Journal: Mechanisms of ageing and development

Article Title: Suppressive Effects of Sirtinol on Human Cytomegalovirus (hCMV) Infection and hCMV-Induced Activation of Molecular Mechanisms of Senescence and Production of Reactive Oxygen Species

doi: 10.1016/j.mad.2015.12.005

Figure Lengend Snippet: Effect of sirtinol treatment on cell viability of low passage WI-38 cells (30PD) (panel A) and effects of hCMV infection alone or hCMV infection with pretreatment of sirtinol on morphological changes of low passage WI-38 cells (panel B). For panel A, WI-38 cells (3×104) were cultured in DEME-0.2% FBS for 48–72 h and then treated with sirtinol at indicated concentration for 48 h and then with MTT for 4 h. The reaction was stopped by DMSO and cell viability was determined spectrophotometrically at 490 nm. For panel B, under the same culture condition, WI-38 cells were pretreated with sirtinol (20μM) for 2h followed by hCMV inoculation at MOI of 0.01 and cultured for additional 7 days. Representative photographs shown are from 4 repeated experiments taken at day 7 post infection (dpi) using a digital camera with experimental conditions stated on each photograph.

Article Snippet: Cell Culture and hCMV infection Primary human embryonic lung diploid fibroblasts (WI-38) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Infection, Cell Culture, Concentration Assay

Journal: Mechanisms of ageing and development

Article Title: Suppressive Effects of Sirtinol on Human Cytomegalovirus (hCMV) Infection and hCMV-Induced Activation of Molecular Mechanisms of Senescence and Production of Reactive Oxygen Species

doi: 10.1016/j.mad.2015.12.005

Figure Lengend Snippet: Effects of sirtinol on hCMV viral replication in WI-38 cells. Low passage WI-38 cells (30PD) (3×104) were cultured in DEME-0.2% FBS for 48–72 h and then treated with sirtinol (20μM) for 2h before hCMV inoculation (MOI of 0.01). Cells were harvested at indicated time points for viral DNA copies determined using qPCR. Data were obtained from three independent experiments. *P<0.001 vs hCMV alone.

Article Snippet: Cell Culture and hCMV infection Primary human embryonic lung diploid fibroblasts (WI-38) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Cell Culture

Journal: Mechanisms of ageing and development

Article Title: Suppressive Effects of Sirtinol on Human Cytomegalovirus (hCMV) Infection and hCMV-Induced Activation of Molecular Mechanisms of Senescence and Production of Reactive Oxygen Species

doi: 10.1016/j.mad.2015.12.005

Figure Lengend Snippet: Effects of sirtinol on expression of hCMV viral immediate early (IE) and early UL44 protein expression in young WI-38 fibroblasts (30 PD). Serum starved low passage WI-38 cells (30PD) were treated with sirtinol at the indicated concentrations added 2h before (−2h) or at 10μM added 2h after (+2h) hCMV inoculation (MOI of 0.01), and then were harvest at 72 hour post infection (hpi) for protein expression using Western blot analysis. Beta-actin expression levels are shown in both panels as loading control.

Article Snippet: Cell Culture and hCMV infection Primary human embryonic lung diploid fibroblasts (WI-38) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Infection, Western Blot, Control

Journal: Mechanisms of ageing and development

Article Title: Suppressive Effects of Sirtinol on Human Cytomegalovirus (hCMV) Infection and hCMV-Induced Activation of Molecular Mechanisms of Senescence and Production of Reactive Oxygen Species

doi: 10.1016/j.mad.2015.12.005

Figure Lengend Snippet: SA-β-Gal staining ofWI-38 cells under the following conditions: replicative senescent cells at 52 population doublings (52 PD, upper right panel) as a positive control; Low passage or young cells at PD30 either with Mock infection control (upper left panel), HCMV infection alone at MOI of 0.01 (lower left panel), or same hCMV infection with pretreatment of sirtinol (20μM, lower right panel), all at 4 dpi. Representative photos shown are from three repeated experiments.

Article Snippet: Cell Culture and hCMV infection Primary human embryonic lung diploid fibroblasts (WI-38) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Staining, Positive Control, Infection, Control

Journal: Mechanisms of ageing and development

Article Title: Suppressive Effects of Sirtinol on Human Cytomegalovirus (hCMV) Infection and hCMV-Induced Activation of Molecular Mechanisms of Senescence and Production of Reactive Oxygen Species

doi: 10.1016/j.mad.2015.12.005

Figure Lengend Snippet: (A) Effects of sirtinol on hCMV-induced expression of Rb, pRb Ser780, p16INK4, and p53 in WI-38 fibroblasts at 72 hours post infection (hpi). Serum starved low passage WI-38 cells (PD30) were treated by sirtinol at indicated concentration added 2h before hCMV inoculation (MOI 0.01) (−2h) and then were harvested at 72 hpi for expression of the above molecules using Western blot analysis. Beta-actin expression level is shown as loading control. Representative images were acquired from three different experiments. (B) Quantitative analysis of the protein levels of Rb, pRbSer780, p16INK4, and p53. Bars represent relative protein levels counted as D1/D0 (the value for Mock was set as 1.0), where D0 and D1 stand for the optical density of beta-actin ladder and sample ladder, respectively. The optical density for each ladder was calculated by Image J soft ware. Data were obtained from three independent experiments. *P < 0.05 versus Mock group; **P < 0.01 versus Mock group; #P < 0.05 versus hCMV alone group; ##P < 0.01 versus hCMV alone group.

Article Snippet: Cell Culture and hCMV infection Primary human embryonic lung diploid fibroblasts (WI-38) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Infection, Concentration Assay, Western Blot, Control

Journal: Mechanisms of ageing and development

Article Title: Suppressive Effects of Sirtinol on Human Cytomegalovirus (hCMV) Infection and hCMV-Induced Activation of Molecular Mechanisms of Senescence and Production of Reactive Oxygen Species

doi: 10.1016/j.mad.2015.12.005

Figure Lengend Snippet: Effects of sirtinol on hCMV-induced ROS production in low passage WI-38 cells (30PD). Serum starved young WI-38 cells (30PD) were mock infection treated, hCMV infection alone (MOI 0.01), or pretreated with sirtinol at indicated concentrations added 2h before hCMV inoculation (MOI 0.01), and then were harvested at 4 dpi for ROS measurement by Flow cytometry analysis using probe H2DCFDA (% of ROS production shown on the Y axis). Data were obtained from three independent experiments. ##P<0.01, vs Mock infection group;*P<0.05, vs hCMV infection alone group.

Article Snippet: Cell Culture and hCMV infection Primary human embryonic lung diploid fibroblasts (WI-38) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Infection, Flow Cytometry