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Figure S1 + p21siRNA a b Ctrl siRNA c p21 β-actin Figure S1. Knockdown of p21 in CD4+ T cells using siRNA. Expression of p21 was analyzed by RT-PCR 36 h post transfection. -actin was used as a house-keeping gene. P21-specific siRNA was performed using 8 nmol/ml (a), or 4nmol/ml (b) and siControl (c). This experiment is representative of repeated experiments using cells from different donors. Figure S2 + Atv (0.25 μg/ml) + Atv (0.5 μg/ml) + Atv (1 μg/ml) CD4 -Atv Annexin V Figure S2. Viability of CD4+ T cells following atorvastatin treatment. Representative dot plot from CD4+ T cells isolated from 1 HIV-1 seronegative individual in the absence of treatment or treated with atorvastatin (0.25 to 1 g/ml) for 4 days. Following treatment surface staining was performed. Gated cells representing percentages of apoptotic CD4+ cells as measured by flow cytometry. Figure S3 Negative CD14 A + Atv (1 μg/ml) + Atv (0.5 μg/ml) - Atv p24 - + - Atorvastatin + B P21 β-actin 1 2 3 4 Figure S3. Atorvastatin treatment limits HIV-1 replication in CD14+ cells. (A) Representative flow cytometry dot plots from CD14+ T cells in the absence or presence of atorvastatin (0.5 and 1g/ml) after infection with X-4-tropic tropic HIV-1 isolates respectively. Data obtained by viral p24 antigen intracellular staining on day 4 post infection. These plots are representative of three repeat experiments using cells from different donors. (B) RT-PCR reflecting -actin and p21 gene expression in monocyte-derived macrophages treated with atorvastatin (1 g/ml) for 48 h compared with untreated cells prior to total RNA isolation. P < 0.0001 100 1.0 80 60 40 B 20 0 0.5 mg/ml 1 mg/ml R5-tropic HIV-1 0.5 mg/ml %CD4+p24+ A %p24 suppression Figure S4 1 mg/ml 0.8 P < 0.0001 0.6 0.4 0.2 0.0 -Atv +Atv (1µg/ml) R5-tropic HIV-1 + Atv (0.5 μg/ml) X4-tropic R5-tropic C + Atv (0.5 μg/ml) + Atv (1 μg/ml) Uninfected 0 X4-tropic D p24 R5-tropic CD4 p24 - Atv +Atv (1µg/ml) X4-tropic HIV-1 + Atv (1 μg/ml) CD4 - Atv -Atv X4-tropic HIV-1 Figure S4. Atorvastatin reduces HIV-1 infection in resting CD4+ T cells. (A) Percentages of p24 suppression in CD4+ T cells that were exposed to atorvastatin (0.5-1g/ml) for 48 h post HIV-1 infection with R5 (HIV-1JR-CSF) and X-4tropic isolates. (B) Percentages of CD4+p24+ cells in non-activated CD4+ T cells in the presence of atorvastatin (1g/ml) 48 h after HIV-1 infection with R5 (HIV-1JR-CSF) and X-4-tropic (HIV-1LAI) isolates. Significance was tested using paired t test. (C) Representative flow cytometry dot plots from CD4+ T cells infected and then treated with 0.5-1g/ml atorvastatin post infection with X-4-tropic or R5-tropic HIV-1 isolates respectively. (D) Representative flow cytometry dot plots from CD4+ T cells treated for 24 h with 0.5-1g/ml atorvastatin post infection with R5 (HIV-1JR-CSF) and X-4-tropic (HIV-1LAI) isolates. CD4+ T cells from 5 HIV-1 seronegative donors were used for each viral isolate. Data are from 5 HIV-1 seronegative individuals infected with both X4-and R5-tropic viral isolates in vitro with duplicate cultures per condition. Error bars indicate mean ± SEM from duplicate cell cultures from 5 HIV-1 seronegative individuals infected 4 separate times with both X4-and R5-tropic viral isolates in vitro. Each point represents an individual. Figure S5 +Atv (1μg/ml) siControl CD4 +Atv (0.5 μg/ml) siRNAp21 CD4 R5-tropic HIV -Atv p24 Figure S5. Atorvastatin mediated upregulation of p21 reduces HIV-1 infection in activated CD4+ T cells. Representative example of dot plots from activated CD4+ T cells infected in the presence of p21-specific or control siRNA with R5-tropic HIV-1 Isolate in the presence (0.5 g/ml or 1 g/ml) or absence of atorvastatin. Viral infection was measured by viral p24 antigen staining on CD4+ T cells using flow cytometry. These plots are representative of independent experiments performed on three different donors for each viral isolate. Figure S6 + Atv (0.5 μg/ml) + Atv (1 μg/ml) + Mevalo. + Atv (0.5 μg/ml) + Mevalo. (1 mM) + Atv (1 μg/ml) + Mevalo. (1 mM) X4-tropic (HIV-1LAI) CD4 X4-tropic (HIV-1LAI) - Atv p24 Figure S6. Atorvastatin reduces infection of CD4+ T cells to HIV-1 infection and upregulates p21 through mevalonate pathway. Representative dot plots of non-activated CD4+ T cells in the presence of atorvastatin (0.5-1g/ml) for 48 h and also in the presence or absence of L. mevalonate (1mM) post in vitro infection with X4-tropic viral isolate. Viral infection was measured by viral p24 antigen staining on CD4+ T cells using flow cytometry. These plots are representative of independent experiments performed on three different donors.