thr 592 p thr592  (ProSci Incorporated)

Journal: Journal of Virology

Article Title: Expression of MDM2 in Macrophages Promotes the Early Postentry Steps of HIV-1 Infection through Inhibition of p53

doi: 10.1128/JVI.01871-18

Figure Lengend Snippet: MDM2 gene silencing induces some p53-regulated genes. (A to C) MDMs were transfected with siCtrl or siMDM2 for 48 h, which corresponds to the time of virus infection in previous figures. (A) qRT-PCR analysis was performed to monitor the relative mRNA expression for MDM2, TP53, CDKN1A, FAS, and BAX genes. Each symbol represents a different donor. The dotted line represents the gene expression of the siCtrl condition. Statistical analyses were performed using the paired Student's t test (ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n = 10). (B) Western blot analyses were carried out to quantify the amount of MDM2, p53, p21, total SAMHD1, p-T592 SAMHD1, and actin. Total SAMHD1 and p-T592 SAMHD1 were probed on a different membrane using the same samples and are shown with their respective actin loading controls. Some samples were treated with MG-132 for 6 h before cell lysis. Donor shown is representative of 4 distinct donors. (C) Apoptotic cells at the time of infection were measured by flow cytometry with annexin V and 7-AAD. siRNAs have only a mild effect on apoptosis compared to that of siCtrl. This donor is representative of a total of 2 donors.

Article Snippet: These membranes were probed overnight at 4°C with antibodies specific for MDM2 (SMP14; Novus Biologicals, Oakville, ON), p53 (DO-1; BioLegend, San Diego, CA), p21 (12D1; Cell Signaling Technology, Danvers, MA), SAMHD1 (I19-18; Millipore, Etobicoke, ON), phosphorylated SAMHD1 (Thr 592) (D702M; Cell Signaling Technology), and actin (C-2; Santa Cruz Biotechnology, Dallas, TX).

Techniques: Transfection, Virus, Infection, Quantitative RT-PCR, Expressing, Gene Expression, Western Blot, Membrane, Lysis, Flow Cytometry

Journal: Journal of Virology

Article Title: Expression of MDM2 in Macrophages Promotes the Early Postentry Steps of HIV-1 Infection through Inhibition of p53

doi: 10.1128/JVI.01871-18

Figure Lengend Snippet: Release of p53 from MDM2 control is sufficient to reduce susceptibility of MDMs to HIV-1. (A, B, and D to F) MDMs were pretreated for 1 h with Nutlin-3, EFV, or RGV before infection with DNase-treated NL/Bal-HSA. The drug pressure was maintained for the entire period of virus infection. (A) The percentage of HSA+ MDMs was estimated by flow cytometry analysis at 3 dpi. (B) Cell viability was measured by flow cytometry at 3 dpi with the fixable viability dye eFluor780. No significant change in cell viability was detected (one-way ANOVA with Tukey’s multiple-comparison test, n = 5). (C) MDMs were treated with Nutlin-3 for 3 days without exposure to HIV-1. The percentage of apoptotic cells was measured by flow cytometry with annexin V and 7-AAD. Treatment with Nutlin-3 induces a small increase in apoptosis compared to the level for the DMSO control. This donor is representative of a total of 2 donors. (D and E) The number of completed reverse transcripts (D) and integrated proviral DNA copies (E) was defined by qPCR. (F) Relative mRNA levels of MDM2, CDKN1A, and Tat were measured at 24 and 72 h posttreatment by qRT-PCR. The dotted line represents the gene expression for the DMSO-treated MDMs. (G) The total protein levels of MDM2, p53, p21, SAMHD1, and phosphorylated SAMHD1 (Thr592) in MDMs after 24 h of treatment with Nutlin-3 without exposure to HIV-1 were determined by a Western blot assay. Indicated samples were treated with MG-132 for 6 h before protein extraction. Some proteins had to be exposed for a different time when treated with MG-132, as shown with the cropped membrane. The donor shown is representative of 5 distinct donors. Statistical analyses were done using the ratio-paired Student's t test (A, D, and E; n = 5) or paired Student's t test (F; n = 4) (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Article Snippet: These membranes were probed overnight at 4°C with antibodies specific for MDM2 (SMP14; Novus Biologicals, Oakville, ON), p53 (DO-1; BioLegend, San Diego, CA), p21 (12D1; Cell Signaling Technology, Danvers, MA), SAMHD1 (I19-18; Millipore, Etobicoke, ON), phosphorylated SAMHD1 (Thr 592) (D702M; Cell Signaling Technology), and actin (C-2; Santa Cruz Biotechnology, Dallas, TX).

Techniques: Control, Infection, Virus, Flow Cytometry, Comparison, Quantitative RT-PCR, Gene Expression, Western Blot, Protein Extraction, Membrane

Journal: Journal of Virology

Article Title: Expression of MDM2 in Macrophages Promotes the Early Postentry Steps of HIV-1 Infection through Inhibition of p53

doi: 10.1128/JVI.01871-18

Figure Lengend Snippet: p53 and p21 knockdown lead to an increase in HIV-1 infection. (A) MDMs from 6 different donors were transfected with siCtrl or an siRNA specific for MDM2, p53, or p21 and next infected for 3 days with NL/Bal-HSA. The percentage of HSA+ cells then was assessed by flow cytometry. One-way ANOVA with Dunnett's multiple-comparison test was performed (*, P ≤ 0.05; **, P ≤ 0.01). (B) The relative mRNA expression levels for MDM2, TP53, and CDKN1A were monitored by qRT-PCR for 5 biological replicates at the time of HIV-1 infection. One-way ANOVA with Holm-Sidak's multiple-comparison test was performed (ns, nonsignificant; *, P ≤ 0.05). (C) MDMs were transfected for 2 days and then exposed to NL/Bal-HSA. Cell viability was measured by flow cytometry at 3 dpi with the fixable viability dye eFluor780. No significant change in cell viability was detected (one-way ANOVA with Tukey’s multiple-comparison test, n = 5). (D) MDMs were transfected for 2 days. Apoptotic cells at the time of infection were measured by flow cytometry with annexin V and 7-AAD. siRNAs have only a mild effect on apoptosis compared to that of the siCtrl. This donor is representative of a total of 2 donors. (E, left) The total protein levels of p53, p21, SAMHD1, and phosphorylated SAMHD1 (Thr592) at the time of virus infection were estimated by Western blot analysis. (Right) Band intensities were measured, normalized on the corresponding actin band, and compared to those of the siCtrl. Donor shown is representative of 4 independents donors. (F) The total protein levels of p53 and actin at the time of virus infection. Samples were treated with MG-132 for 6 h before cell lysis. Band intensities were measured, normalized on the corresponding actin band, and compared to those of the siCtrl. (G) The total number of completed reverse transcripts (left) and integrated proviral DNA copies (right) for each experimental condition was quantified by qPCR analysis in 6 biological replicates at 2 dpi. One-way ANOVA with Dunnett's multiple-comparison test was performed (ns, nonsignificant; *, P ≤ 0.05).

Article Snippet: These membranes were probed overnight at 4°C with antibodies specific for MDM2 (SMP14; Novus Biologicals, Oakville, ON), p53 (DO-1; BioLegend, San Diego, CA), p21 (12D1; Cell Signaling Technology, Danvers, MA), SAMHD1 (I19-18; Millipore, Etobicoke, ON), phosphorylated SAMHD1 (Thr 592) (D702M; Cell Signaling Technology), and actin (C-2; Santa Cruz Biotechnology, Dallas, TX).

Techniques: Knockdown, Infection, Transfection, Flow Cytometry, Comparison, Expressing, Quantitative RT-PCR, Virus, Western Blot, Lysis

Journal: Journal of Virology

Article Title: Expression of MDM2 in Macrophages Promotes the Early Postentry Steps of HIV-1 Infection through Inhibition of p53

doi: 10.1128/JVI.01871-18

Figure Lengend Snippet: p53-mediated change in susceptibility of MDMs to HIV-1 is inhibited by Vpx-dependent degradation of SAMHD1. (A) Flow cytometry analysis showing fold changes of HSA expression on the surface of MDMs compared to that of the control from 5 biological replicates transfected for 48 h with control or gene-specific siRNA before either being left unexposed or being exposed to VLP-Vpx for 2 h, washed, and infected with NL/Bal-HSA for 3 days. One-way ANOVA with Dunnett's multiple-comparison test was performed (ns, nonsignificant; *, P ≤ 0.05; **, P ≤ 0.01). (B) Flow cytometry analysis showing fold changes of HSA expression on the surface of MDMs compared to that of the control from 5 biological replicates left unexposed or exposed to VLP-Vpx for 2 h and then to Nutlin-3 or DMSO for 1 h before being infected with NL/Bal-HSA for 3 days. Ratio-paired Student’s t tests were determined (ns, nonsignificant; **, P ≤ 0.01). (A and B) Data represent fold changes compared to the level of the respective control.

Article Snippet: These membranes were probed overnight at 4°C with antibodies specific for MDM2 (SMP14; Novus Biologicals, Oakville, ON), p53 (DO-1; BioLegend, San Diego, CA), p21 (12D1; Cell Signaling Technology, Danvers, MA), SAMHD1 (I19-18; Millipore, Etobicoke, ON), phosphorylated SAMHD1 (Thr 592) (D702M; Cell Signaling Technology), and actin (C-2; Santa Cruz Biotechnology, Dallas, TX).

Techniques: Flow Cytometry, Expressing, Control, Transfection, Infection, Comparison

Journal: Journal of Virology

Article Title: Expression of MDM2 in Macrophages Promotes the Early Postentry Steps of HIV-1 Infection through Inhibition of p53

doi: 10.1128/JVI.01871-18

Figure Lengend Snippet: Proposed model for MDM2-mediated regulation of HIV-1 infection in human macrophages. Inhibition of MDM2 p53-binding activity with Nutlin-3 before HIV-1 infection stabilizes and increases the level of p53, which in turn results in a higher level of p21 expression, interfering with the phosphorylation/inactivation of SAMHD1 levels by CDK1. This increase in active SAMHD1 leads to a lower number of HIV-1 replication intermediates. Thus, in HIV-1-susceptible MDMs, MDM2 maintains a low level of p53, leading to a high level of phosphorylated SAMHD1 and promoting viral reverse transcription and integration.

Article Snippet: These membranes were probed overnight at 4°C with antibodies specific for MDM2 (SMP14; Novus Biologicals, Oakville, ON), p53 (DO-1; BioLegend, San Diego, CA), p21 (12D1; Cell Signaling Technology, Danvers, MA), SAMHD1 (I19-18; Millipore, Etobicoke, ON), phosphorylated SAMHD1 (Thr 592) (D702M; Cell Signaling Technology), and actin (C-2; Santa Cruz Biotechnology, Dallas, TX).

Techniques: Infection, Inhibition, Binding Assay, Activity Assay, Expressing, Phospho-proteomics, Reverse Transcription