3 a aminocholestane  (Abcam)

Journal: bioRxiv

Article Title: Virion-associated US28 rapidly modulates Akt activity to suppress HCMV lytic replication in monocytes

doi: 10.1101/2023.09.05.556359

Figure Lengend Snippet: (A to F) Monocytes were infected with mock, WT, or US28Δ (MOI = 1). After 30 min, cells were treated with ethanol (EtOH; carrier control) or 3AC (5 µM; SHIP1 inhibitor) and then incubated for (A to C) 30 min, or (D to F) 24 h. (A to C) Total SHIP1, p-SHIP1, total Akt, p-Akt S473, and p-Akt T308 were detected by western blot. IE1 expression was detected by (D, E) western blot or (F) RT-qPCR. All membranes were probed for β-actin as a loading control. Western blots are representative of at least 3 independent experiments. Densitometry was performed using Image Lab software (Bio-Rad) from 3 independent blood donors. Statistical significance was measured using one-way ANOVA.; ****, P < 0.0001, ***P < 0.0005, *P<0.05, ns= not significant.

Article Snippet: Where indicated, the following reagents were used to treat the cells at concentrations noted in the text: Bafilomycin (Baf; inhibitor of lysosomal degradation of proteins ( )), MG132 (MG; proteasome inhibitor ( )), AG-1478 (AG; EGFR inhibitor ( )), 3-α-aminocholestane (3AC; SHIP1 inhibitor ( )) and LY294002 (pan-PI3K inhibitor ( )) all from Calbiochem; MK-2206 (MK; Akt inhibitor ( )) and staurosporine (ST; broad spectrum protein kinase inhibitor/inducer of apoptosis ( )) both from Selleckchem.

Techniques: Infection, Incubation, Western Blot, Expressing, Quantitative RT-PCR, Software

Journal: bioRxiv

Article Title: Virion-associated US28 rapidly modulates Akt activity to suppress HCMV lytic replication in monocytes

doi: 10.1101/2023.09.05.556359

Figure Lengend Snippet: (A, B) Monocytes were infected (MOI = 1) with WT or US28Δ for 30 min to allow for viral entry and then the indicated samples were treated with 3AC (5 µM; SHIP1 inhibitor). (A) After 24 h, monocyte viability was measured by flow cytometry using annexin V and PI staining. Staurosporine (0.5 µM, ST; non-selective protein kinase inhibitor) was used as a positive control for cell death. (B) After 2 h, cells were treated with proteinase K (1.0 mg/ml, 5 min at 37°C) and then washed twice with PBS to remove the viral particles that remained outside of the cell membrane. Samples were then lysed and the viral genome was detected using RT-qPCR (UL123/GAPDH). Statistical significance was measured using one-way ANOVA; **P < 0.005, *P<0.05.

Article Snippet: Where indicated, the following reagents were used to treat the cells at concentrations noted in the text: Bafilomycin (Baf; inhibitor of lysosomal degradation of proteins ( )), MG132 (MG; proteasome inhibitor ( )), AG-1478 (AG; EGFR inhibitor ( )), 3-α-aminocholestane (3AC; SHIP1 inhibitor ( )) and LY294002 (pan-PI3K inhibitor ( )) all from Calbiochem; MK-2206 (MK; Akt inhibitor ( )) and staurosporine (ST; broad spectrum protein kinase inhibitor/inducer of apoptosis ( )) both from Selleckchem.

Techniques: Infection, Flow Cytometry, Staining, Positive Control, Membrane, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Virion-associated US28 rapidly modulates Akt activity to suppress HCMV lytic replication in monocytes

doi: 10.1101/2023.09.05.556359

Figure Lengend Snippet: During WT HCMV infection of monocytes, US28 negatively regulates EGFR phosphorylation. Downstream of EGFR, PI3K phosphorylates PI( , )P 2 to PI( , , )P . Additionally, HCMV infection results in increased SHIP1 activity, which dephosphorylates PI( , , )P to PI( , )P 2 . Recruitment of Akt to PI( , )P 2 leads to a preferential phosphorylation at S473 and establishment of a quiescent infection during WT infection. Contrarily, in US28Δ-infected monocytes, EGFR is robustly phosphorylated without impacting SHIP1 activity. Thus, in the absence of US28, an accumulation of PI( , , )P leads to Akt phosphorylation at both S473 and T308, which is required for IE1 expression and lytic HCMV replication.

Article Snippet: Where indicated, the following reagents were used to treat the cells at concentrations noted in the text: Bafilomycin (Baf; inhibitor of lysosomal degradation of proteins ( )), MG132 (MG; proteasome inhibitor ( )), AG-1478 (AG; EGFR inhibitor ( )), 3-α-aminocholestane (3AC; SHIP1 inhibitor ( )) and LY294002 (pan-PI3K inhibitor ( )) all from Calbiochem; MK-2206 (MK; Akt inhibitor ( )) and staurosporine (ST; broad spectrum protein kinase inhibitor/inducer of apoptosis ( )) both from Selleckchem.

Techniques: Infection, Activity Assay, Expressing

Journal: The EMBO Journal

Article Title: PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

doi: 10.15252/embj.2020105603

Figure Lengend Snippet: A–C A mass ELISA was used to detect specific PIP species after exposure to anti‐FcγRII/III (2.4G2) with or without 3‐a‐aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI‐3K inhibitor). In M‐MOP cells (left panel) and primary microglia (right panel), PI(4,5)P 2 (A), PI(3,4)P 2 (B) and PIP 3 (C) were detected. All data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA (log‐transformed data were used in C) with Sidak’s multiple comparison tests performed. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522).

Article Snippet: Designated wells were inhibited by pre‐exposure for 2 h with 3‐a‐aminocholestane (20 µM, B‐0341), LY294002 (10 µM, B‐0294) or SF1670 (5 µM, B‐0350) (Echelon Biosciences).

Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: The EMBO Journal

Article Title: PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

doi: 10.15252/embj.2020105603

Figure Lengend Snippet: A–C A mass ELISA was used to detect specific PIP species after exposure to anti‐FcγRII/III (2.4G2) with or without 3‐a‐aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI‐3K inhibitor). In M‐MOP cells (left panel) and primary microglia (right panel), PI(4,5)P 2 (A), PI(3,4)P 2 (B) and PIP 3 (C) were detected. All data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA (log‐transformed data were used in C) with Sidak’s multiple comparison tests performed. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522).

Article Snippet: In some assays, M‐MØP cells were pre‐exposed for 2 h with edelfosine (10 µM) (Tocris), U73122 (5 µM) (Sigma), xestospongin C (5 µM) (Abcam), 3‐a‐aminocholestane (20 µM, B‐0341), LY294002 (10 µM, B‐0294) or SF1670 (5 µM, B‐0350) to inhibit phosphoinositide metabolism.

Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: The EMBO Journal

Article Title: PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

doi: 10.15252/embj.2020105603

Figure Lengend Snippet: A–C A mass ELISA was used to detect specific PIP species after exposure to anti‐FcγRII/III (2.4G2) with or without 3‐a‐aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI‐3K inhibitor). In M‐MOP cells (left panel) and primary microglia (right panel), PI(4,5)P 2 (A), PI(3,4)P 2 (B) and PIP 3 (C) were detected. All data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA (log‐transformed data were used in C) with Sidak’s multiple comparison tests performed. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522).

Article Snippet: In some assays, M‐MØP cells were pre‐exposed for 2 h with edelfosine (10 µM) (Tocris), U73122 (5 µM) (Sigma), xestospongin C (5 µM) (Abcam), 3‐a‐aminocholestane (20 µM, B‐0341), LY294002 (10 µM, B‐0294) or SF1670 (5 µM, B‐0350) to inhibit phosphoinositide metabolism.

Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Glycoprotein-Initiated Signaling Mediates the Aberrant Activation of Akt

doi: 10.1128/JVI.00167-20

Figure Lengend Snippet: Phosphorylation of EGFR and integrin β1 results in the activation of the EGFR/PI3K/SHIP1 signaling pathway. (A and B) EGFR, integrin β1, p85, and SHIP1 were immunoprecipitated after monocytes were infected with or without HCMV for 0 min or 5 min (A) or 5 min (B). (C) Cells were treated with 5 μM AG (an EGFR inhibitor) and 20 μM ATN (an integrin inhibitor) for 1 h at 37°C and then infected with HCMV for 15 min. Cells were then lysed followed by Western blot analyses to determine the presence of different proteins using specific antibodies. Results are representative of at least 3 independent experiments using different donors.

Article Snippet: For the inhibitor studies, the following reagents were purchased from the indicated companies: AG-1478 (AG) (an EGFR inhibitor [ 96 ]), 3-α-aminocholestane (3AC) (a SHIP1 inhibitor [ 66 ]), and LY294002 (a pan-PI3K inhibitor [ 97 ]) from Calbiochem (Billerica, MA) and ATN161 (ATN) (an integrin α5β1 inhibitor [ 98 ]) from Selleck Chemicals (Houston, TX).

Techniques: Activation Assay, Immunoprecipitation, Infection, Western Blot

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Glycoprotein-Initiated Signaling Mediates the Aberrant Activation of Akt

doi: 10.1128/JVI.00167-20

Figure Lengend Snippet: HCMV induces a chronic SHIP1 activation required for the survival of infected monocytes. (A) Monocytes were mock or HCMV infected for 15 min, 24 h, or 48 h, and total or p-SHIP1 was detected from whole-cell lysates. (B, C, and D) Monocytes were treated with 5 μM AG (an EGFR inhibitor), 50 μM LY (a PI3K inhibitor), or 5 μM 3AC (a SHIP1 inhibitor) for 1 h and then mock or HCMV infected. (B) After 5 min infection, anti-gH antibody was used to pull down gH glycoprotein (and any other proteins bound to gH) followed by blotting for gH, integrin β1, SHIP1, and p-SHIP1 in the immunoprecipitate using specific antibodies. Input controls were blotted for actin to confirm homogeneous loading of the samples. (C) After 24 h, Western blot analysis was performed to detect Mcl1 and HSP27 from total cell lysates. (D) Survival of monocytes was measured after 48 h of infection with or without the inhibitors by annexin V and PI staining using flow cytometry. Results are representative of at least 3 to 5 independent experiments using different donors. **, P < 0.005.

Article Snippet: For the inhibitor studies, the following reagents were purchased from the indicated companies: AG-1478 (AG) (an EGFR inhibitor [ 96 ]), 3-α-aminocholestane (3AC) (a SHIP1 inhibitor [ 66 ]), and LY294002 (a pan-PI3K inhibitor [ 97 ]) from Calbiochem (Billerica, MA) and ATN161 (ATN) (an integrin α5β1 inhibitor [ 98 ]) from Selleck Chemicals (Houston, TX).

Techniques: Activation Assay, Infection, Western Blot, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: Human Cytomegalovirus Glycoprotein-Initiated Signaling Mediates the Aberrant Activation of Akt

doi: 10.1128/JVI.00167-20

Figure Lengend Snippet: Proposed model for activation of monocyte receptors by HCMV glycoproteins required for Akt phosphorylation and subsequent survival of infected monocytes. During HCMV entry into monocytes, gB and gH directly bind EGFR and integrin β1, respectively. Activation of either receptor can also lead to the cross-activation of the other receptor. Ultimately, activation of EGFR by HCMV results in the recruitment of the p85 and p110β subunits of PI3K to initiate signaling from PI(3,4,5)P3. However, the concomitant recruitment of SHIP1 to integrin β1 diverts signaling away from canonical PI(3,4,5)P3 signaling to a noncanonical PI(3,4)P2-mediated activation of Akt. The nonclassical activation of Akt leads to the upregulation of a select subset of Akt-dependent antiapoptotic proteins required for the survival of HCMV-infected monocytes.

Article Snippet: For the inhibitor studies, the following reagents were purchased from the indicated companies: AG-1478 (AG) (an EGFR inhibitor [ 96 ]), 3-α-aminocholestane (3AC) (a SHIP1 inhibitor [ 66 ]), and LY294002 (a pan-PI3K inhibitor [ 97 ]) from Calbiochem (Billerica, MA) and ATN161 (ATN) (an integrin α5β1 inhibitor [ 98 ]) from Selleck Chemicals (Houston, TX).

Techniques: Activation Assay, Infection

Journal: Viruses

Article Title: Human Cytomegalovirus Mediates Unique Monocyte-to-Macrophage Differentiation through the PI3K/SHIP1/Akt Signaling Network

doi: 10.3390/v12060652

Figure Lengend Snippet: HCMV engages SHIP1 to induce monocyte-to-macrophage differentiation. ( A ) Human peripheral blood monocytes were pretreated for 1 h with a SHIP1 inhibitor (3AC) at 10 μM. Cells were mock- or HCMV-infected for 48 h, and the percent of cells positive for M1 (CD80 and CD86) and M2 (CD16 and CD163) macrophage markers was measured by flow cytometry. ( B ) Monocytes were pretreated for 1 h with 3AC at 10 μM, then mock- or HCMV-infected for 48 h. Viability was measured through PI and annexin V staining by flow cytometry. ( A , B ) Results are representative of 3–6 independent experiments using monocytes from different donors.

Article Snippet: For the inhibitor studies, the following reagents were used: MK-2206 2HCL (MK; an Akt inhibitor), BYL-719 (BYL; a p110α inhibitor), TGX-221 (TGX; a p110β inhibitor), and CAL-101 (CAL; a p110δ inhibitor) from Selleckchem (Houston, TX, USA), LY-294002 (LY; a pan-PI3K inhibitor) from Calbiochem (Billerica, MA, USA), 3-α-aminocholestane (3AC; a SHIP1 inhibitor) from Echelon Biosciences (Salt Lake City, UT, USA), z-DEVD-fmk (a caspase 3 inhibitor) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and z-VAD-fmk (a pan-caspase inhibitor) from Selleckchem (Houston, TX, USA).

Techniques: Infection, Flow Cytometry, Staining