Journal: Scientific Reports

Article Title: Immune Activation Influences SAMHD1 Expression and Vpx-mediated SAMHD1 Degradation during Chronic HIV-1 Infection

doi: 10.1038/srep38162

Figure Lengend Snippet: ( a ) Dot-plots of SAMHD1 expression in THP-1 and Jurkat cell lines. THP-1 cells were treated with 4ng/mL SIV-Vpx for 24 h, or were untreated. SAMHD1-silent THP-1 cells (THP-1-shSAMHD1) or control shRNA (THP-1-shRNA Ctrl) were used as SAMHD1 staining controls. Percentages indicate percentage of SAMHD1 + cells, and digits represent MFI of SAMHD + cells. (b ) SAMHD1 expression was further confirmed using western blotting. Relative protein blot signal intensity analysis was performed using Li-Cor Image Studio software. Numbers represent relative value to the control or blank by normalization with to β-Actin. (c ) PBMCs from an HIV-seronegative donor were stained with an antibody mixture with, or without, SAMHD1 pAb, a histogram illustrating SAMHD1 expression. ( d ) SAMHD1 positive and negative subgroups were sorted and expression of SAMHD1 was confirmed using western blotting. ( e ) Gating strategy results for SAMHD1 expression in activated CD4 + T cells, resting CD4 + T cells, and monocytes of PBMCs from an HIV-seronegative individual are presented. Percentages indicate percentage of SAMHD1 + cells.

Article Snippet: Whole-cell lysates were resolved on SDS-PAGE, transferred to a 0.22 μm polyvinylidene fluoride membrane, and probed using rabbit anti-SAMHD1 polyclonal Ab (clone 1F9, Abcam), rabbit anti-phospho-SAMHD1 (Thr592) Antibody (CST), mouse anti–β-actin mAb (Santa Cruz), and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz).

Techniques: Expressing, Control, shRNA, Staining, Western Blot, Software

Journal: Scientific Reports

Article Title: Immune Activation Influences SAMHD1 Expression and Vpx-mediated SAMHD1 Degradation during Chronic HIV-1 Infection

doi: 10.1038/srep38162

Figure Lengend Snippet: (a ) Representative flow cytometric analysis results for SAMHD1 expression in activated CD4 + T cells, resting CD4 + T cells, and monocytes from HC, cART-naive, and cART patients are presented. Numbers in top right indicate percent SAMHD1 positive cells. (b ) Statistical analysis of data was obtained for n = 25 HC, n = 22 cART-naive individuals, or for n = 17 cART individuals. (c ) Fresh isolated PBMCs were treated with 2 ng/mL SIV-Vpx or SIV-Mock for 48 h, and the expression of SAMHD1 in activated CD4 + T cells, resting CD4 + T cells, and monocytes from HC, cART-naive, and cART cohorts was determined. Representative histograms were shown. Numbers in top left indicate percent SAMHD1 positive cells in SIV-Mock (grey), and SIV-Vpx treatment (black), groups. (d) Shown are summary results for the effects of SIV-Vpx (“∆”) or SIV-Mock (“○”) on SAMHD1 expression in activated CD4 + T cells, resting CD4 + T cells, and monocytes from 10 HC, 22 cART-naive individuals, and 17 cART individuals. Numbers represent the mean value of loss of SAMHD1 positive cells after SIV-Vpx treatment. The significance was measured using the Mann-Whitney test, * p < 0.05, ** p < 0.001, *** p < 0.0001; n.s, not significant.

Article Snippet: Whole-cell lysates were resolved on SDS-PAGE, transferred to a 0.22 μm polyvinylidene fluoride membrane, and probed using rabbit anti-SAMHD1 polyclonal Ab (clone 1F9, Abcam), rabbit anti-phospho-SAMHD1 (Thr592) Antibody (CST), mouse anti–β-actin mAb (Santa Cruz), and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz).

Techniques: Expressing, Isolation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Immune Activation Influences SAMHD1 Expression and Vpx-mediated SAMHD1 Degradation during Chronic HIV-1 Infection

doi: 10.1038/srep38162

Figure Lengend Snippet: ( a ) Results of statistical analysis of the correlation between viral load and SAMHD1 expression in activated CD4 + T cells (“○”), resting CD4 + T cells (“▵”), or monocytes (“×”), in cART-naive patients ( n = 22). Correlation statistics were analyzed using the Spearman correlation. r , Spearman’s correlation coefficient. (b ) The proportions of SAMHD1 positive cells in activated CD4 + T cells, resting CD4 + T cells, and monocytes are presented for the chronic viremia (CHVIR, VL>10000 copies/mL), and viremic controllers (VC, VL<2000 copies/mL), cohorts, CHVIR, n = 8; VC, n = 6. Comparisons were made using the Mann-Whitney test, p -value is not indicated when p > 0.05. ( c , d ) There was no correlation for activated CD4 + T cells, whereas there was a negative correlation, between the frequency of cells susceptible to SAMHD1 degradation in monocytes and plasma viral loads from cART-naive subjects ( n = 22). Correlation statistics were analyzed using the Spearman correlation. r , Spearman’s correlation coefficient. (e , f ) The frequency of SAMHD1 degradation by SIV-Vpx in activated CD4 + T cells, and monocytes was compared between CHVIR and VC cohorts. Numbers represent the mean value of loss of SAMHD1 positive cells after SIV-Vpx treatment. p values were evaluated using the Mann-Whitney test, * p < 0.05, ** p < 0.001, *** p < 0.0001; n.s , not significant. SIV-Vpx (“▵”), SIV-Mock (“○”).

Article Snippet: Whole-cell lysates were resolved on SDS-PAGE, transferred to a 0.22 μm polyvinylidene fluoride membrane, and probed using rabbit anti-SAMHD1 polyclonal Ab (clone 1F9, Abcam), rabbit anti-phospho-SAMHD1 (Thr592) Antibody (CST), mouse anti–β-actin mAb (Santa Cruz), and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz).

Techniques: Expressing, MANN-WHITNEY, Clinical Proteomics

Journal: Scientific Reports

Article Title: Immune Activation Influences SAMHD1 Expression and Vpx-mediated SAMHD1 Degradation during Chronic HIV-1 Infection

doi: 10.1038/srep38162

Figure Lengend Snippet: The relationship of SAMHD1 degradation by SIV-Vpx in activated CD4 + T cells with CD4 counts ( a ), the number of activated CD4 + T cells ( c ), or blood IFN-α level ( e ). Correlation between SAMHD1 degradation by SIV-Vpx in CD14 + monocytes and CD4 + T-cell counts ( b ), the number of activated CD4 + T cells ( d ), or blood IFN-α level ( f ). Subjects with CD4 counts and the number of activated CD4 + T cells were from cART-naive populations ( n = 22); subjects with blood IFN-α level were from HIV-1 infected individuals ( n = 15). r , Spearman’s correlation coefficient; p < 0.05 was considered to indicate a statistically significant result.

Article Snippet: Whole-cell lysates were resolved on SDS-PAGE, transferred to a 0.22 μm polyvinylidene fluoride membrane, and probed using rabbit anti-SAMHD1 polyclonal Ab (clone 1F9, Abcam), rabbit anti-phospho-SAMHD1 (Thr592) Antibody (CST), mouse anti–β-actin mAb (Santa Cruz), and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz).

Techniques: Infection

Journal: Scientific Reports

Article Title: Immune Activation Influences SAMHD1 Expression and Vpx-mediated SAMHD1 Degradation during Chronic HIV-1 Infection

doi: 10.1038/srep38162

Figure Lengend Snippet: ( a ) PBMC cells were stimulated with PI (1 μM PMA plus 1 μg/mL Ionomycin) for 24 h. Then, SAMHD1 expression in CD4 + T cells was assessed. Representative dot-plot results for SAMHD1 expression are presented. Percentages indicate absolute SAMHD1 + cell numbers. (b ) Statistical analysis of data obtained as described in a was performed using the Mann-Whitney test. (c) PBMCs were pre-stimulated with IFN-α, or were not stimulated, for 24 h. Cells were then treated for another 48 h with SIV-Vpx or SIV-Mock. Histograms of SAMHD1 expression in activated CD4 + T cells, resting CD4 + T cells, and monocytes are presented. (d ) Statistical analysis of data was performed using the Wilcoxon matched pairs test, data from HIV-1 infected individuals (n = 15). ( e ) Purified CD4 + T cells and monocytes were separated from PBMCs, pre-stimulated with PI or IFN-α, then treated with SIV-Vpx or SIV-Mock; whole cell lysates were examined by western blot analysis. Relative protein blot signal intensity analysis was performed using Li-Cor Image Studio software, numbers represent relative value normalized to β-Actin.

Article Snippet: Whole-cell lysates were resolved on SDS-PAGE, transferred to a 0.22 μm polyvinylidene fluoride membrane, and probed using rabbit anti-SAMHD1 polyclonal Ab (clone 1F9, Abcam), rabbit anti-phospho-SAMHD1 (Thr592) Antibody (CST), mouse anti–β-actin mAb (Santa Cruz), and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz).

Techniques: Expressing, MANN-WHITNEY, Infection, Purification, Western Blot, Software