Journal: mBio

Article Title: Nuclear Herpesvirus Capsid Motility Is Not Dependent on F-Actin

doi: 10.1128/mBio.01909-14

Figure Lengend Snippet: Representative members of all Herpesviridae subfamilies show intranuclear capsid motility. MEF cells constitutively expressing Lifeact-EGFP were infected with HSV-1 mRFP-VP26, PRV mRFP-VP26, MCMV S-mCherry-SCP, or MHV-68 mCherry-ORF65 at a multiplicity of infection (MOI) of at least 5. Live cell spinning disk microscopy with at least 3.7 frames per second was performed in one confocal section after the first capsids appeared in the nucleus at 6 hpi, 4 hpi, 20 hpi, or 8 hpi, respectively. The right column depicts a temporal maximum projection of at least 20 frames representing 5.4 s. The degree of signal smearing indicates the degree of capsid motility. Scale bar indicates 5 µm.

Article Snippet: MEF cells as well as PK15 cells (ATCC CCL-33) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum under standard conditions.

Techniques: Expressing, Infection, Microscopy

Journal: mBio

Article Title: Nuclear Herpesvirus Capsid Motility Is Not Dependent on F-Actin

doi: 10.1128/mBio.01909-14

Figure Lengend Snippet: Latrunculin A is the only drug that inhibits nuclear capsid motility. (A) MEF cells constitutively expressing Lifeact-EGFP were infected with HSV-1 mRFP-VP26, PRV mRFP-VP26, MCMV S-mCherry-SCP, or MHV-68 mCherry-ORF65 at an MOI of at least 5. LatA at 4.7 µM (2 µg/ml) (red) was added for 1 h or cells were mock treated (black) at 6 hpi, 4 hpi, 20 hpi, or 8 hpi. (B) MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. Cells at 4 hpi were either mock treated (black) or incubated for 1 h with 4.7 µM (2 µg/ml) LatA (red), 1 µM Jaspla (green), or 2 µM MLB (blue). Oblique laser microscopy was performed with 100 frames per second for 10 s, and resulting time-lapse videos of infected nuclei were subjected to autocorrelation analysis. Solid lines indicate mean values; dashed lines indicate the means ± standard deviations. (A) HSV-1, 19 cells mock, 15 cells LatA; PRV, 23 cells mock, 13 cells LatA; MCMV, 21 cells mock, 8 cells LatA; MHV-68, 21 cells mock, 17 cells LatA. (B) Twenty-three cells mock, 14 cells LatA, 29 cells Jaspla, 30 cells MLB. Scale bar indicates 5 µm.

Article Snippet: MEF cells as well as PK15 cells (ATCC CCL-33) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum under standard conditions.

Techniques: Expressing, Infection, Incubation, Microscopy

Journal: mBio

Article Title: Nuclear Herpesvirus Capsid Motility Is Not Dependent on F-Actin

doi: 10.1128/mBio.01909-14

Figure Lengend Snippet: Latrunculin A induces nuclear actin rods which coincide with an inhibition of intranuclear capsid dynamics. MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. Cells at 4 hpi were either mock treated or incubated for 1 h with 4.7 µM (2 µg/ml) LatA. Cells were fixed and nuclei were stained with DAPI and imaged by confocal laser scanning microscopy (A) or cells were subjected to live-cell spinning disk microscopy (B). A maximum intensity projection of about 1 µm each is shown in the first row of panel A, and a projection of about 8 µm is shown in the second row. (B) One confocal section; the right column represents a temporal maximum projection of 50 frames at 3.7 frames/s. The degree of signal smearing indicates capsid motility. Scale bars indicate 5 µm.

Article Snippet: MEF cells as well as PK15 cells (ATCC CCL-33) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum under standard conditions.

Techniques: Inhibition, Expressing, Infection, Incubation, Staining, Confocal Laser Scanning Microscopy, Microscopy

Journal: mBio

Article Title: Nuclear Herpesvirus Capsid Motility Is Not Dependent on F-Actin

doi: 10.1128/mBio.01909-14

Figure Lengend Snippet: Actin rod formation causes intranuclear capsid motility to stop. MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. (A) Cells at 4 hpi were either mock treated (black) or incubated for 1 h with 4.7 µM (2 µg/ml) LatA (red) or 2 µM of CytD as well as 4.7 µM (2 µg/ml) LatA (green). Oblique laser microscopy was performed with 100 frames/s for 10 s, and resulting time-lapse videos of infected nuclei were subjected to autocorrelation analysis. Solid lines indicate mean values; dashed lines indicate the means ± standard deviations (23 cells mock, 7 cells CytD and LatA). (B) Cells treated with LatA or with LatA and after 1 h with CytD were fixed, stained with DAPI and fluorescent phalloidin, and analyzed by confocal laser scanning microscopy. One confocal section is shown. Scale bar indicates 5 µm.

Article Snippet: MEF cells as well as PK15 cells (ATCC CCL-33) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum under standard conditions.

Techniques: Expressing, Infection, Incubation, Microscopy, Staining, Confocal Laser Scanning Microscopy

Journal: mBio

Article Title: Nuclear Herpesvirus Capsid Motility Is Not Dependent on F-Actin

doi: 10.1128/mBio.01909-14

Figure Lengend Snippet: Latrunculin A-induced actin rods bind the major capsid protein. MEF cells constitutively expressing Lifeact-EGFP were infected with PRV mRFP-VP26. Cells at 4 hpi were either mock treated (mock) or incubated for 1 h with 4.7 µM (2 µg/ml) LatA (LatA). Cells were lysed, and the precleared lysates were incubated with antibodies directed against glutathione S -transferase (GST) or GFP, detecting Lifeact-EGFP (LA-GFP) and protein A/G beads for 1.5 h at 4°C. Bead-bound immunoprecipitates were separated from supernatant and washed extensively. Ten percent of lysate (input) and supernatants (sup) and 50% of immunoprecipitated protein (pellet) were subjected to SDS-PAGE and Western blotting using antiactin, anti-GFP, and anti-VP5 antibodies.

Article Snippet: MEF cells as well as PK15 cells (ATCC CCL-33) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum under standard conditions.

Techniques: Expressing, Infection, Incubation, Immunoprecipitation, SDS Page, Western Blot

Journal: Molecular Medicine Reports

Article Title: Effect of cellular mass on chondrogenic differentiation during embryoid body formation

doi: 10.3892/mmr.2018.9272

Figure Lengend Snippet: Size of EBs and time (days) in the suspension culture affects the expression of germ layer specific genes. During suspension cell culture, the pluripotency markers, (A) NANOG and (B) SOX2, decreased among all studied variants. Gene expression analysis indicated that the mesoderm, (C) Brachyury and (D) MIXL1, germ layer was favoured in EBs formed from 500 and 1,000 cell wells when compared with the 1,500 and 2,000 cell wells at the end of suspension culture (day 15). The mesenchymal marker (E) SMA and endoderm marker (F) FOXA2 did not predominantly express in the studied variants. Gene expression analysis indicated that the ectoderm germ layer, (G) VIM and (H) PAX6, was favoured in EBs formed from 500 and 1,000 cell wells when compared with the 1,500 and 2,000 cell wells at the end of the suspension culture (day 15). The y-axis represents the relative expression of analysed genes, normalized to the BG01V cell line. Data are presented as the mean ± standard deviation. *P<0.05, as indicated. EBs, embryoid bodies; SOX2, sex determining region Y-box 2; MIXL1, mix paired-like homeobox; α-SMA, α-smooth muscle actin; FOXA2, forkhead box protein A2; VIM, vimentin; PAX6, paired box 6.

Article Snippet: The BG01V hESC line (American Type Culture Collection, Manassas, VA; USA) was cultured in mitomycin-C-treated mouse embryonic fibroblasts (MEFs; passage 3; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) seeded on a culture dish previously coated with MatrigelTM (Corning Incorporated, Corning, NY, USA).

Techniques: Suspension, Expressing, Cell Culture, Gene Expression, Marker, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Effect of cellular mass on chondrogenic differentiation during embryoid body formation

doi: 10.3892/mmr.2018.9272

Figure Lengend Snippet: Smaller EBs exhibit more prochondrogenic outcomes. Following collection of EBs from suspension culture, the 3 week chondrogenic differentiation protocol was performed and gene expression was analysed. Expression of (A) NANOG and (B) SOX2 decreased in all studied variants. Expression of (C) T, (D) SOX9 and (E) FGFR3 was significantly higher in EBs formed in 500-cell wells when compared with 2,000-cell wells. (F) CD44 was significantly expressed in differentiated 2,000-cell wells EB collected on the fifth day of suspension culture. (G) The low level of COL1A2 expression was observed in all studied variants following chondrogenic differentiation. (H) Expression of COL2A1 was significantly higher in EBs formed in 500-cell wells when compared with 2,000-cell wells. (I) ACAN expression increased only in EBs collected on day 5. Overall, 15 days of suspension culture resulted in a lower expression of analysed genes in chondrogenically differentiated EBs formed from 500- and 2,000-cell wells. The y-axis represents the relative expression of analysed genes, normalized to the BG01V or HC-402-05a cell line. Data are presented as the mean ± standard deviation. *P<0,05, as indicated. EBs, embryoid bodies; hESC, human embryonic stem cells; SOX, sex determining region Y-box; T, brachyury gene; FGFR3, fibroblast growth factor receptor 3; CD44, cluster of differentiation 44; COL, collagen; ACAN, aggrecan.

Article Snippet: The BG01V hESC line (American Type Culture Collection, Manassas, VA; USA) was cultured in mitomycin-C-treated mouse embryonic fibroblasts (MEFs; passage 3; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) seeded on a culture dish previously coated with MatrigelTM (Corning Incorporated, Corning, NY, USA).

Techniques: Suspension, Gene Expression, Expressing, Standard Deviation

Journal: The Journal of infectious diseases

Article Title: Immunomodulatory Function of Interleukin 28B during primary infection with cytomegalovirus.

doi: 10.1093/infdis/jiu144

Figure Lengend Snippet: Figure 2. IL-28B SNP in fibroblasts reduces CMV replication by increased CMV-induced IFN-α and reduced IFN-λ responses. HFF cells with different IL-28B genotypes (HS97FS rs12979860: CC, rs8099917: TT; SCRC1041 rs12979860: CT, rs8099917: TT; and CCD1112SK rs12979860: TT, rs8099917: TT) were infected with human CMV (Towne strain, MOI 0.3). mRNA expression is relative to HPRT and non-infected controls, and 3 independent experiments were performed unless otherwise indicated. Bars or symbols indicate median value, whiskers the interquartile ranges unless otherwise indicated. The rs8099917 was the same for all tested cell lines (major allele genotype). A, CMV replication in HFF cells measured with plaque assays. Supernatants were collected at day 4 from 9 individual experiments. Viral growth was determined using plaque assays as described, and viral load is expressed as plaque- forming units (PFU) per mL of supernatant. B, IFN-λ mRNA-expression of infected fibroblasts. IL-28A, IL-28B, and IL-29 mRNA is expressed at 24 hours. C, IFN-α2 mRNA expression of stimulated or infected fibroblasts. In addition to infection (as previously described), a stimulation with poly I:C (50 μg/mL) was performed. mRNA expression of IFN-α2 is shown at 12 hours. D, Proinflammatory ISG response (IFIT2 and OAS1) during CMV infection of HFF cells. E, Proinflammatory ISG response (Mx1 an ISG15) during CMV infection of HFF cells. F, Anti-inflammatory ISG response (SOCS1 and USP18) during infection and stimulation of HFF cells. Same infection condition as previously describe were used, but in this experiment stimulation was performed with transfected poly I:C (7.5 μg/mL). Abbreviations: CMV, cytomegalovirus; HFF, human foreskin fibroblast; IFN, interferon; IL, interleukin; ISG, IFN-stimulated gene; mRNA, messenger RNA.

Article Snippet: Human foreskin fibroblasts (HFF) from ATCC (HS97FS, SCRC1041, and CCD1112SK, Supplementary Table 1A) and peripheral blood mononuclear cells (PBMCs) were grown in standard media.

Techniques: Infection, Expressing, Transfection

Journal: PLoS ONE

Article Title: Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

doi: 10.1371/journal.pone.0039329

Figure Lengend Snippet: (A) Phase-contrast images of mESCs treated with LIF (1,000 U/mL), or 10, 15, and 20 µM SB-216763 for more than a month. All doses of SB-216763 maintained mESCs as compact, elliptical or circular colonies characteristic of pluripotent cells. Morphology for mESC colonies maintained with SB-216763 was comparable to those in LIF supplemented medium. Scale bars represent 100 µm. (B) Twenty thousand mESCs from each treatment were seeded in a 6-well chamber at each passage and monitored for the percentage of pluripotent-like colonies two days after each passage. Data are expressed as the mean of five fields analyzed ± SEM. The percentage of pluripotent-like colonies in vehicle-treated cultures was significantly different from 15 µM and 20 µM SB-216763 samples at passage 4 (p<0.05) and from all treated samples from passage 5 onwards (p<0.05). (C) The average number of the total colonies and total pluripotent-like colonies observed between passages 4–7. The number of pluripotent-like colonies in vehicle-treated cultures was significantly lower than that for all other treatments (p<0.05). None of the SB-216763-treated samples were significantly different from LIF-treated mESCs for either the total number of colonies or the total number of pluripotent-like colonies.

Article Snippet: J1 mESC (SCRC-1010, ATCC) and MilliTrace Nanog GFP Reporter mESC (SCR089, Millipore) cell lines were cultured on MEFs that were mitotically inactivated using 10 μg/mL mitomycin C (Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

doi: 10.1371/journal.pone.0039329

Figure Lengend Snippet: The graph also shows expression of these markers in EBs derived from month-old LIF and SB-216763 mESCs. SYBR Green qPCR results for these four genes were normalized to β-actin housekeeping gene using qbasePLUS software. SB-216763 treated mESCs expressed Lrh-1 and Sox2 about 3-fold higher than LIF treated mESCs. Nanog expression was also moderately higher in the SB-216763-treated mESCs, whereas Oct-4 expression was nearly 50% higher in LIF-treated mESC. Expression of these genes in differentiated cells was generally lower than the mESCs maintained with either LIF or 10 µM SB-216763. The data are average ± S.D. of technical replicates from one of the two experiments.

Article Snippet: J1 mESC (SCRC-1010, ATCC) and MilliTrace Nanog GFP Reporter mESC (SCR089, Millipore) cell lines were cultured on MEFs that were mitotically inactivated using 10 μg/mL mitomycin C (Sigma).

Techniques: Expressing, Derivative Assay, SYBR Green Assay, Software

Journal: PLoS ONE

Article Title: Glycogen Synthase Kinase 3 (GSK3) Inhibitor, SB-216763, Promotes Pluripotency in Mouse Embryonic Stem Cells

doi: 10.1371/journal.pone.0039329

Figure Lengend Snippet: MESCs maintained with either LIF or 10 µM SB-216763 for more than a month yielded pluripotent-like colonies. Immunostaining revealed that Nanog expression (green fluorescence) was homogeneous in mESC colonies maintained with SB-216763, whereas LIF-treated mESC colonies often displayed incomplete Nanog staining. Three representative colonies from each treatment are shown. Scale bars represent 40 µm.

Article Snippet: J1 mESC (SCRC-1010, ATCC) and MilliTrace Nanog GFP Reporter mESC (SCR089, Millipore) cell lines were cultured on MEFs that were mitotically inactivated using 10 μg/mL mitomycin C (Sigma).

Techniques: Immunostaining, Expressing, Fluorescence, Staining