .0001; 2-way analysis of variance (ANOVA). (I) Binding of SOX11 HMG to SAMHD1 measured by MST. The fluorescence change in labeled SAMHD1 upon titration with SOX11 HMG is plotted; error bars are standard deviation of the mean values from 3 independent experiments. Fitting of the data to a hyperbolic binding isotherm gives KD = 3.2 ± 0.6 μM. CETSA, cellular thermal shift assay.

SOX11 binds to SAMHD1 in MCL. (A) Identification of SOX11 protein interactors by mass spectrometry–based proteomics analysis of coimmunoprecipitation of SOX1 in Granta-519 and JeKo-1 cell lines. Three biological replicates were analyzed per cell line, and the mean number of peptide spectrum matches (PSMs) across the 3 replicates per protein are displayed on the axes. Proteins with 0 PSMs indicate absence of interaction in that cell line. See “Methods” for defining SOX11 interacting proteins. (B) PLA performed on 2 primary MCL cells with different SOX11 expression levels (supplemental Table 1) using rabbit polyclonal anti-SOX11 and mouse monoclonal anti-SAMHD1. The DAPI channel represents stained nuclei, whereas the red channel (TRITC) represents SOX11-SAMHD1 colocalization. Original magnification, 60×; scale bar, 50 μm; and the pinhole was set at 1.2. The red fluorescent foci represent the colocalization between SOX11 and SAMHD1. (C) PLA performed on Granta-519, JeKo-1, and Rec1 cells using rabbit polyclonal anti-SOX11 and mouse monoclonal anti-SAMHD1. Original magnification, 60×; and pinhole set at 1.2. (D) Representative western blot showing the efficiency of doxycycline-induced expression SOX11 in JVM-2vector and JVM-2iSOX11 at 72 hours after doxycycline treatment. SOX11 band is detected at 74 kDa and GAPDH at 37 kDa. (E) Representative images of PLA performed on JVM-2vector and JVM-2iSOX11 using rabbit polyclonal anti-SOX11 and mouse monoclonal anti-SAMHD1; original magnification, 60×; scale bar, 10 μm; and pinhole set at 1.2. Cells were treated with doxycycline 0.1 μM for 72 hours. (F) Scatterplot shows number of fluorescent foci per cell. The number of foci per cell were analyzed in total 350 cells of JVM-2vector or JVM-2iSOX11 using CellProfiler software. The data are represented as mean ± standard error of the mean (SEM) of 3 independent biological replicates. P < .0001 was calculated by unpaired, 2-tailed t test with Welch correction. (G) CETSA performed using JVM-2vector and JVM-2iSOX11 72 hours after treatment with 0.1 μM doxycycline. A representative western blot showing band intensities of SAMHD1 (71 kDa) and thermostable superoxide dismutase-1 (SOD-1) (20 kDa). (H) Sigmoidal Boltzmann curve of percentage of remaining SAMHD1 protein on y-axis and log10 temperature on x-axis. Data are represented as mean ± SEM of 3 independent biological replicates. ∗∗∗∗P < .0001; 2-way analysis of variance (ANOVA). (I) Binding of SOX11 HMG to SAMHD1 measured by MST. The fluorescence change in labeled SAMHD1 upon titration with SOX11 HMG is plotted; error bars are standard deviation of the mean values from 3 independent experiments. Fitting of the data to a hyperbolic binding isotherm gives KD = 3.2 ± 0.6 μM. CETSA, cellular thermal shift assay.

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