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  • 99
    ATCC hela cells
    SF3A2 and PRP31 interact with <t>HEC1</t> only during the M phase in the presence of MTs. ( A ) Co-IP analysis showing that SF3A2 and PRP31 interact with HEC1 (input was 8% in all experiments). HC and LC, IgG heavy and light chains, respectively. ( B ) Reciprocal Co-IPs showing that SF3A2 and PRP31 interact with each other. ( C ) HEC1-SF interaction in <t>HeLa</t> cells synchronized using a double thymidine block. Cells were examined at different times after release from the block (see Materials and methods for details). As, Asynchronous cells; S (0 hr), G2 (5 hr) and M (8 hr) indicate the phases of the cell cycle; PHH3 is the mitotic marker phosphohistone H3; H3, histone H3; LC and HC are the IgG light and heavy chains. Tubulin and actin are loading controls. Note that SF3A2 and PRP31 coprecipitate with HEC1 only when the sample contains mitotic cells. ( D ) SF-interaction in HeLa cells synchronized using nocodazole treatment. Cells were collected by mitotic shake-off at 0 hr or 6 hr after nocodazole removal; Ad, Adherent cells. Note that SF3A2 and PRP31 do not interact with HEC1 in cells with depolymerized MTs (0 hr).
    Hela Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 25295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore hela cells
    S. aureus induced dose-dependent <t>G2/M</t> phase transition delay in the subsequent G2/M phase. A. <t>HeLa</t> cells were synchronized by DTB and were exposed to S. aureus bacteria (MW2) at MOI 20∶1 or to the media alone for 2 h, followed by incubation in cDMEM-Gent100 for 2 h, and subsequently incubated for an additional 12 h, 14 h, 18 h, 20 h and 24 h. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. Each experiment was performed four times. The number of cells in different phases is presented on the histograms. The values shown are those of a representative assay out of the four assays performed. Exposure of the cells to S. aureus bacteria induced a G2/M phase transition delay in a time-dependent manner. B . HeLa cells were synchronized by DTB and were exposed to S. aureus bacteria (MW2) at different MOIs ranging from 5∶1 to 20∶1, followed by incubation in cDMEM-Gent100 for 2 h, and further incubated for an additional 20 h. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. The percentage of cells is presented on the graph as a function of bacterial concentration. The data correspond to a representative experiment out of the three assays performed and are presented as mean +/− SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. (*) P-values
    Hela Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hela cells
    <t>Hec1</t> S69 phosphorylation is reduced at kinetochores in response to microtubule depolymerization but not in response to microtubule stabilization. (A–C) Immunofluorescence images showing kinetochore localization of pS69 in <t>HeLa</t> cells after incubation with 50 nM taxol for 5 h (A), 10 µM taxol for 30 min (B), and 1 µM nocodazole for 1 h (C). (D) Immunofluorescence images of control cells and cells treated with 1 µM nocodazole for 1 h and stained with an antibody to active Aurora A kinase phosphorylated at T288 (pT288). Shown on the right of each panel is the quantification of either pS69 kinetochore fluorescence intensity (A–C) or total cellular pAAK fluorescence intensity (D). For all conditions in A–C, ≥200 kinetochores were measured from ≥20 cells. For the experiment shown in D, total fluorescence was measured from ≥20 cells per condition. Error bars indicate SD. Bars, 10 µm.
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    93
    Becton Dickinson hela cells
    Effects of ligase inhibitors on <t>DNA</t> synthesis, cell viability and DNA damage The effects of L67, (closed circles) L82, (closed squares) or L82-G17 (closed triangles) on BrdU incorporation, cell viability and formation of γH2AX foci were determined as described in Materials and Methods. A . Asynchronous <t>HeLa</t> cells were treated with the ligase inhibitors for 4 hours and were then assayed for BrdU incorporation. Results of three independent assays are shown graphically. B . HeLa cells were incubated with the inhibitors for 5 days prior to the determination of cell viability using the MTT assay. Results of three independent assays are shown graphically. Data points at 20 and 30 µM are significant at p
    Hela Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 2551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hela s3 cells
    Analysis of extracts of <t>HeLa</t> <t>S3</t> cells by reverse-phase HPLC. (A) Analysis of pure JA28-TP including the full UV spectrum of the JA28-TP peak at 3.6 min (inlay). mAU, milli-absorbance units. (B) Analysis of extracts of untreated HeLa S3 cells including
    Hela S3 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    MatTek hela cells
    RASSF4 regulates the levels of PI(4)P and PIP5Ks at the PM. (A) Confocal images of <t>HeLa</t> cells expressing N-PH-ORP5-GFP and treated with either siControl of siRASSF4. Bar, 5 µm. Relative N-PH-ORP5-GFP fluorescence intensity in the PM from > 30 cells across three independent experiments was evaluated. Means ± SEM are shown. (B) Relative PM PI(4)P levels were detected by anti-PI(4)P antibody in HeLa cells treated with either siControl or siRASSF4 from ∼30 cells across three independent experiments. Means ± SEM are shown. (C) Confocal images of HeLa cells expressing YFP-PIP5K1B, treated with either siRASSF4 or siControl. Bar, 5 µm. Relative YFP-PIP5K1B fluorescence intensity in the PM from > 30 cells across two independent experiments was evaluated. Means ± SEM are shown. (D) A diagram of the rapamycin-inducible approach that selectively increases PI(4,5)P 2 at the PM. (E) Relative PM PI(4,5)P 2 levels in HeLa cells transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki (control) were monitored by PI(4,5)P 2 immunostaining. More than 30 cells for each condition across three independent experiments were evaluated. Means ± SEM are plotted. (C–E) Cells were treated with 5 µM rapamycin for 10 min after the <t>transfection</t> and before the experiments. (F) The density of stable ER puncta in HeLa cells treated with the indicated siRNA and expressing YFP-KDEL, mCherry-KRAS-tail, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. More than 15 cells for each condition across two independent experiments were analyzed. Plotted are means ± SEM. (G) HeLa cells were transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. Cells were stimulated with 100 µM His, 1 µM TG, and 2 mM EGTA; 2 mM Ca 2+ was added 6 min after stimulation. Mean peak values of Fura-2 ratio ± SEM of the Ca 2+ add-back phase are plotted from > 300 cells for each condition across three independent experiments. **, P
    Hela Cells, supplied by MatTek, used in various techniques. Bioz Stars score: 93/100, based on 1758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery hela cells
    Partial nuclear distribution of Ago2 in <t>HeLa</t> and HaCaT cells. A , specificity of anti-Ago2 antibody in Western blotting of Ago2 expressed from HeLa cells <t>transfected</t> with a nonspecific (−) or Ago2-specific (+ si-Ago2 ) siRNA. Total cell extract
    Hela Cells, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 1648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences hela cells
    Interaction of intracellular <t>salusin-β</t> with a membrane-associated β-actin-profilin complex. ( a ) Whole cell lysates obtained from the human cell lines <t>HeLa,</t> NB-1, THP-1 and U937 were immunoprecipitated with anti-profilin1/2 or anti-β-actin IgG and immunoblotted with an anti-salusin-β antibody. The upper and lower panels represent 3- and 30-min exposures, respectively, of the same immunoblot. ( b ) Whole cell lysates were immunoprecipitated with anti-profilin1/2 IgG and immunoblotted with the anti-salusin-β antibody (left panel), or immunoprecipitated with anti-PIP 2 IgG and immunoblotted with anti-profilin1/2 IgG. ( c ) Immunofluorescence staining of NB-1 cells with the anti-salusin-β antibody reveals marked staining in the cell periphery. ( d ) Quiescent HeLa and THP-1 cells stimulated by 10% fetal bovine serum and endothelin-1 (10 −9 M), respectively (upper and middle panels) and growing THP-1 cells stimulated by TNF-α (100 ng/ml, lower panel) for the indicated times were fractionated by differential centrifugation. Plasma membrane (PM)-, low-density microsome (LDM)-, mitochondria/nucleotide (M/N)-, and high-density microsome (HDM)-enriched fractions were immunoprecipitated with anti-profilin1/2 IgG, separated by SDS-PAGE and analyzed by immunoblotting with the anti-salusin-β antibody.
    Hela Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 1077 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa hela tet off cells
    The predicted LARP4 ARE confers instability on β-globin reporter mRNA. <t>HeLa</t> <t>Tet-Off</t> cells were cotransfected with a vector containing GFP under the control of a constitutive CMV promoter, along with β-globin constructs under the control
    Hela Tet Off Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 829 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Mirus Bio hela cells
    Time course analysis for luciferase activity and quantitative RT-PC R. (A and B) Time course of luciferase protein and mRNA expression in <t>transfected</t> <t>HeLa</t> cells. HeLa cells were transfected with SV40 or proximal plasmids respectively and RNA was extracted at the indicated time point and reverse transcribed. Another series of wells was used for standard luciferase assays. Real-time PC R was performed using primers specific for luciferase ORF. Luciferase mRNA expression was calculated using the 2 −ΔΔCt method as fold over the SV40 mRNA at 12 hr after normalized with GAPDH. Values shown are the mean ± SD (n = 3), and standardized to the zero time point. (C) Quantitative RT-PC R for proximal and distal 3′ UTR luciferase mRNAs. HeLa cells were transfected with plasmids as indicated for 48 hours. Luciferase mRNA expression was determined by real-time quantitative RT-PC R. The relative differences in luciferase expression were calculated using the 2 −ΔΔCt method by comparing with the mRNA expression of the SV40 plasmid after normalization with GAPDH. The values shown are the mean ± SD (n = 3). p
    Hela Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 94/100, based on 1200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polyplus Transfection hela cells
    <t>Atf4</t> but not Xbp-1s stimulates NLRP1 gene expression during ER stress. (A) <t>HeLa</t> cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.
    Hela Cells, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 1144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DSMZ hela cells
    Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) <t>HCT116</t> wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in <t>HeLa</t> cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( 12 , 15 ), genes upregulated by HPV E7 ( 64 ) and genes downregulated by HPV E2 proteins repressing E7 ( 65 , 66 ).
    Hela Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 92/100, based on 451 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology hela cells
    Activation of the ERK or the JNK pathway inhibits GR transcriptional activation. ( A ) Selective activation of ERK and JNK in <t>HeLa</t> cells by f-sos and racQ61L. pRK5-HA ERK (1.5 μg), pRK5-M2 JNK (1.5 μg), along with pRK5-HA f-sos (4 μg) or pCMV5-racQ61L (4 μg) or an empty pRK5 vector (4 μg), where indicated, was transfected into HeLa cells (2.5 × 10 5 cells/60-mm dish) using Lipofectamine. Transfected cells were treated with 100 nM Dex where indicated and harvested 8 h later. To visualize HA ERK and HA f-sos, WCE from ERK-transfected cells were immunoblotted with anti-HA mouse monoclonal antibody (left panel). FLAG-tagged JNK was immunoprecipitated from WCE by using the FLAG-specific mouse monoclonal antibody M2 and used in vitro to phosphorylate recombinant purified GST–c-Jun. Phosphorylated proteins were separated on 10% <t>SDS/polyacrylamide</t> gels, stained with Coomassie blue to verify equal amount of GST–c-Jun in each lane, and autoradiographed (right panel). ( B ) Inhibition of GR transcriptional enhancement by activated ERK and JNK. HeLa cells were transfected as described in A ) and normalized to β-Gal activity. ( C ) Level of GR protein is unaffected by JNK or ERK activation. WCE were prepared from a parallel set of transfected cells. Equal amounts of total protein (50 μg/lane) were separated by SDS/PAGE, transferred to Immobilon paper, probed with a GR-specific polyclonal antibody, and visualized by enhanced chemiluminescence.
    Hela Cells, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 969 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Carl Zeiss hela cells
    FRET analysis reveals SBM-dependent interaction of La with nucleolin in the nucleolus. (A) Constructs used for the interaction analysis below were first examined for expression in <t>HeLa</t> cells prior to FRET analysis. Column I shows the yellow channel fluorescence of the <t>YFP-hLa</t> constructs, column II shows the cyan channel fluorescence of the CFP-nucleolin constructs, and column III shows the overlaid views for the indicated constructs. Note that the last row shows CFP-fibrillarin. (B) Acceptor photobleaching was used to monitor FRET between the YFP-hLa (acceptor) constructs and the CFP-nucleolin or CFP-fibrillarin (donor) after specific bleaching of the acceptor with YFP-wavelength light. FRET efficiencies reflective of positive interactions were plotted as bars above the horizontal line (see Materials and Methods). Bars below the horizontal line reflect control measurements in the same cells in nucleolar areas in which there was no prebleaching of the acceptor. At least 30 measurements were collected and quantitated for each bar in the graph. CFP/YFP, CFP alone plus YFP alone; CFP-YFP, CFP-YFP fusion protein.
    Hela Cells, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 93/100, based on 965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hela 229 cells
    Effect of substrate availability on maintenance of infectivity. P. amoebophila and C. trachomatis cells were harvested from infected amoeba and <t>HeLa</t> 229 cell cultures, respectively, and incubated for indicated periods of time in different host-free media. Subsequently, incubated bacteria were used to infect amoebae ( P. amoebophila ) or HeLa 229 cells ( C. trachomatis ), which were then fixed at 48 h or 24 h p.i., respectively. Bacteria were detected by FISH ( P. amoebophila ) or immunostaining ( C. trachomatis ). The observed infectivity, relative to that observed for 2 h incubation in DGM-D ( P. amoebophila ) or 30 min incubation in DGM-D6P ( C. trachomatis ) is depicted in (A) and (C), respectively. Data represent means and standard deviations from at least three replicate host-free incubations. For each sample a minimum of 600 amoebae (A) or 300 HeLa 229 cells (C) was counted. Statistically significant differences compared to the values obtained for DGM-D (A) or DGM-D6P (C) are indicated (ANOVA; ***, p≤0.001; **, p≤0.01; *, p≤0.05). In (B) representative fluorescence and DIC images of amoebae infected with P. amoebophila after 48 h host-free incubation in the indicated media are shown (FISH, red). The bar indicates 10 µm. In (D) representative confocal fluorescence images of HeLa 229 cells infected with C. trachomatis after 2 h host-free incubation in the indicated media are shown. Bacteria were detected by immunostaining (red), host cells and DNA were stained using HCS cytoplasmic stain (grey) and DAPI (blue), respectively. The bar indicates 25 µm.
    Hela 229 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 363 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Olympus hela cells
    The effect of Enhancer nanobody binding on the repeated <t>photoswitching</t> behavior of rsGreens in <t>HeLa</t> cells (500 cycles): ( A ) representative fluorescence photoswitching trace of rsGreen1, inset shows the initial five switching cycles; ( B ) signal of the maximally on-switched frame for each switching cycle scaled to the initial on-state fluorescence; and ( C ) baseline fluorescence (maximally off-switched frame) as fraction of the initial on-state fluorescence.
    Hela Cells, supplied by Olympus, used in various techniques. Bioz Stars score: 93/100, based on 986 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ibidi hela cells
    Confocal microscopy of <t>HeLa</t> cells after <t>transfection</t> with DNA2aD1 – DNA3rD5 (row 1), DNA2rD1 – DNA3aD8 (row 2), DNA2aD2–DNA3aD8 (row 3) and DNA2rD4 – DNA3aD8 (row 4). The visualization was performed using a Leica TCS-SPE (DMi8) inverted microscope with an ACS APO 63×/1.30 oil objective. For DNA2aD1 – DNA3rD5 λ exc = 405 nm (UV laser), λ em = 435–470 nm (blue) and 575–750 nm (yellow), for DNA2rD1 – DNA3aD8 λ exc = 405 nm (UV laser), λ em = 415–550 nm (blue) and 575–750 nm (red), for DNA2aD2–DNA3aD8 λ exc = 488 nm (argon ion laser), λ em = 490–550 nm (green) and 550–675 nm (red), for DNA2rD4 – DNA3aD8 λ exc = 488 nm (argon ion laser), λ em = 490–550 nm (green) and 675–800 nm (red), scale bar = 20 µm.
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    Addgene inc hela cells
    Species-specific differences in primate CMV TRS1 <t>PKR</t> antagonism. (A) <t>HeLa</t> PKR KO, HeLa (human), or BSC40 (Agm) cells were mock infected or infected (MOI 0.1) with WT VacV, VacVΔE3L, or VacVΔE3L recombinants containing HCMV TRS1 , AgmCMV TRS1 , RhCMV TRS1 , or SmCMV TRS1 . At 48 hpi, viral replication was quantified by measuring β-gal activity (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected HeLa PKR KO cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length.
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    93
    Greiner Bio hela cells
    Effect of <t>DNA</t> sequences susceptible or unsusceptible to AAV Rep-mediated nicking on the stable transfection levels with AAVS1 -targeting vectors. ( A ) Structures of the nicking-competent targeting plasmids pA1.p5.GFP.A2, pA1.RBE/trs.GFP.A2 and pA1.trs/RBE.GFP.A2 and that of the nicking-resistant donor constructs pA1.GFP.A2, pA1.RBE/Δtrs.GFP.A2 and pA1.RBEst/trs.GFP.A2. The nucleotide sequence corresponding to the wild-type RBE is shown in black uppercase letters whereas the trs (i.e. the position at which Rep78/68-mediated nicking takes place) is indicated by black lowercase letters and a vertical arrow. The DNA sequence between the RBE and the trs is called the spacer. Targeting plasmid pA1.RBE/Δtrs.GFP.A2 has the dinucleotide at which Rep endonuclease-mediated nicking occurs mutated from TT to AA while in pA1.RBE.st/trs.GFP.A2 a 21-bp stuffer positions the trs at a bigger distance from RBE-bound AAV Rep molecules. For an explanation of the other symbols and elements see the legend of Figure 3 A. ( B ) Representative dot plots of flow cytometric analysis of the frequency of GFP -modified <t>HeLa</t> cells at 37 days post-transfection in cultures initially exposed to pA1.p5.GFP.A2 and ‘empty’ plasmid (p5 + +empty), pA1.p5.GFP.A2 and pGAPDH.Rep68(Y156F) (p5 + +Y156F), pA1.GFP.A2 and pGAPDH.Rep68 (p5 − +Rep68), pA1.RBE/Δtrs.GFP.A2 and pGAPDH.Rep68 (RBE/Δtrs+Rep68), pA1.RBEst/trs.GFP.A2 and pGAPDH.Rep68 (RBEst/trs+Rep68), pA1.p5.GFP.A2 and pGAPDH.Rep68 (p5 + +Rep68), pA1.RBE/trs.GFP.A2 and pGAPDH.Rep68 (RBE/trs+Rep68) or to pA1.trs/RBE.GFP.A2 and pGAPDH.Rep68 (trs/RBE+Rep68). Untransfected HeLa cells were used to set the background of the assay at 0.00% GFP-positive cells (HeLa). For each sample, 10 5 viable single cells were analyzed. ( C ) PCR analysis using primer set #649/#651 on chromosomal DNA extracted from untransfected HeLa cells (HeLa) and from HeLa cells co-transfected with pGAPDH.Rep68 (Rep68) and targeting constructs pA1.GFP.A2 (p5 − ), pA1.RBE/Δtrs.GFP.A2 (RBE/Δtrs), pA1.RBEst/trs.GFP.A2 (RBEst/trs), pA1.p5.GFP.A2 (p5 + ), pA1.RBE/trs.GFP.A2 (RBE/trs) or pA1.trs/RBE.GFP.A2 (trs/RBE). HeLa cells were also co-transfected with pA1.p5.GFP.A2 (p5 + ) and pGAPDH.Rep68(Y156F) or with an ‘empty’ control plasmid (empty). The genomic DNA was isolated at 43 days post-transfection. HPRT1 -specific PCRs served as control for the integrity of the input DNA. ( D ) In vivo nicking assay based on Southern blot analysis of DpnI-resistant extrachromosomal DNA. Episomal DNA was isolated at 4 days post-transfection from 911 cells co-transfected with pGAPDH.Rep68 and pA1.p5.GFP.A2 (lanes 1 and 2), pGAPDH.Rep68 and pA1.RBE/trs.GFP.A2 (lane 4) or pGAPDH.Rep68 and pA1.RBE/Δtrs.GFP.A2 (lane 5). Episomal DNA isolated from 911 cells co-transfected with pGAPDH.Rep68(Y156F) and pA1.p5.GFP.A2 (lane 3) served as a negative control. All cell cultures except for the one represented by lane 2 were exposed to Ad.floxedΨ.F50 at a multiplicity of infection of 25 infectious units per cell. Lane M, Gene Ruler DNA Ladder Mix. Prior to Southern blot analysis, the DNA was digested with DpnI (to fragment the input prokaryotic DNA) and with NcoI. Southern blots were exposed to a radiolabeled GFP -specific probe. The position of the 4.9-kb GFP -containing NcoI fragments derived from de novo synthesized DNA is indicated by an arrow at the right of the autoradiogram.
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    Image Search Results


    SF3A2 and PRP31 interact with HEC1 only during the M phase in the presence of MTs. ( A ) Co-IP analysis showing that SF3A2 and PRP31 interact with HEC1 (input was 8% in all experiments). HC and LC, IgG heavy and light chains, respectively. ( B ) Reciprocal Co-IPs showing that SF3A2 and PRP31 interact with each other. ( C ) HEC1-SF interaction in HeLa cells synchronized using a double thymidine block. Cells were examined at different times after release from the block (see Materials and methods for details). As, Asynchronous cells; S (0 hr), G2 (5 hr) and M (8 hr) indicate the phases of the cell cycle; PHH3 is the mitotic marker phosphohistone H3; H3, histone H3; LC and HC are the IgG light and heavy chains. Tubulin and actin are loading controls. Note that SF3A2 and PRP31 coprecipitate with HEC1 only when the sample contains mitotic cells. ( D ) SF-interaction in HeLa cells synchronized using nocodazole treatment. Cells were collected by mitotic shake-off at 0 hr or 6 hr after nocodazole removal; Ad, Adherent cells. Note that SF3A2 and PRP31 do not interact with HEC1 in cells with depolymerized MTs (0 hr).

    Journal: eLife

    Article Title: Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

    doi: 10.7554/eLife.40325

    Figure Lengend Snippet: SF3A2 and PRP31 interact with HEC1 only during the M phase in the presence of MTs. ( A ) Co-IP analysis showing that SF3A2 and PRP31 interact with HEC1 (input was 8% in all experiments). HC and LC, IgG heavy and light chains, respectively. ( B ) Reciprocal Co-IPs showing that SF3A2 and PRP31 interact with each other. ( C ) HEC1-SF interaction in HeLa cells synchronized using a double thymidine block. Cells were examined at different times after release from the block (see Materials and methods for details). As, Asynchronous cells; S (0 hr), G2 (5 hr) and M (8 hr) indicate the phases of the cell cycle; PHH3 is the mitotic marker phosphohistone H3; H3, histone H3; LC and HC are the IgG light and heavy chains. Tubulin and actin are loading controls. Note that SF3A2 and PRP31 coprecipitate with HEC1 only when the sample contains mitotic cells. ( D ) SF-interaction in HeLa cells synchronized using nocodazole treatment. Cells were collected by mitotic shake-off at 0 hr or 6 hr after nocodazole removal; Ad, Adherent cells. Note that SF3A2 and PRP31 do not interact with HEC1 in cells with depolymerized MTs (0 hr).

    Article Snippet: The kinetochore localization of HEC1 after Sf3A2 and Prp31 RNAi in HeLa cells should also be investigated in cells treated with nocodazole (to standardize the conditions) and properly quantified relative to constitutive kinetochore components (e.g. using ACA serum from CREST patients).

    Techniques: Co-Immunoprecipitation Assay, Blocking Assay, Marker

    SF3A2 localization in HeLa cells Immunolocalization of SF3A2 (red) during mitosis of HeLa cells stained for tubulin (green) and DNA (blue). Note the nuclear localization of SF3A2 in prophase and late telophase cells. Scale bar, 5 μm.

    Journal: eLife

    Article Title: Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

    doi: 10.7554/eLife.40325

    Figure Lengend Snippet: SF3A2 localization in HeLa cells Immunolocalization of SF3A2 (red) during mitosis of HeLa cells stained for tubulin (green) and DNA (blue). Note the nuclear localization of SF3A2 in prophase and late telophase cells. Scale bar, 5 μm.

    Article Snippet: The kinetochore localization of HEC1 after Sf3A2 and Prp31 RNAi in HeLa cells should also be investigated in cells treated with nocodazole (to standardize the conditions) and properly quantified relative to constitutive kinetochore components (e.g. using ACA serum from CREST patients).

    Techniques: Staining

    Localization of HEC1 in HeLa cells depleted of SF3A2 and PRP31 and stained for HURP. ( A ) Representative control (mock), SF3A2 RNAi and PRP31 RNAi cells fixed after pre-extraction with Triton X-100 (see Materials and methods), and stained with DAPI (DNA, blue), anti-HURP (red) and anti-HEC1 (green) antibodies.

    Journal: eLife

    Article Title: Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

    doi: 10.7554/eLife.40325

    Figure Lengend Snippet: Localization of HEC1 in HeLa cells depleted of SF3A2 and PRP31 and stained for HURP. ( A ) Representative control (mock), SF3A2 RNAi and PRP31 RNAi cells fixed after pre-extraction with Triton X-100 (see Materials and methods), and stained with DAPI (DNA, blue), anti-HURP (red) and anti-HEC1 (green) antibodies.

    Article Snippet: The kinetochore localization of HEC1 after Sf3A2 and Prp31 RNAi in HeLa cells should also be investigated in cells treated with nocodazole (to standardize the conditions) and properly quantified relative to constitutive kinetochore components (e.g. using ACA serum from CREST patients).

    Techniques: Staining

    Star-PAP negatively regulates the invasive potential of breast cancer cells. (A) Western blot showing endogenous levels of Star-PAP and PIPKIα, along with Star-PAP targets KISS1R, NME1, and BIK and loading control β-tubulin from HEK 293, MCF7, HeLa, and MDA-MB-231 cells, as indicated. Numbers on the right indicate molecular mass in kilodaltons. (Quantification of the blots is shown in Fig. S1C to E in the supplemental material.) (B) Analysis of wound closure on collagen-treated plates after blocking cell proliferation with mitomycin C at various time points (0 to 60 h) postscratch in MCF7 cells after lentivirus-based stable knockdown of Star-PAP and PIPKIα and combined knockdown of both, as indicated (representative of 3 independent experiments). (Protein levels of Star-PAP and PIPKIα at various time points are shown in panel G.) (C) Wound healing assay, as in panel B, of MDA-MB-231 cells after Star-PAP or PIPKIα knockdown, as indicated. (D) Quantification of wound closure in panel B expressed relative to that at time zero postscratch. (The actual measurements of wound gaps [in micrometers] are plotted in Fig. S4A in the supplemental material.) The error bars represent SEM of 3 independent experiments. (E) Quantification of wound closure in panel C expressed relative to that at time zero. (The actual wound gaps are plotted in Fig. S4B in the supplemental material.) The error bars represent SEM of 3 independent experiments. (F) Western blot of Star-PAP, PIPKIα, CKIα, and Star-PAP targets KISS1R and NME1 from MCF7 cell lysates after Star-PAP or PIPKIα knockdown. − shRNA indicates that control shRNA was used. Numbers on the right indicate molecular mass in kilodaltons. (G) Western blots of Star-PAP and PIPKIα at various time points postscratch (in panel B). Numbers on the left indicate molecular mass in kilodaltons. (H) Wound healing assay as in panel C with ectopic expression of FLAG epitope-tagged Star-PAP sm and -PIPKIα in MDA-MB-231 cells. (Quantification of wound closure relative to that at time zero postwounding and actual wound gaps [in micrometers] are shown in Fig. S1G and S4C in the supplemental material, respectively.) (I) Western blot analysis of FLAG-Star-PAP and -PIPKIα after overexpression in MDA-MB-231 cells. Numbers on the left indicate molecular mass in kilodaltons. VC, vector control. (J) qRT-PCR analysis of various Star-PAP targets, as indicated, after individual and combined knockdown of Star-PAP and PIPKIα ( n = 3). The error bars represent SEM.

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion

    doi: 10.1128/MCB.00457-17

    Figure Lengend Snippet: Star-PAP negatively regulates the invasive potential of breast cancer cells. (A) Western blot showing endogenous levels of Star-PAP and PIPKIα, along with Star-PAP targets KISS1R, NME1, and BIK and loading control β-tubulin from HEK 293, MCF7, HeLa, and MDA-MB-231 cells, as indicated. Numbers on the right indicate molecular mass in kilodaltons. (Quantification of the blots is shown in Fig. S1C to E in the supplemental material.) (B) Analysis of wound closure on collagen-treated plates after blocking cell proliferation with mitomycin C at various time points (0 to 60 h) postscratch in MCF7 cells after lentivirus-based stable knockdown of Star-PAP and PIPKIα and combined knockdown of both, as indicated (representative of 3 independent experiments). (Protein levels of Star-PAP and PIPKIα at various time points are shown in panel G.) (C) Wound healing assay, as in panel B, of MDA-MB-231 cells after Star-PAP or PIPKIα knockdown, as indicated. (D) Quantification of wound closure in panel B expressed relative to that at time zero postscratch. (The actual measurements of wound gaps [in micrometers] are plotted in Fig. S4A in the supplemental material.) The error bars represent SEM of 3 independent experiments. (E) Quantification of wound closure in panel C expressed relative to that at time zero. (The actual wound gaps are plotted in Fig. S4B in the supplemental material.) The error bars represent SEM of 3 independent experiments. (F) Western blot of Star-PAP, PIPKIα, CKIα, and Star-PAP targets KISS1R and NME1 from MCF7 cell lysates after Star-PAP or PIPKIα knockdown. − shRNA indicates that control shRNA was used. Numbers on the right indicate molecular mass in kilodaltons. (G) Western blots of Star-PAP and PIPKIα at various time points postscratch (in panel B). Numbers on the left indicate molecular mass in kilodaltons. (H) Wound healing assay as in panel C with ectopic expression of FLAG epitope-tagged Star-PAP sm and -PIPKIα in MDA-MB-231 cells. (Quantification of wound closure relative to that at time zero postwounding and actual wound gaps [in micrometers] are shown in Fig. S1G and S4C in the supplemental material, respectively.) (I) Western blot analysis of FLAG-Star-PAP and -PIPKIα after overexpression in MDA-MB-231 cells. Numbers on the left indicate molecular mass in kilodaltons. VC, vector control. (J) qRT-PCR analysis of various Star-PAP targets, as indicated, after individual and combined knockdown of Star-PAP and PIPKIα ( n = 3). The error bars represent SEM.

    Article Snippet: Human embryonic kidney (HEK 293) cells, HeLa cell lines, MCF7 (estrogen receptor (ER)/progesterone receptor (PR)-positive human breast cancer) cells, and MDA-MB-231 (triple-negative human breast cancer) cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin-streptomycin (50 U/ml) at 37°C in 5% CO2 .

    Techniques: Western Blot, Multiple Displacement Amplification, Blocking Assay, Wound Healing Assay, shRNA, Expressing, FLAG-tag, Over Expression, Plasmid Preparation, Quantitative RT-PCR

    rEETI-II is internalized in part via a macropinocytosis and a clathrin-mediated endocytic pathway. ( a ) rEETI-II is internalized via membrane-bound vesicles that are derived from the plasma membrane. HeLa cells were labeled with the membrane marker PKH26 (4 μM) for 4 min, washed extensively with complete media and then incubated with rEETI-II-A488 (20 μM) for 45 min at 37 °C. Media were aspirated and the cells were fixed with 4% PFA and then imaged by fluorescence microscopy. Shown are the top, center and bottom planes of a representative 3D cell volume. Scale bar, 10 μm. Representative images from two independent experiments are shown. ( b ) rEETI-II-A488 is co-localized with transferrin-positive (Tf-A555, shown in red) vesicles in the early/recycling endosomes. ( c ) rEETI-II-A488 co-localizes with EGF-positive puncta (EGF-A647, shown in red, false colored). ( d ) rEETI-II-A488 does not co-localize with internalized cholera toxin (CTxB-A647, shown in red, false colored). HeLa (panels a–c) or NIH 3T3 (panel d) cells were incubated with rEETI-II-A488 (30 μM, 60 min) at 37 °C and endosomal markers (Tf-A555, 0.2 mg/ml; EGF-A647, 5 μg/ml; CTxB-A647, 25 μg/ml) were added in the last 10 min of incubation (the short incubation with transferrin and EFG was used to mainly target the probes to early endosome compartments). In panels b–d, cells were fixed with 4% PFA and processed as described in methods. Fluorescence images were captured on an upright LEICA SPE laser scanning confocal microscope (Leica Microsystems). Representative images from at least three independent experiments are shown. Scale bar, 15 μm. ( e ) rEETI-II-A488 (30 μM, 1 h) co-localizes with internalized 3 K dextran-TR (Texas red-conjugated, 0.1 mg/ml, 1 h, shown in red). HeLa cells were fixed with 4% PFA and processed as described in methods. Fluorescence images were captured on a high throughput ImageXpress Micro XL imaging system (Molecular Devices). Representative images from at least two independent experiments are shown. Scale bar, 10 μm.

    Journal: Scientific Reports

    Article Title: Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking

    doi: 10.1038/srep35179

    Figure Lengend Snippet: rEETI-II is internalized in part via a macropinocytosis and a clathrin-mediated endocytic pathway. ( a ) rEETI-II is internalized via membrane-bound vesicles that are derived from the plasma membrane. HeLa cells were labeled with the membrane marker PKH26 (4 μM) for 4 min, washed extensively with complete media and then incubated with rEETI-II-A488 (20 μM) for 45 min at 37 °C. Media were aspirated and the cells were fixed with 4% PFA and then imaged by fluorescence microscopy. Shown are the top, center and bottom planes of a representative 3D cell volume. Scale bar, 10 μm. Representative images from two independent experiments are shown. ( b ) rEETI-II-A488 is co-localized with transferrin-positive (Tf-A555, shown in red) vesicles in the early/recycling endosomes. ( c ) rEETI-II-A488 co-localizes with EGF-positive puncta (EGF-A647, shown in red, false colored). ( d ) rEETI-II-A488 does not co-localize with internalized cholera toxin (CTxB-A647, shown in red, false colored). HeLa (panels a–c) or NIH 3T3 (panel d) cells were incubated with rEETI-II-A488 (30 μM, 60 min) at 37 °C and endosomal markers (Tf-A555, 0.2 mg/ml; EGF-A647, 5 μg/ml; CTxB-A647, 25 μg/ml) were added in the last 10 min of incubation (the short incubation with transferrin and EFG was used to mainly target the probes to early endosome compartments). In panels b–d, cells were fixed with 4% PFA and processed as described in methods. Fluorescence images were captured on an upright LEICA SPE laser scanning confocal microscope (Leica Microsystems). Representative images from at least three independent experiments are shown. Scale bar, 15 μm. ( e ) rEETI-II-A488 (30 μM, 1 h) co-localizes with internalized 3 K dextran-TR (Texas red-conjugated, 0.1 mg/ml, 1 h, shown in red). HeLa cells were fixed with 4% PFA and processed as described in methods. Fluorescence images were captured on a high throughput ImageXpress Micro XL imaging system (Molecular Devices). Representative images from at least two independent experiments are shown. Scale bar, 10 μm.

    Article Snippet: Cell culture HeLa cells (ATCC no. CCL-2) or NIH 3T3 cells (ATCC no. CRL-1658) were grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 2 mM Glutamax™.

    Techniques: Derivative Assay, Labeling, Marker, Incubation, Fluorescence, Microscopy, High Throughput Screening Assay, Imaging

    Post endocytic trafficking of rEETI-II is dependent on intact microtubules. ( a–d ) Treatment with nocodazole (microtubule polymerization inhibitor) alters the cellular distribution of internalized rEET-II-A488, transferrin, EGF and 3 K dextran. rEETI-II-A488 is co-localized with transferrin ( a ), EGF ( b ), 3 K dextran ( d ) but not cholera toxin subunit B ( c ), in the presence of the nocodazole. HeLa (panels a,b,d) or NIH 3T3 (panel c) cells were treated with nocodazole (10 μM) for 30 min then with rEETI-II-A488 (30 μM) for 60 min in the presence of nocodazole (10 μM) at 37 °C, with endosomal markers (Tf-A555, 0.2 mg/ml; EGF-A647, 5 μg/ml; CTxB-A647, 25 μg/ml) were added in the last 10 min of incubation. 3 K Dextran-TR (0.1 mg/ml) was added with rEETI-II-A488 at the same time to cells for 60 min. Cells were then fixed with 4% PFA and processed as described in methods. In panels a–c, fluorescence images were captured on an upright LEICA SPE laser scanning confocal microscope (Leica Microsystems). Representative images from at least three independent experiments are shown. Scale bar, 15 μm. In panel d, fluorescence images were captured on a high throughput ImageXpress Micro XL imaging system (Molecular Devices). Representative images from at least two independent experiments are shown. Scale bar, 10 μm.

    Journal: Scientific Reports

    Article Title: Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking

    doi: 10.1038/srep35179

    Figure Lengend Snippet: Post endocytic trafficking of rEETI-II is dependent on intact microtubules. ( a–d ) Treatment with nocodazole (microtubule polymerization inhibitor) alters the cellular distribution of internalized rEET-II-A488, transferrin, EGF and 3 K dextran. rEETI-II-A488 is co-localized with transferrin ( a ), EGF ( b ), 3 K dextran ( d ) but not cholera toxin subunit B ( c ), in the presence of the nocodazole. HeLa (panels a,b,d) or NIH 3T3 (panel c) cells were treated with nocodazole (10 μM) for 30 min then with rEETI-II-A488 (30 μM) for 60 min in the presence of nocodazole (10 μM) at 37 °C, with endosomal markers (Tf-A555, 0.2 mg/ml; EGF-A647, 5 μg/ml; CTxB-A647, 25 μg/ml) were added in the last 10 min of incubation. 3 K Dextran-TR (0.1 mg/ml) was added with rEETI-II-A488 at the same time to cells for 60 min. Cells were then fixed with 4% PFA and processed as described in methods. In panels a–c, fluorescence images were captured on an upright LEICA SPE laser scanning confocal microscope (Leica Microsystems). Representative images from at least three independent experiments are shown. Scale bar, 15 μm. In panel d, fluorescence images were captured on a high throughput ImageXpress Micro XL imaging system (Molecular Devices). Representative images from at least two independent experiments are shown. Scale bar, 10 μm.

    Article Snippet: Cell culture HeLa cells (ATCC no. CCL-2) or NIH 3T3 cells (ATCC no. CRL-1658) were grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 2 mM Glutamax™.

    Techniques: Incubation, Fluorescence, Microscopy, High Throughput Screening Assay, Imaging

    Isochorismatase is required for the clearance of A. baumannii in vitro and in vivo . A − C ) HeLa ( A ), MH-S ( B ), and THP-1 cells ( C ) were infected with strain 98-37-09, ACJ6, or ΔACJ6 for different time [multiplicity of infection (MOI) = 10:1]. Then, infected cells were cultured in medium containing 100 μg gentamicin ml −1 for 1 h to remove extracellular bacteria. Intracellular bacteria were counted and expressed as CFUs. D ) The ratio of the intracellular bacteria at 2 or 6 h postinfection to the intracellular bacteria at 60 min postinfection. E ) Mice peritoneal macrophages were pretreated with rapamycin (Rapa) or 3-MA and were then infected with A. baumannii strain 98-37-09, ACJ6, or ΔACJ6 for 2 h (MOI = 10:1), and the number of intracellular bacteria in each group was counted and expressed as CFUs. F ) Autophagy was induced by A. baumannii in a mouse infection model. Bacteria burden in spleen were counted and showed as CFU. Data are representative of 3 experiments with similar results. * P

    Journal: The FASEB Journal

    Article Title: Critical role of bacterial isochorismatase in the autophagic process induced by Acinetobacter baumannii in mammalian cells

    doi: 10.1096/fj.201500019R

    Figure Lengend Snippet: Isochorismatase is required for the clearance of A. baumannii in vitro and in vivo . A − C ) HeLa ( A ), MH-S ( B ), and THP-1 cells ( C ) were infected with strain 98-37-09, ACJ6, or ΔACJ6 for different time [multiplicity of infection (MOI) = 10:1]. Then, infected cells were cultured in medium containing 100 μg gentamicin ml −1 for 1 h to remove extracellular bacteria. Intracellular bacteria were counted and expressed as CFUs. D ) The ratio of the intracellular bacteria at 2 or 6 h postinfection to the intracellular bacteria at 60 min postinfection. E ) Mice peritoneal macrophages were pretreated with rapamycin (Rapa) or 3-MA and were then infected with A. baumannii strain 98-37-09, ACJ6, or ΔACJ6 for 2 h (MOI = 10:1), and the number of intracellular bacteria in each group was counted and expressed as CFUs. F ) Autophagy was induced by A. baumannii in a mouse infection model. Bacteria burden in spleen were counted and showed as CFU. Data are representative of 3 experiments with similar results. * P

    Article Snippet: Cells and cell cultures HeLa, THP-1, and MH-S cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI 1640 medium that was supplemented with 10% fetal bovine serum.

    Techniques: In Vitro, In Vivo, Infection, Cell Culture, Mouse Assay

    A. baumannii induced Beclin-1–dependent autophagy via the AMPK/ERK/mTOR pathway. A ) HeLa, THP-1, and MH-S cells were infected with strain 98-37-09 (multiplicity of infection = 10:1). Western blotting for AMPK, p-AMPK, Akt, p-Akt, mTOR, p-mTOR, p70S6K, p-70S6K, 4E-BP1, p-4E-BP1, ERK, pERK, p-JNK, JNK, p-P38, P38, and LC3 was performed by using proteins from the 98-37-09–infected HeLa, THP-1, and MH-S cells. B ) HeLa cells were infected with strain 98-37-09 for 2 h and pretreated with U0126 (10 μM) for 1 h. Western blotting for p-AMPKβ, p-ERK, and p-mTOR was performed. C ) The ratio of LC3II/I was calculated. D ) The ratio of p-AMPKβ, p-ERK, and p-mTOR to β-actin was calculated. E ) Western blotting of Beclin-1 in 98-37-09–infected HeLa, THP-1, and MH-S cells. F , G ) Western blotting of Beclin-1 ( F ) and Atg7 ( G ) in HeLa cells. H ) HeLa cells were transfected with pmRFP-LC3 plasmid for 24 h and then transfected with the shRNA negative control, shBeclin-1, or shAtg7 plasmids for 48 h before cells were infected with strain 98-37-09 for 2 h. Confocal images show the induction of the LC3 puncta. I ) The number of puncta in each cell was determined, and cells with > 10 puncta were considered LC3-RFP puncta cells. Data are representative of 100 cells. J , K ) Western blotting for LC3 was performed. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Data are representative of 3 experiments with similar results. * P

    Journal: The FASEB Journal

    Article Title: Critical role of bacterial isochorismatase in the autophagic process induced by Acinetobacter baumannii in mammalian cells

    doi: 10.1096/fj.201500019R

    Figure Lengend Snippet: A. baumannii induced Beclin-1–dependent autophagy via the AMPK/ERK/mTOR pathway. A ) HeLa, THP-1, and MH-S cells were infected with strain 98-37-09 (multiplicity of infection = 10:1). Western blotting for AMPK, p-AMPK, Akt, p-Akt, mTOR, p-mTOR, p70S6K, p-70S6K, 4E-BP1, p-4E-BP1, ERK, pERK, p-JNK, JNK, p-P38, P38, and LC3 was performed by using proteins from the 98-37-09–infected HeLa, THP-1, and MH-S cells. B ) HeLa cells were infected with strain 98-37-09 for 2 h and pretreated with U0126 (10 μM) for 1 h. Western blotting for p-AMPKβ, p-ERK, and p-mTOR was performed. C ) The ratio of LC3II/I was calculated. D ) The ratio of p-AMPKβ, p-ERK, and p-mTOR to β-actin was calculated. E ) Western blotting of Beclin-1 in 98-37-09–infected HeLa, THP-1, and MH-S cells. F , G ) Western blotting of Beclin-1 ( F ) and Atg7 ( G ) in HeLa cells. H ) HeLa cells were transfected with pmRFP-LC3 plasmid for 24 h and then transfected with the shRNA negative control, shBeclin-1, or shAtg7 plasmids for 48 h before cells were infected with strain 98-37-09 for 2 h. Confocal images show the induction of the LC3 puncta. I ) The number of puncta in each cell was determined, and cells with > 10 puncta were considered LC3-RFP puncta cells. Data are representative of 100 cells. J , K ) Western blotting for LC3 was performed. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Data are representative of 3 experiments with similar results. * P

    Article Snippet: Cells and cell cultures HeLa, THP-1, and MH-S cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI 1640 medium that was supplemented with 10% fetal bovine serum.

    Techniques: Infection, Western Blot, Transfection, Plasmid Preparation, shRNA, Negative Control

    A. baumannii infection up-regulated autophagic degradation. A ) HeLa cells were transfected with ptfLC3 plasmid for 24 h, infected with strain 98-37-09 for 2 h [multiplicity of infection (MOI) = 10:1], and observed by using confocal microscopy. Arrows indicate the LC3 puncta, which could only be detected in the RFP channel. Red and green LC3 puncta were counted in 50 LC3 puncta cells respectively. B ) HeLa cells were infected with FITC-labeled strain 98-37-09 for 2 h (MOI = 10:1) and were then immunostained for LAMP-1 and analyzed by confocal microscopy. Arrows indicate the bacteria inside the autophagic vesicles (AVs) marked with LAMP-1. C ) HeLa cells were infected with strain 98-37-09 for 2 h (MOI = 10:1), incubated with 50 nM of LysoTracker for 1 h at 37°C, and then immunostained for LAMP-1 and analyzed by confocal microscopy. Arrows indicate the acidic LysoTracker and bacteria inside the AV marked with LAMP-1. D ) HeLa, THP-1, and MH-S cells were infected with strain 98-37-09 for 2 and 3 h. Then, Western blotting for LC3 and p62 was performed and ratios of LC3-II/I and p62/β-actin were calculated. Data are representative of 3 experiments with similar results. * P

    Journal: The FASEB Journal

    Article Title: Critical role of bacterial isochorismatase in the autophagic process induced by Acinetobacter baumannii in mammalian cells

    doi: 10.1096/fj.201500019R

    Figure Lengend Snippet: A. baumannii infection up-regulated autophagic degradation. A ) HeLa cells were transfected with ptfLC3 plasmid for 24 h, infected with strain 98-37-09 for 2 h [multiplicity of infection (MOI) = 10:1], and observed by using confocal microscopy. Arrows indicate the LC3 puncta, which could only be detected in the RFP channel. Red and green LC3 puncta were counted in 50 LC3 puncta cells respectively. B ) HeLa cells were infected with FITC-labeled strain 98-37-09 for 2 h (MOI = 10:1) and were then immunostained for LAMP-1 and analyzed by confocal microscopy. Arrows indicate the bacteria inside the autophagic vesicles (AVs) marked with LAMP-1. C ) HeLa cells were infected with strain 98-37-09 for 2 h (MOI = 10:1), incubated with 50 nM of LysoTracker for 1 h at 37°C, and then immunostained for LAMP-1 and analyzed by confocal microscopy. Arrows indicate the acidic LysoTracker and bacteria inside the AV marked with LAMP-1. D ) HeLa, THP-1, and MH-S cells were infected with strain 98-37-09 for 2 and 3 h. Then, Western blotting for LC3 and p62 was performed and ratios of LC3-II/I and p62/β-actin were calculated. Data are representative of 3 experiments with similar results. * P

    Article Snippet: Cells and cell cultures HeLa, THP-1, and MH-S cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI 1640 medium that was supplemented with 10% fetal bovine serum.

    Techniques: Infection, Transfection, Plasmid Preparation, Confocal Microscopy, Labeling, Incubation, Western Blot

    Transfection with siRNA reduces targeted protein over time. HeLa cells were untransfected (Un) or transfected with 25 nM siRNA to the indicated gene. Protein samples were subjected to Western immunoblotting and membranes probed for the protein of interest. Membranes were probed for Ran-GTP (Ran) as a loading control. Size markers in kDa are indicated to the left of each blot. At 24, 48, 72 and 96 hours post transfection (hpt), total protein was collected (A) or cells were fractionated into cytoplasmic (cyt) and nuclear (nuc) fractions (B). Panel C, HeLa cells were transfected with 0–25 nM siRNA to Wee1 and total protein collected at 24 hpt.

    Journal: PLoS ONE

    Article Title: A Genome-Wide siRNA Screen to Identify Host Factors Necessary for Growth of the Parasite Toxoplasma gondii

    doi: 10.1371/journal.pone.0068129

    Figure Lengend Snippet: Transfection with siRNA reduces targeted protein over time. HeLa cells were untransfected (Un) or transfected with 25 nM siRNA to the indicated gene. Protein samples were subjected to Western immunoblotting and membranes probed for the protein of interest. Membranes were probed for Ran-GTP (Ran) as a loading control. Size markers in kDa are indicated to the left of each blot. At 24, 48, 72 and 96 hours post transfection (hpt), total protein was collected (A) or cells were fractionated into cytoplasmic (cyt) and nuclear (nuc) fractions (B). Panel C, HeLa cells were transfected with 0–25 nM siRNA to Wee1 and total protein collected at 24 hpt.

    Article Snippet: The HeLa cell line (ATCC) was used for siRNA experiments because immortalized cell lines are more consistent and easily transformed than primary cells such as HFFs.

    Techniques: Transfection, Western Blot

    siRNA against TJP2 reduces protein but does not inhibit T. gondii growth. (A) HeLa cells were transfected with 25 nM siRNA to TJP2 (diamonds), CKLF (square), GRIN-1 (triangle), PKD-1 (inverted triangle), AllStars Hs Cell Death Control siRNA (death, closed circles), or AllStars Negative Control siRNA (negative, open circles) for 24 hours, then infected with 2.5×10 4 mCherry-expressing parasites. Replication of T. gondii over time was measured by fluorescence. Asterisks indicate a significant (p

    Journal: PLoS ONE

    Article Title: A Genome-Wide siRNA Screen to Identify Host Factors Necessary for Growth of the Parasite Toxoplasma gondii

    doi: 10.1371/journal.pone.0068129

    Figure Lengend Snippet: siRNA against TJP2 reduces protein but does not inhibit T. gondii growth. (A) HeLa cells were transfected with 25 nM siRNA to TJP2 (diamonds), CKLF (square), GRIN-1 (triangle), PKD-1 (inverted triangle), AllStars Hs Cell Death Control siRNA (death, closed circles), or AllStars Negative Control siRNA (negative, open circles) for 24 hours, then infected with 2.5×10 4 mCherry-expressing parasites. Replication of T. gondii over time was measured by fluorescence. Asterisks indicate a significant (p

    Article Snippet: The HeLa cell line (ATCC) was used for siRNA experiments because immortalized cell lines are more consistent and easily transformed than primary cells such as HFFs.

    Techniques: Transfection, Negative Control, Infection, Expressing, Fluorescence

    siRNAs, not transfection alone, induces host cell toxicity over time. (A) At 24, 48, 72 and 96 hours post transfection, medium from HeLa cells transfected with 25 nM siRNA pools was mixed with an equal volume CytoToxONE assay reagent. Fluorescence of samples was measured and data plotted as a percentage of the maximum LDH release obtained from lysed cells (% max release). (B) HeLa cells were treated with transfection medium alone (white) or transfected with AllStars Negative Control (black) or AllStars Hs Cell Death Control (blue) siRNAs. At the indicated times, cells were trypsinized and the number of viable cells determined by trypan blue exclusion. Asterisks indicate a significant (p

    Journal: PLoS ONE

    Article Title: A Genome-Wide siRNA Screen to Identify Host Factors Necessary for Growth of the Parasite Toxoplasma gondii

    doi: 10.1371/journal.pone.0068129

    Figure Lengend Snippet: siRNAs, not transfection alone, induces host cell toxicity over time. (A) At 24, 48, 72 and 96 hours post transfection, medium from HeLa cells transfected with 25 nM siRNA pools was mixed with an equal volume CytoToxONE assay reagent. Fluorescence of samples was measured and data plotted as a percentage of the maximum LDH release obtained from lysed cells (% max release). (B) HeLa cells were treated with transfection medium alone (white) or transfected with AllStars Negative Control (black) or AllStars Hs Cell Death Control (blue) siRNAs. At the indicated times, cells were trypsinized and the number of viable cells determined by trypan blue exclusion. Asterisks indicate a significant (p

    Article Snippet: The HeLa cell line (ATCC) was used for siRNA experiments because immortalized cell lines are more consistent and easily transformed than primary cells such as HFFs.

    Techniques: Transfection, Fluorescence, Negative Control

    siRNA against human genes inhibits T. gondii growth. HeLa cells were transfected with siRNA pools to the indicated gene for 24 hours, then infected with 2.5×10 4 mCherry-expressing parasites. Fluorescent measurements were taken at 24, 48, and 72 hours post infection. (A) Replication of T. gondii as measured by fluorescence in HeLa cells transfected with DDX48 siRNA (DDX48, squares), AllStars Negative Control siRNA (negative, circles), or AllStars Hs Cell Death Control siRNA (death, triangles). (B) Replication of T. gondii graphed as the percent growth of wells treated with AllStars Negative Control siRNA (% negative siRNA) at 24 (black), 48 (red), and 72 (blue) hours post infection. Asterisks indicate a significant (p

    Journal: PLoS ONE

    Article Title: A Genome-Wide siRNA Screen to Identify Host Factors Necessary for Growth of the Parasite Toxoplasma gondii

    doi: 10.1371/journal.pone.0068129

    Figure Lengend Snippet: siRNA against human genes inhibits T. gondii growth. HeLa cells were transfected with siRNA pools to the indicated gene for 24 hours, then infected with 2.5×10 4 mCherry-expressing parasites. Fluorescent measurements were taken at 24, 48, and 72 hours post infection. (A) Replication of T. gondii as measured by fluorescence in HeLa cells transfected with DDX48 siRNA (DDX48, squares), AllStars Negative Control siRNA (negative, circles), or AllStars Hs Cell Death Control siRNA (death, triangles). (B) Replication of T. gondii graphed as the percent growth of wells treated with AllStars Negative Control siRNA (% negative siRNA) at 24 (black), 48 (red), and 72 (blue) hours post infection. Asterisks indicate a significant (p

    Article Snippet: The HeLa cell line (ATCC) was used for siRNA experiments because immortalized cell lines are more consistent and easily transformed than primary cells such as HFFs.

    Techniques: Transfection, Infection, Expressing, Fluorescence, Negative Control

    Nr3c2 reporter is regulated by miR-135a. (A) High probability miRNA target sequences in the mouse Nr3c2 3’ UTR are drawn. Candidate miRNAs are predicted by microT v 3.0, TargetScan 5.2, and PicTar algoritms. Positions of mir-135a and miR-124 target sequences in the mouse annotated Nr3c2 3’ UTR and details of miRNA/mRNA base pairing are indicated. Beneath miRNA sequences, nucleotides mutated at the level of miRNA binding sites in the mutant constructs Nr3c2 m135a (mutated at both miR-135a seed binding sites) and Nr3c2 m124 (mutated at both miR-124 seed binding sites), are indicated (nts in bold). (B) Nr3c2 luciferase reporter (Nr3c2), and mutant reporter constructs were co-transfected into Hela cells together with empty vector or miRNA expression vectors (p135a and p124). Luciferase activity was measured 24 hours post-transfection. Values are expressed relatively to the internal renilla luciferase activity and presented as percentage of the activity achieved in the presence of the empty control vector. Results are shown as means ± SE (n=6). ** P

    Journal: PLoS ONE

    Article Title: Acute Stress Alters Amygdala microRNA miR-135a and miR-124 Expression: Inferences for Corticosteroid Dependent Stress Response

    doi: 10.1371/journal.pone.0073385

    Figure Lengend Snippet: Nr3c2 reporter is regulated by miR-135a. (A) High probability miRNA target sequences in the mouse Nr3c2 3’ UTR are drawn. Candidate miRNAs are predicted by microT v 3.0, TargetScan 5.2, and PicTar algoritms. Positions of mir-135a and miR-124 target sequences in the mouse annotated Nr3c2 3’ UTR and details of miRNA/mRNA base pairing are indicated. Beneath miRNA sequences, nucleotides mutated at the level of miRNA binding sites in the mutant constructs Nr3c2 m135a (mutated at both miR-135a seed binding sites) and Nr3c2 m124 (mutated at both miR-124 seed binding sites), are indicated (nts in bold). (B) Nr3c2 luciferase reporter (Nr3c2), and mutant reporter constructs were co-transfected into Hela cells together with empty vector or miRNA expression vectors (p135a and p124). Luciferase activity was measured 24 hours post-transfection. Values are expressed relatively to the internal renilla luciferase activity and presented as percentage of the activity achieved in the presence of the empty control vector. Results are shown as means ± SE (n=6). ** P

    Article Snippet: Cell culture and transfection Hela cells and Neuro-2a (N2a) mouse neuroblastoma cells were obtained from ATCC ( http://www.lgcstandards-atcc.org/ ).

    Techniques: Binding Assay, Mutagenesis, Construct, Luciferase, Transfection, Plasmid Preparation, Expressing, Activity Assay

    BRD4 inhibition leads to changes in the percentages of S and G2/M phase cells. Representative images and quantification of flow cytometry cell cycle plots in a HeLa, b HCT116, and c RNase H-inducible HeLa cells following 48 h of JQ1 treatment. Data presented as mean ± SEM ( n = 3 separate experiments). Significance assessed using ANOVA followed by Sidak’s test in a (%S DMSO vs JQ1 ***Adjusted P = 0.0002; % G2/M DMSO vs JQ1 ***Adjusted P = 0.0004); b (%S DMSO vs JQ1 ***Adjusted P = 0.0003; % G2/M DMSO vs JQ1 *Adjusted P = 0.0188), and c (%S DMSO RNase H1 negative vs JQ1 RNase H1 negative ***Adjusted P = 0.0008; %G2/M DMSO RNase H1 negative vs JQ1 RNase H1 negative *Adjusted P = 0.0155). d Growth curves of RNase H-inducible HeLa cells in the absence (−RNase H) or presence (+RNase H) of DMSO or JQ1. Data presented as mean ( n = 2 independent experiments). Please refer to Supplementary Fig. 12 for flow cytometry gating strategy. Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: BRD4 prevents the accumulation of R-loops and protects against transcription–replication collision events and DNA damage

    doi: 10.1038/s41467-020-17503-y

    Figure Lengend Snippet: BRD4 inhibition leads to changes in the percentages of S and G2/M phase cells. Representative images and quantification of flow cytometry cell cycle plots in a HeLa, b HCT116, and c RNase H-inducible HeLa cells following 48 h of JQ1 treatment. Data presented as mean ± SEM ( n = 3 separate experiments). Significance assessed using ANOVA followed by Sidak’s test in a (%S DMSO vs JQ1 ***Adjusted P = 0.0002; % G2/M DMSO vs JQ1 ***Adjusted P = 0.0004); b (%S DMSO vs JQ1 ***Adjusted P = 0.0003; % G2/M DMSO vs JQ1 *Adjusted P = 0.0188), and c (%S DMSO RNase H1 negative vs JQ1 RNase H1 negative ***Adjusted P = 0.0008; %G2/M DMSO RNase H1 negative vs JQ1 RNase H1 negative *Adjusted P = 0.0155). d Growth curves of RNase H-inducible HeLa cells in the absence (−RNase H) or presence (+RNase H) of DMSO or JQ1. Data presented as mean ( n = 2 independent experiments). Please refer to Supplementary Fig. 12 for flow cytometry gating strategy. Source data are provided as a Source data file.

    Article Snippet: Cell culture HeLa (ATCC CCL-2) and HCT116 (ATCC CCL-247) cells were purchased directly from the American Type Culture Collection and used without further authentication.

    Techniques: Inhibition, Flow Cytometry

    S. aureus induced dose-dependent G2/M phase transition delay in the subsequent G2/M phase. A. HeLa cells were synchronized by DTB and were exposed to S. aureus bacteria (MW2) at MOI 20∶1 or to the media alone for 2 h, followed by incubation in cDMEM-Gent100 for 2 h, and subsequently incubated for an additional 12 h, 14 h, 18 h, 20 h and 24 h. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. Each experiment was performed four times. The number of cells in different phases is presented on the histograms. The values shown are those of a representative assay out of the four assays performed. Exposure of the cells to S. aureus bacteria induced a G2/M phase transition delay in a time-dependent manner. B . HeLa cells were synchronized by DTB and were exposed to S. aureus bacteria (MW2) at different MOIs ranging from 5∶1 to 20∶1, followed by incubation in cDMEM-Gent100 for 2 h, and further incubated for an additional 20 h. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. The percentage of cells is presented on the graph as a function of bacterial concentration. The data correspond to a representative experiment out of the three assays performed and are presented as mean +/− SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. (*) P-values

    Journal: PLoS ONE

    Article Title: Staphylococcus aureus-Induced G2/M Phase Transition Delay in Host Epithelial Cells Increases Bacterial Infective Efficiency

    doi: 10.1371/journal.pone.0063279

    Figure Lengend Snippet: S. aureus induced dose-dependent G2/M phase transition delay in the subsequent G2/M phase. A. HeLa cells were synchronized by DTB and were exposed to S. aureus bacteria (MW2) at MOI 20∶1 or to the media alone for 2 h, followed by incubation in cDMEM-Gent100 for 2 h, and subsequently incubated for an additional 12 h, 14 h, 18 h, 20 h and 24 h. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. Each experiment was performed four times. The number of cells in different phases is presented on the histograms. The values shown are those of a representative assay out of the four assays performed. Exposure of the cells to S. aureus bacteria induced a G2/M phase transition delay in a time-dependent manner. B . HeLa cells were synchronized by DTB and were exposed to S. aureus bacteria (MW2) at different MOIs ranging from 5∶1 to 20∶1, followed by incubation in cDMEM-Gent100 for 2 h, and further incubated for an additional 20 h. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. The percentage of cells is presented on the graph as a function of bacterial concentration. The data correspond to a representative experiment out of the three assays performed and are presented as mean +/− SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. (*) P-values

    Article Snippet: Analysis of the bacterial population in HeLa cells synchronized in G1 or G2 cell cycle phases CDK4/6 inhibitor IV (Calbiochem, Darmstadt, Germany), which prevents G1/S transition , was used to enrich cell culture with cells in the G1 phase.

    Techniques: Sublimation, Incubation, Staining, FACS, Software, Concentration Assay

    Blocking HeLa cells in the G2 phase results in an increased internalization and in an increased intracellular replication of S. aureus. . HeLa cells were treated with either 10 µM CDK4/6 inhibitor IV (enrichment in the G1 phase) or 9 µM of RO-3306 (enrichment in the G2 phase) 3 h before the addition of S. aureus at MOI 10∶1 and 50∶1 for 2 h (T0). The cells then were incubated in cDMEM-Gent100 for an additional 2 h, 4 h (T4) or 20 h (T20). The proportions of cells in the different cell cycle stages at T2, T4 and T20 were determined by FACS (A). The CFU of intracellular S. aureus at T2, T4 and T20 was determined by plate count. CFU values were normalized to 10 5 HeLa cells (B). Experiments were performed in triplicate. The data are presented as mean +/− SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. (*) P

    Journal: PLoS ONE

    Article Title: Staphylococcus aureus-Induced G2/M Phase Transition Delay in Host Epithelial Cells Increases Bacterial Infective Efficiency

    doi: 10.1371/journal.pone.0063279

    Figure Lengend Snippet: Blocking HeLa cells in the G2 phase results in an increased internalization and in an increased intracellular replication of S. aureus. . HeLa cells were treated with either 10 µM CDK4/6 inhibitor IV (enrichment in the G1 phase) or 9 µM of RO-3306 (enrichment in the G2 phase) 3 h before the addition of S. aureus at MOI 10∶1 and 50∶1 for 2 h (T0). The cells then were incubated in cDMEM-Gent100 for an additional 2 h, 4 h (T4) or 20 h (T20). The proportions of cells in the different cell cycle stages at T2, T4 and T20 were determined by FACS (A). The CFU of intracellular S. aureus at T2, T4 and T20 was determined by plate count. CFU values were normalized to 10 5 HeLa cells (B). Experiments were performed in triplicate. The data are presented as mean +/− SD. Tukey's Honestly Significant Difference test was applied for comparison of means between the groups. (*) P

    Article Snippet: Analysis of the bacterial population in HeLa cells synchronized in G1 or G2 cell cycle phases CDK4/6 inhibitor IV (Calbiochem, Darmstadt, Germany), which prevents G1/S transition , was used to enrich cell culture with cells in the G1 phase.

    Techniques: Blocking Assay, Incubation, FACS

    S. aureus induced a G2/M phase transition delay. HeLa cells were synchronized by DTB and were exposed to live or heat-killed bacteria (MW2) at MOI 20∶1, to the latex beads or to the media alone for 2 h, followed by incubation in cDMEM-Gent100 for 2 h, and further incubated for 24 h. Some cells were exposed to etoposide for the same length of time as the exposure to bacteria. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. The average percentage of cell cycle phase ± SD is indicated. Each experiment was performed at least five times. Exposure of the cells to live S. aureus bacteria induced an increase in the number of cells in the G2/M phase, (*) p

    Journal: PLoS ONE

    Article Title: Staphylococcus aureus-Induced G2/M Phase Transition Delay in Host Epithelial Cells Increases Bacterial Infective Efficiency

    doi: 10.1371/journal.pone.0063279

    Figure Lengend Snippet: S. aureus induced a G2/M phase transition delay. HeLa cells were synchronized by DTB and were exposed to live or heat-killed bacteria (MW2) at MOI 20∶1, to the latex beads or to the media alone for 2 h, followed by incubation in cDMEM-Gent100 for 2 h, and further incubated for 24 h. Some cells were exposed to etoposide for the same length of time as the exposure to bacteria. Detached cells were then combined with adherent cells and stained with PI. Cell cycle phases of PI-stained cells were monitored by FACS. The data were collected from 20,000 cells and analysis was performed with Cell Quest software. The average percentage of cell cycle phase ± SD is indicated. Each experiment was performed at least five times. Exposure of the cells to live S. aureus bacteria induced an increase in the number of cells in the G2/M phase, (*) p

    Article Snippet: Analysis of the bacterial population in HeLa cells synchronized in G1 or G2 cell cycle phases CDK4/6 inhibitor IV (Calbiochem, Darmstadt, Germany), which prevents G1/S transition , was used to enrich cell culture with cells in the G1 phase.

    Techniques: Sublimation, Incubation, Staining, FACS, Software

    Hec1 S69 phosphorylation is reduced at kinetochores in response to microtubule depolymerization but not in response to microtubule stabilization. (A–C) Immunofluorescence images showing kinetochore localization of pS69 in HeLa cells after incubation with 50 nM taxol for 5 h (A), 10 µM taxol for 30 min (B), and 1 µM nocodazole for 1 h (C). (D) Immunofluorescence images of control cells and cells treated with 1 µM nocodazole for 1 h and stained with an antibody to active Aurora A kinase phosphorylated at T288 (pT288). Shown on the right of each panel is the quantification of either pS69 kinetochore fluorescence intensity (A–C) or total cellular pAAK fluorescence intensity (D). For all conditions in A–C, ≥200 kinetochores were measured from ≥20 cells. For the experiment shown in D, total fluorescence was measured from ≥20 cells per condition. Error bars indicate SD. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

    doi: 10.1083/jcb.201707160

    Figure Lengend Snippet: Hec1 S69 phosphorylation is reduced at kinetochores in response to microtubule depolymerization but not in response to microtubule stabilization. (A–C) Immunofluorescence images showing kinetochore localization of pS69 in HeLa cells after incubation with 50 nM taxol for 5 h (A), 10 µM taxol for 30 min (B), and 1 µM nocodazole for 1 h (C). (D) Immunofluorescence images of control cells and cells treated with 1 µM nocodazole for 1 h and stained with an antibody to active Aurora A kinase phosphorylated at T288 (pT288). Shown on the right of each panel is the quantification of either pS69 kinetochore fluorescence intensity (A–C) or total cellular pAAK fluorescence intensity (D). For all conditions in A–C, ≥200 kinetochores were measured from ≥20 cells. For the experiment shown in D, total fluorescence was measured from ≥20 cells per condition. Error bars indicate SD. Bars, 10 µm.

    Article Snippet: For silence and rescue experiments in HeLa cells, at the time of siRNA treatment, Hec1-GFP constructs were simultaneously transfected (4 µg) using oligofectamine.

    Techniques: Immunofluorescence, Incubation, Staining, Fluorescence

    Aurora A kinase binds and localizes to INCENP. (A) Volcano plot of quantitative liquid chromatography–tandem mass spectrometry experiments identifying interactors of INCENP. Plotted are the differences in label-free quantification intensity between the INCENP-GFP (right) and control group against the transformed (−log 10 ) p-value of a Student’s t test. The red line indicates the permutation-based false discovery rate (FDR) threshold (0.001) to correct for multiple testing. (B) Western blot analysis of an anti-GFP immunoprecipitation (IP) performed in mitotic cell lysates, using either nocodazole (N) or STLC (S) from HeLa cells expressing either LAP or INCENP-LAP. After SDS-PAGE, the Western blot was probed for GFP and Aurora A kinase (AAK). 10% of input was loaded. (C) Western blot analysis of an anti-GFP immunoprecipitation performed in cell lysates from HEK-293T cells transiently expressing either GFP, full-length (FL) INCENP-GFP, or various truncation mutants of INCENP-GFP (1–820, 1–878, and 1–897). The Western blot was probed for GFP and Aurora A kinase and reprobed for Aurora B kinase (ABK). 10% of input was loaded. (D) ). Cells were immunostained with an Aurora A kinase antibody. Enlarged images show recruitment of Aurora A kinase to the LacI-mCherry-ΔCEN-INCENP spot. Quantification of Aurora A kinase intensity is shown on the right. (E) Immunofluorescence images of U2OS cells expressing VSV-INCENP-GFP under a doxycycline-inducible promoter, either noninduced or induced. Cells were immunostained with GFP and Aurora A kinase antibodies. Enlarged images show colocalization of INCENP-GFP and Aurora A kinase upon overexpression of INCENP-GFP. (F) Line graphs of kinetochore pairs highlighted in E. (G) Immunofluorescence images showing kinetochore localization of pS69 in HeLa cells treated with siRNA to either luciferase (control) or INCENP (siINCENP). Quantification of pS69 and INCENP fluorescence intensity is shown on the right. For pS69 quantification, n = 3 experiments of 15–20 cells; for INCENP quantification, n = 1 experiment of 15–20 cells. Error bars indicate SD. ****, P

    Journal: The Journal of Cell Biology

    Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

    doi: 10.1083/jcb.201707160

    Figure Lengend Snippet: Aurora A kinase binds and localizes to INCENP. (A) Volcano plot of quantitative liquid chromatography–tandem mass spectrometry experiments identifying interactors of INCENP. Plotted are the differences in label-free quantification intensity between the INCENP-GFP (right) and control group against the transformed (−log 10 ) p-value of a Student’s t test. The red line indicates the permutation-based false discovery rate (FDR) threshold (0.001) to correct for multiple testing. (B) Western blot analysis of an anti-GFP immunoprecipitation (IP) performed in mitotic cell lysates, using either nocodazole (N) or STLC (S) from HeLa cells expressing either LAP or INCENP-LAP. After SDS-PAGE, the Western blot was probed for GFP and Aurora A kinase (AAK). 10% of input was loaded. (C) Western blot analysis of an anti-GFP immunoprecipitation performed in cell lysates from HEK-293T cells transiently expressing either GFP, full-length (FL) INCENP-GFP, or various truncation mutants of INCENP-GFP (1–820, 1–878, and 1–897). The Western blot was probed for GFP and Aurora A kinase and reprobed for Aurora B kinase (ABK). 10% of input was loaded. (D) ). Cells were immunostained with an Aurora A kinase antibody. Enlarged images show recruitment of Aurora A kinase to the LacI-mCherry-ΔCEN-INCENP spot. Quantification of Aurora A kinase intensity is shown on the right. (E) Immunofluorescence images of U2OS cells expressing VSV-INCENP-GFP under a doxycycline-inducible promoter, either noninduced or induced. Cells were immunostained with GFP and Aurora A kinase antibodies. Enlarged images show colocalization of INCENP-GFP and Aurora A kinase upon overexpression of INCENP-GFP. (F) Line graphs of kinetochore pairs highlighted in E. (G) Immunofluorescence images showing kinetochore localization of pS69 in HeLa cells treated with siRNA to either luciferase (control) or INCENP (siINCENP). Quantification of pS69 and INCENP fluorescence intensity is shown on the right. For pS69 quantification, n = 3 experiments of 15–20 cells; for INCENP quantification, n = 1 experiment of 15–20 cells. Error bars indicate SD. ****, P

    Article Snippet: For silence and rescue experiments in HeLa cells, at the time of siRNA treatment, Hec1-GFP constructs were simultaneously transfected (4 µg) using oligofectamine.

    Techniques: Liquid Chromatography, Mass Spectrometry, Transformation Assay, Western Blot, Immunoprecipitation, Expressing, SDS Page, Immunofluorescence, Over Expression, Luciferase, Fluorescence

    Hec1 S69 is phosphorylated by Aurora A kinase in cells. (A) Immunofluorescence images of HeLa cells treated with 2 µM ZM447439 (ZM) to inhibit Aurora B kinase (ABK), 0.5 µM MLN8054 (MLN) to inhibit Aurora A kinase (AAK), or both inhibitors, and stained with phosphospecific antibodies to Hec1 S69, S55, and S44. (B) Quantification of the experiment represented in A. For each condition, ≥300 kinetochores were measured from ≥30 cells. Asterisks indicate respective p-values (each condition is compared with the corresponding untreated control; unpaired t test). (C) Fluorescence images showing cells immunostained with antibodies to active Aurora B kinase phosphorylated at T232 (pT232) and active Aurora A kinase phosphorylated at T288 (pT288) in HeLa cells treated with either 0.5 µM MLN8054, 2 µM ZM447439, or no inhibitor. Quantification is shown on the right. For each condition shown, ≥300 kinetochores from ≥30 cells were measured. Error bars indicate SD. Bars, 10 µm. ACA, anticentromere antibody.

    Journal: The Journal of Cell Biology

    Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

    doi: 10.1083/jcb.201707160

    Figure Lengend Snippet: Hec1 S69 is phosphorylated by Aurora A kinase in cells. (A) Immunofluorescence images of HeLa cells treated with 2 µM ZM447439 (ZM) to inhibit Aurora B kinase (ABK), 0.5 µM MLN8054 (MLN) to inhibit Aurora A kinase (AAK), or both inhibitors, and stained with phosphospecific antibodies to Hec1 S69, S55, and S44. (B) Quantification of the experiment represented in A. For each condition, ≥300 kinetochores were measured from ≥30 cells. Asterisks indicate respective p-values (each condition is compared with the corresponding untreated control; unpaired t test). (C) Fluorescence images showing cells immunostained with antibodies to active Aurora B kinase phosphorylated at T232 (pT232) and active Aurora A kinase phosphorylated at T288 (pT288) in HeLa cells treated with either 0.5 µM MLN8054, 2 µM ZM447439, or no inhibitor. Quantification is shown on the right. For each condition shown, ≥300 kinetochores from ≥30 cells were measured. Error bars indicate SD. Bars, 10 µm. ACA, anticentromere antibody.

    Article Snippet: For silence and rescue experiments in HeLa cells, at the time of siRNA treatment, Hec1-GFP constructs were simultaneously transfected (4 µg) using oligofectamine.

    Techniques: Immunofluorescence, Staining, Fluorescence

    Hec1 S69 is phosphorylated throughout mitosis. (A) ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

    Journal: The Journal of Cell Biology

    Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

    doi: 10.1083/jcb.201707160

    Figure Lengend Snippet: Hec1 S69 is phosphorylated throughout mitosis. (A) ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

    Article Snippet: For silence and rescue experiments in HeLa cells, at the time of siRNA treatment, Hec1-GFP constructs were simultaneously transfected (4 µg) using oligofectamine.

    Techniques: Immunofluorescence, Staining, Derivative Assay

    Inhibition of Hec1 S69 phosphorylation results in chromosome segregation errors and altered mitotic timing. (A) HeLa Flp-In T-REx cells depleted of endogenous Hec1 and induced to express either WT- or S69A-Hec1-GFP were fixed and scored for segregation errors. Errors included for quantification were lagging anaphase chromosomes and anaphase bridges. Examples of WT- and S69A-Hec1-GFP–expressing cells in anaphase are shown (chromosomes stained with DAPI are in red, and Hec1-GFP is in green). The arrow points to lagging chromosomes; this region is shown magnified to the right. For the quantification, ≥400 cells from four experiments were analyzed. Error bars represent SD. Bars: (main image) 10 µm; (magnified image) 1 µm. (B) Quantification of mitotic timing of HeLa Flp-In T-REx cells depleted of endogenous Hec1 and induced to express either WT- or S69A-Hec1-GFP. Mitotic progression was scored from nuclear envelope breakdown (NEB) to metaphase (M) and from metaphase to anaphase onset (AO). Total time in mitosis was scored from nuclear envelope breakdown to anaphase onset. For each condition, ≥75 cells were analyzed.

    Journal: The Journal of Cell Biology

    Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

    doi: 10.1083/jcb.201707160

    Figure Lengend Snippet: Inhibition of Hec1 S69 phosphorylation results in chromosome segregation errors and altered mitotic timing. (A) HeLa Flp-In T-REx cells depleted of endogenous Hec1 and induced to express either WT- or S69A-Hec1-GFP were fixed and scored for segregation errors. Errors included for quantification were lagging anaphase chromosomes and anaphase bridges. Examples of WT- and S69A-Hec1-GFP–expressing cells in anaphase are shown (chromosomes stained with DAPI are in red, and Hec1-GFP is in green). The arrow points to lagging chromosomes; this region is shown magnified to the right. For the quantification, ≥400 cells from four experiments were analyzed. Error bars represent SD. Bars: (main image) 10 µm; (magnified image) 1 µm. (B) Quantification of mitotic timing of HeLa Flp-In T-REx cells depleted of endogenous Hec1 and induced to express either WT- or S69A-Hec1-GFP. Mitotic progression was scored from nuclear envelope breakdown (NEB) to metaphase (M) and from metaphase to anaphase onset (AO). Total time in mitosis was scored from nuclear envelope breakdown to anaphase onset. For each condition, ≥75 cells were analyzed.

    Article Snippet: For silence and rescue experiments in HeLa cells, at the time of siRNA treatment, Hec1-GFP constructs were simultaneously transfected (4 µg) using oligofectamine.

    Techniques: Inhibition, Expressing, Staining

    Effects of ligase inhibitors on DNA synthesis, cell viability and DNA damage The effects of L67, (closed circles) L82, (closed squares) or L82-G17 (closed triangles) on BrdU incorporation, cell viability and formation of γH2AX foci were determined as described in Materials and Methods. A . Asynchronous HeLa cells were treated with the ligase inhibitors for 4 hours and were then assayed for BrdU incorporation. Results of three independent assays are shown graphically. B . HeLa cells were incubated with the inhibitors for 5 days prior to the determination of cell viability using the MTT assay. Results of three independent assays are shown graphically. Data points at 20 and 30 µM are significant at p

    Journal: DNA repair

    Article Title: Structure-activity relationships among DNA ligase inhibitors; characterization of a selective uncompetitive DNA ligase I inhibitor

    doi: 10.1016/j.dnarep.2017.10.002

    Figure Lengend Snippet: Effects of ligase inhibitors on DNA synthesis, cell viability and DNA damage The effects of L67, (closed circles) L82, (closed squares) or L82-G17 (closed triangles) on BrdU incorporation, cell viability and formation of γH2AX foci were determined as described in Materials and Methods. A . Asynchronous HeLa cells were treated with the ligase inhibitors for 4 hours and were then assayed for BrdU incorporation. Results of three independent assays are shown graphically. B . HeLa cells were incubated with the inhibitors for 5 days prior to the determination of cell viability using the MTT assay. Results of three independent assays are shown graphically. Data points at 20 and 30 µM are significant at p

    Article Snippet: Asynchronous populations of HeLa cells were incubated with the DNA ligase inhibitors for 4 h prior to pulse labeling with BrdU (10 µM) for 45 min. Immunofluorescent staining of cells was performed using a BD BrdU flow Kit (BD Pharmingen) according to the manufacturer’s instructions and then quantitated by flow cytometry using an LSR Fortessa in the UNMCCC Shared Flow Cytometry and High Throughput Screening Resource.

    Techniques: DNA Synthesis, BrdU Incorporation Assay, Incubation, MTT Assay

    Analysis of extracts of HeLa S3 cells by reverse-phase HPLC. (A) Analysis of pure JA28-TP including the full UV spectrum of the JA28-TP peak at 3.6 min (inlay). mAU, milli-absorbance units. (B) Analysis of extracts of untreated HeLa S3 cells including

    Journal:

    Article Title: Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues ▿Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues ▿ †

    doi: 10.1128/AAC.01056-07

    Figure Lengend Snippet: Analysis of extracts of HeLa S3 cells by reverse-phase HPLC. (A) Analysis of pure JA28-TP including the full UV spectrum of the JA28-TP peak at 3.6 min (inlay). mAU, milli-absorbance units. (B) Analysis of extracts of untreated HeLa S3 cells including

    Article Snippet: HeLa S3 cells were pretreated with each nucleoside for 1 h prior to infection to allow for accumulation of intracellular metabolites.

    Techniques: High Performance Liquid Chromatography

    RASSF4 regulates the levels of PI(4)P and PIP5Ks at the PM. (A) Confocal images of HeLa cells expressing N-PH-ORP5-GFP and treated with either siControl of siRASSF4. Bar, 5 µm. Relative N-PH-ORP5-GFP fluorescence intensity in the PM from > 30 cells across three independent experiments was evaluated. Means ± SEM are shown. (B) Relative PM PI(4)P levels were detected by anti-PI(4)P antibody in HeLa cells treated with either siControl or siRASSF4 from ∼30 cells across three independent experiments. Means ± SEM are shown. (C) Confocal images of HeLa cells expressing YFP-PIP5K1B, treated with either siRASSF4 or siControl. Bar, 5 µm. Relative YFP-PIP5K1B fluorescence intensity in the PM from > 30 cells across two independent experiments was evaluated. Means ± SEM are shown. (D) A diagram of the rapamycin-inducible approach that selectively increases PI(4,5)P 2 at the PM. (E) Relative PM PI(4,5)P 2 levels in HeLa cells transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki (control) were monitored by PI(4,5)P 2 immunostaining. More than 30 cells for each condition across three independent experiments were evaluated. Means ± SEM are plotted. (C–E) Cells were treated with 5 µM rapamycin for 10 min after the transfection and before the experiments. (F) The density of stable ER puncta in HeLa cells treated with the indicated siRNA and expressing YFP-KDEL, mCherry-KRAS-tail, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. More than 15 cells for each condition across two independent experiments were analyzed. Plotted are means ± SEM. (G) HeLa cells were transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. Cells were stimulated with 100 µM His, 1 µM TG, and 2 mM EGTA; 2 mM Ca 2+ was added 6 min after stimulation. Mean peak values of Fura-2 ratio ± SEM of the Ca 2+ add-back phase are plotted from > 300 cells for each condition across three independent experiments. **, P

    Journal: The Journal of Cell Biology

    Article Title: RASSF4 controls SOCE and ER–PM junctions through regulation of PI(4,5)P2

    doi: 10.1083/jcb.201606047

    Figure Lengend Snippet: RASSF4 regulates the levels of PI(4)P and PIP5Ks at the PM. (A) Confocal images of HeLa cells expressing N-PH-ORP5-GFP and treated with either siControl of siRASSF4. Bar, 5 µm. Relative N-PH-ORP5-GFP fluorescence intensity in the PM from > 30 cells across three independent experiments was evaluated. Means ± SEM are shown. (B) Relative PM PI(4)P levels were detected by anti-PI(4)P antibody in HeLa cells treated with either siControl or siRASSF4 from ∼30 cells across three independent experiments. Means ± SEM are shown. (C) Confocal images of HeLa cells expressing YFP-PIP5K1B, treated with either siRASSF4 or siControl. Bar, 5 µm. Relative YFP-PIP5K1B fluorescence intensity in the PM from > 30 cells across two independent experiments was evaluated. Means ± SEM are shown. (D) A diagram of the rapamycin-inducible approach that selectively increases PI(4,5)P 2 at the PM. (E) Relative PM PI(4,5)P 2 levels in HeLa cells transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki (control) were monitored by PI(4,5)P 2 immunostaining. More than 30 cells for each condition across three independent experiments were evaluated. Means ± SEM are plotted. (C–E) Cells were treated with 5 µM rapamycin for 10 min after the transfection and before the experiments. (F) The density of stable ER puncta in HeLa cells treated with the indicated siRNA and expressing YFP-KDEL, mCherry-KRAS-tail, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. More than 15 cells for each condition across two independent experiments were analyzed. Plotted are means ± SEM. (G) HeLa cells were transfected with the indicated siRNA, Lyn-FRB, and either CFP-FRBP-PIP5K or CFP-FRBP-PIP5Ki. Cells were stimulated with 100 µM His, 1 µM TG, and 2 mM EGTA; 2 mM Ca 2+ was added 6 min after stimulation. Mean peak values of Fura-2 ratio ± SEM of the Ca 2+ add-back phase are plotted from > 300 cells for each condition across three independent experiments. **, P

    Article Snippet: After transfection of the HRP-ER plasmid with either siRNAs or DNA plasmids, HeLa cells were plated onto coverslips (MatTek Corporation) and fixed with 2% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) for 20 min at room temperature.

    Techniques: Expressing, Fluorescence, Transfection, Immunostaining

    RASSF4 regulates the formation and stability of ER–PM junctions. (A and B, left) EM micrographs of HRP-KDEL–expressing HeLa cells treated with the indicated siRNA or plasmid for 2 d. Red arrows indicate ER–PM junctions. Bar, 2 µm. (Right) The percentage of PM length engaged in contact with the ER is shown. Means ± SEM of six to eight cells are plotted. N indicates the cell nucleus. (C and D) Densities of ER–PM junctions determined using a TIRF image series of YFP-KDEL–expressing HeLa cells that were treated with siRASSF4 or siControl (C) or transfected with RASSF4 or a control vector (D). More than 24 cells for each condition across three independent experiments were analyzed. Means ± SEM are plotted. (E) TIRF images of ER–PM junctions labeled by MAPPER in cells treated with siControl or siRASSF4. Red arrows indicate unstable junctions. Bar, 1 µm. The percentage of stable ER–PM junctions labeled by MAPPER was quantified. More than 500 puncta in six different cells for each condition were evaluated. Means ± SEM are shown. *, P

    Journal: The Journal of Cell Biology

    Article Title: RASSF4 controls SOCE and ER–PM junctions through regulation of PI(4,5)P2

    doi: 10.1083/jcb.201606047

    Figure Lengend Snippet: RASSF4 regulates the formation and stability of ER–PM junctions. (A and B, left) EM micrographs of HRP-KDEL–expressing HeLa cells treated with the indicated siRNA or plasmid for 2 d. Red arrows indicate ER–PM junctions. Bar, 2 µm. (Right) The percentage of PM length engaged in contact with the ER is shown. Means ± SEM of six to eight cells are plotted. N indicates the cell nucleus. (C and D) Densities of ER–PM junctions determined using a TIRF image series of YFP-KDEL–expressing HeLa cells that were treated with siRASSF4 or siControl (C) or transfected with RASSF4 or a control vector (D). More than 24 cells for each condition across three independent experiments were analyzed. Means ± SEM are plotted. (E) TIRF images of ER–PM junctions labeled by MAPPER in cells treated with siControl or siRASSF4. Red arrows indicate unstable junctions. Bar, 1 µm. The percentage of stable ER–PM junctions labeled by MAPPER was quantified. More than 500 puncta in six different cells for each condition were evaluated. Means ± SEM are shown. *, P

    Article Snippet: After transfection of the HRP-ER plasmid with either siRNAs or DNA plasmids, HeLa cells were plated onto coverslips (MatTek Corporation) and fixed with 2% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) for 20 min at room temperature.

    Techniques: Expressing, Plasmid Preparation, Transfection, Labeling

    RASSF4 is a positive regulator of SOCE. (A) A diagram of the domain structure of RASSF4. The regions targeted by siRASSF4_N’ used in C, siRASSF4_C’ used in C, and siRASSF4 used in B are indicated. RA, RAS association. (B) Intracellular Ca 2+ levels determined by analysis of Fura-2 fluorescence ratios in HeLa cells treated with a control siRNA (siControl), siSTIM1, or siRASSF4 and stimulated with 1 µM TG and 100 µM histamine (His). Shown are mean Fura-2 ratios ± SEM of > 300 cells for each condition. Similar results were obtained from > 10 independent experiments. (C) Fura-2 ratios of HeLa cells treated with the indicated siRNAs. Cells were stimulated with 1 µM TG, 100 µM His, and 2 mM EGTA; 2 mM Ca 2+ was added 6 min after stimulation. Shown are mean Fura-2 ratios ± SEM derived from > 1,000 cells for each condition across two independent experiments. (D) Fura-2 ratios of siRNA-treated HUVECs stimulated as described in C. Shown are mean Fura-2 ratios ± SEM derived from > 500 cells for each condition across two independent experiments. (E) Fura-2 ratios of RPE-1 cells treated with the indicated siRNAs. Cells were stimulated with 1 µM TG and 2 mM EGTA; 2 mM Ca 2+ was added 11.5 min after stimulation. Shown are mean Fura-2 ratios ± SEM of > 280 cells for each condition. Similar results were obtained from three independent experiments. (F) Mn 2+ influx measured by Fura-2 quenching in siRNA-treated HeLa cells stimulated with 1 µM TG. Shown are means ± SEM derived from > 200 cells for each condition across two independent experiments. (G) HeLa cells were sequentially transfected with NFAT-YFP, and either siControl or siRASSF4 was treated with 1 µM TG for 10 min and scored for NFAT by fluorescence imaging. Percentage of cells with nuclear translocation of NFAT was calculated from 80–100 cells across three independent experiments. Means ± SEM are plotted. ***, P

    Journal: The Journal of Cell Biology

    Article Title: RASSF4 controls SOCE and ER–PM junctions through regulation of PI(4,5)P2

    doi: 10.1083/jcb.201606047

    Figure Lengend Snippet: RASSF4 is a positive regulator of SOCE. (A) A diagram of the domain structure of RASSF4. The regions targeted by siRASSF4_N’ used in C, siRASSF4_C’ used in C, and siRASSF4 used in B are indicated. RA, RAS association. (B) Intracellular Ca 2+ levels determined by analysis of Fura-2 fluorescence ratios in HeLa cells treated with a control siRNA (siControl), siSTIM1, or siRASSF4 and stimulated with 1 µM TG and 100 µM histamine (His). Shown are mean Fura-2 ratios ± SEM of > 300 cells for each condition. Similar results were obtained from > 10 independent experiments. (C) Fura-2 ratios of HeLa cells treated with the indicated siRNAs. Cells were stimulated with 1 µM TG, 100 µM His, and 2 mM EGTA; 2 mM Ca 2+ was added 6 min after stimulation. Shown are mean Fura-2 ratios ± SEM derived from > 1,000 cells for each condition across two independent experiments. (D) Fura-2 ratios of siRNA-treated HUVECs stimulated as described in C. Shown are mean Fura-2 ratios ± SEM derived from > 500 cells for each condition across two independent experiments. (E) Fura-2 ratios of RPE-1 cells treated with the indicated siRNAs. Cells were stimulated with 1 µM TG and 2 mM EGTA; 2 mM Ca 2+ was added 11.5 min after stimulation. Shown are mean Fura-2 ratios ± SEM of > 280 cells for each condition. Similar results were obtained from three independent experiments. (F) Mn 2+ influx measured by Fura-2 quenching in siRNA-treated HeLa cells stimulated with 1 µM TG. Shown are means ± SEM derived from > 200 cells for each condition across two independent experiments. (G) HeLa cells were sequentially transfected with NFAT-YFP, and either siControl or siRASSF4 was treated with 1 µM TG for 10 min and scored for NFAT by fluorescence imaging. Percentage of cells with nuclear translocation of NFAT was calculated from 80–100 cells across three independent experiments. Means ± SEM are plotted. ***, P

    Article Snippet: After transfection of the HRP-ER plasmid with either siRNAs or DNA plasmids, HeLa cells were plated onto coverslips (MatTek Corporation) and fixed with 2% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) for 20 min at room temperature.

    Techniques: Fluorescence, Derivative Assay, Transfection, Imaging, Translocation Assay

    Partial nuclear distribution of Ago2 in HeLa and HaCaT cells. A , specificity of anti-Ago2 antibody in Western blotting of Ago2 expressed from HeLa cells transfected with a nonspecific (−) or Ago2-specific (+ si-Ago2 ) siRNA. Total cell extract

    Journal: The Journal of Biological Chemistry

    Article Title: Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2 *

    doi: 10.1074/jbc.C115.695049

    Figure Lengend Snippet: Partial nuclear distribution of Ago2 in HeLa and HaCaT cells. A , specificity of anti-Ago2 antibody in Western blotting of Ago2 expressed from HeLa cells transfected with a nonspecific (−) or Ago2-specific (+ si-Ago2 ) siRNA. Total cell extract

    Article Snippet: HeLa cells or primary HFKs were transfected twice or thrice, respectively, at an interval of 48 h with 20 n m (HeLa) or 40 n m (HFKs) of SMARTpool human EIF2C2 siRNAs targeting Ago2 (L-004639-00, Dharmacon, GE Healthcare) or non-targeting siRNA negative control (D-001810-01, Dharmacon, GE Healthcare) using LipoJet transfection reagent (SignaGen Laboratories, Gaithersburg, MD).

    Techniques: Western Blot, Transfection

    Interaction of intracellular salusin-β with a membrane-associated β-actin-profilin complex. ( a ) Whole cell lysates obtained from the human cell lines HeLa, NB-1, THP-1 and U937 were immunoprecipitated with anti-profilin1/2 or anti-β-actin IgG and immunoblotted with an anti-salusin-β antibody. The upper and lower panels represent 3- and 30-min exposures, respectively, of the same immunoblot. ( b ) Whole cell lysates were immunoprecipitated with anti-profilin1/2 IgG and immunoblotted with the anti-salusin-β antibody (left panel), or immunoprecipitated with anti-PIP 2 IgG and immunoblotted with anti-profilin1/2 IgG. ( c ) Immunofluorescence staining of NB-1 cells with the anti-salusin-β antibody reveals marked staining in the cell periphery. ( d ) Quiescent HeLa and THP-1 cells stimulated by 10% fetal bovine serum and endothelin-1 (10 −9 M), respectively (upper and middle panels) and growing THP-1 cells stimulated by TNF-α (100 ng/ml, lower panel) for the indicated times were fractionated by differential centrifugation. Plasma membrane (PM)-, low-density microsome (LDM)-, mitochondria/nucleotide (M/N)-, and high-density microsome (HDM)-enriched fractions were immunoprecipitated with anti-profilin1/2 IgG, separated by SDS-PAGE and analyzed by immunoblotting with the anti-salusin-β antibody.

    Journal: Scientific Reports

    Article Title: Identification of the salusin-β receptor using proteoliposomes embedded with endogenous membrane proteins

    doi: 10.1038/s41598-018-35740-6

    Figure Lengend Snippet: Interaction of intracellular salusin-β with a membrane-associated β-actin-profilin complex. ( a ) Whole cell lysates obtained from the human cell lines HeLa, NB-1, THP-1 and U937 were immunoprecipitated with anti-profilin1/2 or anti-β-actin IgG and immunoblotted with an anti-salusin-β antibody. The upper and lower panels represent 3- and 30-min exposures, respectively, of the same immunoblot. ( b ) Whole cell lysates were immunoprecipitated with anti-profilin1/2 IgG and immunoblotted with the anti-salusin-β antibody (left panel), or immunoprecipitated with anti-PIP 2 IgG and immunoblotted with anti-profilin1/2 IgG. ( c ) Immunofluorescence staining of NB-1 cells with the anti-salusin-β antibody reveals marked staining in the cell periphery. ( d ) Quiescent HeLa and THP-1 cells stimulated by 10% fetal bovine serum and endothelin-1 (10 −9 M), respectively (upper and middle panels) and growing THP-1 cells stimulated by TNF-α (100 ng/ml, lower panel) for the indicated times were fractionated by differential centrifugation. Plasma membrane (PM)-, low-density microsome (LDM)-, mitochondria/nucleotide (M/N)-, and high-density microsome (HDM)-enriched fractions were immunoprecipitated with anti-profilin1/2 IgG, separated by SDS-PAGE and analyzed by immunoblotting with the anti-salusin-β antibody.

    Article Snippet: For quantification of fluorescent salusin-β-binding, HeLa cells cultured in non-coated 96 well black plates (Corning ® ) were plated to be confluent at the time of assay, replaced with serum starved media with or without the indicated concentrations of synthetic salusin-β in 0.05% NP40/HBSS for 30 min, and further incubated for 60 min after addition of FAM-labeled salusin-β in 0.05% NP40/HBSS.

    Techniques: Immunoprecipitation, Immunofluorescence, Staining, Centrifugation, SDS Page

    Salusin-β binding to the HeLa cell surface and immunofluorescence colocalization of the ATP synthase β-subunit. ( a – d ) Confocal microscopic images of fluorescent salusin-β peptides bound to cultured HeLa cells and immunofluorescent staining for the ATP synthase-β subunit. Salusin-β-FAM ( a ): 10 −8 M, ( b – d : 10 −7 M) was overlaid for 1 min on HeLa cells pretreated without ( a , b ) or with excess β-casomorphin 7 ( c ) or enterostatin ( d ) for 30 min, fixed and stained with specific antibody against the β-chain of ATP synthase (×1000). The green signals correspond to the cell surface binding sites of salusin-β-FAM. The red signals corresponding to the localization of the β-chain of ATP synthase were obtained with Alexa Fluor ® 594 secondary antibody (×3000). The nuclei were conterstained with DAPI (blue). Overlay resulted in yellow signals indicative of colocalization. ( e,f ) Quantification of salusin-β-FAM peptide bound to HeLa cell surface. Salusin-β-FAM (10 −6 M) was overlayed on confluent HeLa cells in 96-well culture plates for the indicated time ( e ) or the indicated concentrations of salusin-β-FAM was added for 180 min ( f ) and, after extensive washing, cell-bound immunofluorescence was measured.

    Journal: Scientific Reports

    Article Title: Identification of the salusin-β receptor using proteoliposomes embedded with endogenous membrane proteins

    doi: 10.1038/s41598-018-35740-6

    Figure Lengend Snippet: Salusin-β binding to the HeLa cell surface and immunofluorescence colocalization of the ATP synthase β-subunit. ( a – d ) Confocal microscopic images of fluorescent salusin-β peptides bound to cultured HeLa cells and immunofluorescent staining for the ATP synthase-β subunit. Salusin-β-FAM ( a ): 10 −8 M, ( b – d : 10 −7 M) was overlaid for 1 min on HeLa cells pretreated without ( a , b ) or with excess β-casomorphin 7 ( c ) or enterostatin ( d ) for 30 min, fixed and stained with specific antibody against the β-chain of ATP synthase (×1000). The green signals correspond to the cell surface binding sites of salusin-β-FAM. The red signals corresponding to the localization of the β-chain of ATP synthase were obtained with Alexa Fluor ® 594 secondary antibody (×3000). The nuclei were conterstained with DAPI (blue). Overlay resulted in yellow signals indicative of colocalization. ( e,f ) Quantification of salusin-β-FAM peptide bound to HeLa cell surface. Salusin-β-FAM (10 −6 M) was overlayed on confluent HeLa cells in 96-well culture plates for the indicated time ( e ) or the indicated concentrations of salusin-β-FAM was added for 180 min ( f ) and, after extensive washing, cell-bound immunofluorescence was measured.

    Article Snippet: For quantification of fluorescent salusin-β-binding, HeLa cells cultured in non-coated 96 well black plates (Corning ® ) were plated to be confluent at the time of assay, replaced with serum starved media with or without the indicated concentrations of synthetic salusin-β in 0.05% NP40/HBSS for 30 min, and further incubated for 60 min after addition of FAM-labeled salusin-β in 0.05% NP40/HBSS.

    Techniques: Binding Assay, Immunofluorescence, Cell Culture, Staining

    Suppression of cell surface ATP synthase activity and tumor-induced angiogenesis by salusin-β. ( a – c ) Salusin-β modestly and concentration-dependently inhibits cell surface ATP generation in HeLa cells ( a ) and hMVECs ( b ) cultured at pH 7.4. Pretreatment with casomorphin (10 −6 M) abrogates the effect of salusin-β ( a ). The effect is more profound on hMVECs cultured at pH 6.7 ( c ) Extracellular ATP generation was measured by a bioluminescence surface assay following 20 s of incubation with medium in the presence and absence of ADP. Piceatannol is a stilbene phytochemical that inhibits F 1 F o ATP synthase. ( d ) Sarcoma 180 cells were implanted into mouse dorsal air sacs and the indicated doses of salusin-β were administered subcutaneously once per day for 5 days. Tumor-induced angiogenesis in the skin was evaluated by counting the numbers of neovessels microscopically. Each column with a bar represents the mean ± SEM for eight mice. * P

    Journal: Scientific Reports

    Article Title: Identification of the salusin-β receptor using proteoliposomes embedded with endogenous membrane proteins

    doi: 10.1038/s41598-018-35740-6

    Figure Lengend Snippet: Suppression of cell surface ATP synthase activity and tumor-induced angiogenesis by salusin-β. ( a – c ) Salusin-β modestly and concentration-dependently inhibits cell surface ATP generation in HeLa cells ( a ) and hMVECs ( b ) cultured at pH 7.4. Pretreatment with casomorphin (10 −6 M) abrogates the effect of salusin-β ( a ). The effect is more profound on hMVECs cultured at pH 6.7 ( c ) Extracellular ATP generation was measured by a bioluminescence surface assay following 20 s of incubation with medium in the presence and absence of ADP. Piceatannol is a stilbene phytochemical that inhibits F 1 F o ATP synthase. ( d ) Sarcoma 180 cells were implanted into mouse dorsal air sacs and the indicated doses of salusin-β were administered subcutaneously once per day for 5 days. Tumor-induced angiogenesis in the skin was evaluated by counting the numbers of neovessels microscopically. Each column with a bar represents the mean ± SEM for eight mice. * P

    Article Snippet: For quantification of fluorescent salusin-β-binding, HeLa cells cultured in non-coated 96 well black plates (Corning ® ) were plated to be confluent at the time of assay, replaced with serum starved media with or without the indicated concentrations of synthetic salusin-β in 0.05% NP40/HBSS for 30 min, and further incubated for 60 min after addition of FAM-labeled salusin-β in 0.05% NP40/HBSS.

    Techniques: Activity Assay, Concentration Assay, Cell Culture, Incubation, Mouse Assay

    The predicted LARP4 ARE confers instability on β-globin reporter mRNA. HeLa Tet-Off cells were cotransfected with a vector containing GFP under the control of a constitutive CMV promoter, along with β-globin constructs under the control

    Journal: Molecular and Cellular Biology

    Article Title: LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

    doi: 10.1128/MCB.00804-15

    Figure Lengend Snippet: The predicted LARP4 ARE confers instability on β-globin reporter mRNA. HeLa Tet-Off cells were cotransfected with a vector containing GFP under the control of a constitutive CMV promoter, along with β-globin constructs under the control

    Article Snippet: HeLa Tet-Off cells (Clontech) were maintained in Dulbecco's modified Eagle's medium (DMEM) plus Glutamax (Gibco) supplemented with 100 μg/ml G418 and 10% heat-inactivated fetal bovine serum (FBS) in a humidified 37°C, 5% CO2 incubator.

    Techniques: Plasmid Preparation, Construct

    Time course analysis for luciferase activity and quantitative RT-PC R. (A and B) Time course of luciferase protein and mRNA expression in transfected HeLa cells. HeLa cells were transfected with SV40 or proximal plasmids respectively and RNA was extracted at the indicated time point and reverse transcribed. Another series of wells was used for standard luciferase assays. Real-time PC R was performed using primers specific for luciferase ORF. Luciferase mRNA expression was calculated using the 2 −ΔΔCt method as fold over the SV40 mRNA at 12 hr after normalized with GAPDH. Values shown are the mean ± SD (n = 3), and standardized to the zero time point. (C) Quantitative RT-PC R for proximal and distal 3′ UTR luciferase mRNAs. HeLa cells were transfected with plasmids as indicated for 48 hours. Luciferase mRNA expression was determined by real-time quantitative RT-PC R. The relative differences in luciferase expression were calculated using the 2 −ΔΔCt method by comparing with the mRNA expression of the SV40 plasmid after normalization with GAPDH. The values shown are the mean ± SD (n = 3). p

    Journal: RNA biology

    Article Title: Alternative polyadenylation of MeCP2

    doi:

    Figure Lengend Snippet: Time course analysis for luciferase activity and quantitative RT-PC R. (A and B) Time course of luciferase protein and mRNA expression in transfected HeLa cells. HeLa cells were transfected with SV40 or proximal plasmids respectively and RNA was extracted at the indicated time point and reverse transcribed. Another series of wells was used for standard luciferase assays. Real-time PC R was performed using primers specific for luciferase ORF. Luciferase mRNA expression was calculated using the 2 −ΔΔCt method as fold over the SV40 mRNA at 12 hr after normalized with GAPDH. Values shown are the mean ± SD (n = 3), and standardized to the zero time point. (C) Quantitative RT-PC R for proximal and distal 3′ UTR luciferase mRNAs. HeLa cells were transfected with plasmids as indicated for 48 hours. Luciferase mRNA expression was determined by real-time quantitative RT-PC R. The relative differences in luciferase expression were calculated using the 2 −ΔΔCt method by comparing with the mRNA expression of the SV40 plasmid after normalization with GAPDH. The values shown are the mean ± SD (n = 3). p

    Article Snippet: The luciferase vectors were transfected using the FuGENE 6 transfection reagent (Roche) or LT-1 (Mirus) into HeLa cells at 60% confluency grown on 6 well plates.

    Techniques: Luciferase, Activity Assay, Expressing, Transfection, Plasmid Preparation

    Luciferase assays. (A) Relative luciferase activity of MeCP 2 proximal and distal polyadenylation signals transfected into HeLa cells compared to a luciferase construct with an SV40 polyadenylation signal (SV40) or untransfected cells (control). Constructs used are indicated below the graph. Luciferase activity was normalized to Renilla luciferase activity and to total protein concentration (n = 3). (B) Relative luciferase activity of MeCP 2 proximal and distal polyadenylation signals transfected into neuronal cells (SK-N-SH) compared to a luciferase construct with an SV40 polyadenylation signal (SV40) or untransfected cells (control). Luciferase activity was normalized to Renilla luciferase activity and to total protein concentration (n = 3).

    Journal: RNA biology

    Article Title: Alternative polyadenylation of MeCP2

    doi:

    Figure Lengend Snippet: Luciferase assays. (A) Relative luciferase activity of MeCP 2 proximal and distal polyadenylation signals transfected into HeLa cells compared to a luciferase construct with an SV40 polyadenylation signal (SV40) or untransfected cells (control). Constructs used are indicated below the graph. Luciferase activity was normalized to Renilla luciferase activity and to total protein concentration (n = 3). (B) Relative luciferase activity of MeCP 2 proximal and distal polyadenylation signals transfected into neuronal cells (SK-N-SH) compared to a luciferase construct with an SV40 polyadenylation signal (SV40) or untransfected cells (control). Luciferase activity was normalized to Renilla luciferase activity and to total protein concentration (n = 3).

    Article Snippet: The luciferase vectors were transfected using the FuGENE 6 transfection reagent (Roche) or LT-1 (Mirus) into HeLa cells at 60% confluency grown on 6 well plates.

    Techniques: Luciferase, Activity Assay, Transfection, Construct, Protein Concentration

    Atf4 but not Xbp-1s stimulates NLRP1 gene expression during ER stress. (A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0130635

    Figure Lengend Snippet: Atf4 but not Xbp-1s stimulates NLRP1 gene expression during ER stress. (A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.

    Article Snippet: Cell lysates from wild-type or ATF4 −/− HeLa cells, untreated or treated with BFA for various times, were normalized for total protein content.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, SDS Page

    NLRP1 mRNA and protein are up-regulated upon ER stress. (A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or NLRP1 −/− HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)

    Journal: PLoS ONE

    Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0130635

    Figure Lengend Snippet: NLRP1 mRNA and protein are up-regulated upon ER stress. (A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or NLRP1 −/− HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)

    Article Snippet: Cell lysates from wild-type or ATF4 −/− HeLa cells, untreated or treated with BFA for various times, were normalized for total protein content.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, SDS Page, Immunoprecipitation

    The transcription factor ATF4 binds and regulates the NLRP1 gene promoter. (A) Different NLRP1 promoter regions were cloned into a luciferase reporter vector. At 24 hours after transfection, HeLa cells were treated with BFA for 20 hours before measuring luciferase activity. (B) HeLa cells were transfected with wild-type NLRP1 promoter or a version carrying mutations in the ATF4-binding motif. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (C) The indicated NLRP1 promoter-luciferase vectors were transfected into either wild-type or ATF4 −/− HeLa cells. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (D) ChIP analysis of un-stimulated (DMSO) or BFA-stimulated HeLa cells, followed by qPCR analysis of ATF4 occupancy at the NLRP1 and ATF3 promoter. ATF3 was used as positive control. Each panel is representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0130635

    Figure Lengend Snippet: The transcription factor ATF4 binds and regulates the NLRP1 gene promoter. (A) Different NLRP1 promoter regions were cloned into a luciferase reporter vector. At 24 hours after transfection, HeLa cells were treated with BFA for 20 hours before measuring luciferase activity. (B) HeLa cells were transfected with wild-type NLRP1 promoter or a version carrying mutations in the ATF4-binding motif. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (C) The indicated NLRP1 promoter-luciferase vectors were transfected into either wild-type or ATF4 −/− HeLa cells. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (D) ChIP analysis of un-stimulated (DMSO) or BFA-stimulated HeLa cells, followed by qPCR analysis of ATF4 occupancy at the NLRP1 and ATF3 promoter. ATF3 was used as positive control. Each panel is representative of at least three independent experiments.

    Article Snippet: Cell lysates from wild-type or ATF4 −/− HeLa cells, untreated or treated with BFA for various times, were normalized for total protein content.

    Techniques: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control

    NLRP1 mRNA up-regulation is dependent on both IRE1α and PERK pathways. (A) IRE1α, PERK and ATF6 levels were reduced using siRNA. Upon treatment with ER stress, mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. (B) Stably transduced HeLa cells were cultured in presence or absence of doxycycline (Dox) for 24 hours and then treated overnight with 2μM BFA. mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

    doi: 10.1371/journal.pone.0130635

    Figure Lengend Snippet: NLRP1 mRNA up-regulation is dependent on both IRE1α and PERK pathways. (A) IRE1α, PERK and ATF6 levels were reduced using siRNA. Upon treatment with ER stress, mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. (B) Stably transduced HeLa cells were cultured in presence or absence of doxycycline (Dox) for 24 hours and then treated overnight with 2μM BFA. mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.

    Article Snippet: Cell lysates from wild-type or ATF4 −/− HeLa cells, untreated or treated with BFA for various times, were normalized for total protein content.

    Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, SDS Page, Stable Transfection, Cell Culture

    Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) HCT116 wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in HeLa cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( 12 , 15 ), genes upregulated by HPV E7 ( 64 ) and genes downregulated by HPV E2 proteins repressing E7 ( 65 , 66 ).

    Journal: Nucleic Acids Research

    Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

    doi: 10.1093/nar/gkt849

    Figure Lengend Snippet: Deregulation of PLK4 expression after expression of HPV-16 E7 depends on loss of DREAM binding to the CDE and CHR elements. ( A ) HCT116 wt and HCT116 stably transfected with a plasmid encoding HPV-16 E7 wt or its ΔDLYC mutant. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( B ) Luciferase reporter assays from lysates of HCT116 wt; p53 −/− or p21 −/− cells transfected with plasmids expressing wt or CDE/CHR mutant Plk4 promoter constructs. Plasmids expressing HPV-16 E7 wt or its ΔDLYC mutant were cotransfected. Results are given as relative light units. ( C ) Nuclear extracts from HCT116 E7 wt and E7 ΔDLYC cells were analyzed by DNA affinity purification with wt and CDE/CHR mutant Plk4 promoter probes followed by western blot. Band intensities were quantified by densitometric analyses. Relative intensities are given below the bands. Intensities of input bands were normalized to E7 ΔDLYC. Binding intensities to Plk4 promoters were normalized to CDE/CHR mutant probes from E7 ΔDLYC extracts. ( D ) Protein binding to the PLK4 promoter in HeLa cells was assessed by ChIP. Protein binding to the GAPDHS promoter served as a negative control. ( E ) HCT116 E7 wt and HeLa cells were treated with doxorubicin and roscovitine for 24 h. Cells without treatment served as control. Relative expression of PLK4 mRNA was quantified by RT-PCR and normalized to U6 RNA levels. ( F ) Luciferase reporter assays with wt and CDE/CHR mutant Plk4 promoter reporter constructs in HCT116 p53 −/− cells. Cells were cotransfected with p53mut, p53wt, p21mut or p21wt expression vectors and HPV-16 E7 wt or ΔDLYC mutant. Results are given as relative light units. ( G ) Venn diagram from DREAM-bound genes ( 12 , 15 ), genes upregulated by HPV E7 ( 64 ) and genes downregulated by HPV E2 proteins repressing E7 ( 65 , 66 ).

    Article Snippet: HCT116 E7 wt cells and HeLa cells were treated with doxorubicin and roscovitine yielding no significant repression of Plk4 mRNA ( E, Supplementary Figure S4C ).

    Techniques: Expressing, Binding Assay, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Luciferase, Construct, Affinity Purification, Western Blot, Protein Binding, Chromatin Immunoprecipitation, Negative Control

    Activation of the ERK or the JNK pathway inhibits GR transcriptional activation. ( A ) Selective activation of ERK and JNK in HeLa cells by f-sos and racQ61L. pRK5-HA ERK (1.5 μg), pRK5-M2 JNK (1.5 μg), along with pRK5-HA f-sos (4 μg) or pCMV5-racQ61L (4 μg) or an empty pRK5 vector (4 μg), where indicated, was transfected into HeLa cells (2.5 × 10 5 cells/60-mm dish) using Lipofectamine. Transfected cells were treated with 100 nM Dex where indicated and harvested 8 h later. To visualize HA ERK and HA f-sos, WCE from ERK-transfected cells were immunoblotted with anti-HA mouse monoclonal antibody (left panel). FLAG-tagged JNK was immunoprecipitated from WCE by using the FLAG-specific mouse monoclonal antibody M2 and used in vitro to phosphorylate recombinant purified GST–c-Jun. Phosphorylated proteins were separated on 10% SDS/polyacrylamide gels, stained with Coomassie blue to verify equal amount of GST–c-Jun in each lane, and autoradiographed (right panel). ( B ) Inhibition of GR transcriptional enhancement by activated ERK and JNK. HeLa cells were transfected as described in A ) and normalized to β-Gal activity. ( C ) Level of GR protein is unaffected by JNK or ERK activation. WCE were prepared from a parallel set of transfected cells. Equal amounts of total protein (50 μg/lane) were separated by SDS/PAGE, transferred to Immobilon paper, probed with a GR-specific polyclonal antibody, and visualized by enhanced chemiluminescence.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase

    doi:

    Figure Lengend Snippet: Activation of the ERK or the JNK pathway inhibits GR transcriptional activation. ( A ) Selective activation of ERK and JNK in HeLa cells by f-sos and racQ61L. pRK5-HA ERK (1.5 μg), pRK5-M2 JNK (1.5 μg), along with pRK5-HA f-sos (4 μg) or pCMV5-racQ61L (4 μg) or an empty pRK5 vector (4 μg), where indicated, was transfected into HeLa cells (2.5 × 10 5 cells/60-mm dish) using Lipofectamine. Transfected cells were treated with 100 nM Dex where indicated and harvested 8 h later. To visualize HA ERK and HA f-sos, WCE from ERK-transfected cells were immunoblotted with anti-HA mouse monoclonal antibody (left panel). FLAG-tagged JNK was immunoprecipitated from WCE by using the FLAG-specific mouse monoclonal antibody M2 and used in vitro to phosphorylate recombinant purified GST–c-Jun. Phosphorylated proteins were separated on 10% SDS/polyacrylamide gels, stained with Coomassie blue to verify equal amount of GST–c-Jun in each lane, and autoradiographed (right panel). ( B ) Inhibition of GR transcriptional enhancement by activated ERK and JNK. HeLa cells were transfected as described in A ) and normalized to β-Gal activity. ( C ) Level of GR protein is unaffected by JNK or ERK activation. WCE were prepared from a parallel set of transfected cells. Equal amounts of total protein (50 μg/lane) were separated by SDS/PAGE, transferred to Immobilon paper, probed with a GR-specific polyclonal antibody, and visualized by enhanced chemiluminescence.

    Article Snippet: The GR derivative isolated from HeLa cells was purified on a 9% SDS/polyacrylamide gel and transferred to Immobilon paper for autoradiography and Western blotting with the GR-specific rabbit polyclonal antibody, sc-1002 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Plasmid Preparation, Transfection, Immunoprecipitation, In Vitro, Recombinant, Purification, Staining, Inhibition, Activity Assay, SDS Page

    Activation of the JNK but not ERK pathway in vivo increases GR phosphorylation. HeLa cells (1 × 10 6 ) were transiently transfected in 100-mm dishes with pCMV-GR S224A S232A (2.3 μg) and ( A ) pRK5-M2 JNK (4.5 μg) with pCMV5-racQ61L (12 μg) or an empty pCMV5 vector (12 μg) or ( B ) pRK5-HA ERK (4.5 μg) with pRK5-HA f-sos (12 μg) or an empty pRK5 vector (12 μg) by using Lipofectamine. After 18 h, GR was metabolically labeled for 2 h with 1 mCi/ml of [ 32 P]orthophosphate in the presence of 100 nM Dex. In vivo labeled receptor was immunoprecipitated from cell lysates with the rat GR-specific monoclonal antibody, BuGR2. Immunoprecipitates were separated on 9% SDS/PAGE gels, transferred to Immobilon membrane, and exposed to film to visualize the phosphorylated GR ( Upper ). To normalize to GR protein expression, Western blotting of the same membrane with anti-GR rabbit polyclonal antibody was performed ( Lower ). The densitometric value obtained in the absence of cotransfected racQ61L ( A ) or f-sos ( B ) is arbitrarily set as 1.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase

    doi:

    Figure Lengend Snippet: Activation of the JNK but not ERK pathway in vivo increases GR phosphorylation. HeLa cells (1 × 10 6 ) were transiently transfected in 100-mm dishes with pCMV-GR S224A S232A (2.3 μg) and ( A ) pRK5-M2 JNK (4.5 μg) with pCMV5-racQ61L (12 μg) or an empty pCMV5 vector (12 μg) or ( B ) pRK5-HA ERK (4.5 μg) with pRK5-HA f-sos (12 μg) or an empty pRK5 vector (12 μg) by using Lipofectamine. After 18 h, GR was metabolically labeled for 2 h with 1 mCi/ml of [ 32 P]orthophosphate in the presence of 100 nM Dex. In vivo labeled receptor was immunoprecipitated from cell lysates with the rat GR-specific monoclonal antibody, BuGR2. Immunoprecipitates were separated on 9% SDS/PAGE gels, transferred to Immobilon membrane, and exposed to film to visualize the phosphorylated GR ( Upper ). To normalize to GR protein expression, Western blotting of the same membrane with anti-GR rabbit polyclonal antibody was performed ( Lower ). The densitometric value obtained in the absence of cotransfected racQ61L ( A ) or f-sos ( B ) is arbitrarily set as 1.

    Article Snippet: The GR derivative isolated from HeLa cells was purified on a 9% SDS/polyacrylamide gel and transferred to Immobilon paper for autoradiography and Western blotting with the GR-specific rabbit polyclonal antibody, sc-1002 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, In Vivo, Transfection, Plasmid Preparation, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Expressing, Western Blot

    FRET analysis reveals SBM-dependent interaction of La with nucleolin in the nucleolus. (A) Constructs used for the interaction analysis below were first examined for expression in HeLa cells prior to FRET analysis. Column I shows the yellow channel fluorescence of the YFP-hLa constructs, column II shows the cyan channel fluorescence of the CFP-nucleolin constructs, and column III shows the overlaid views for the indicated constructs. Note that the last row shows CFP-fibrillarin. (B) Acceptor photobleaching was used to monitor FRET between the YFP-hLa (acceptor) constructs and the CFP-nucleolin or CFP-fibrillarin (donor) after specific bleaching of the acceptor with YFP-wavelength light. FRET efficiencies reflective of positive interactions were plotted as bars above the horizontal line (see Materials and Methods). Bars below the horizontal line reflect control measurements in the same cells in nucleolar areas in which there was no prebleaching of the acceptor. At least 30 measurements were collected and quantitated for each bar in the graph. CFP/YFP, CFP alone plus YFP alone; CFP-YFP, CFP-YFP fusion protein.

    Journal: Molecular and Cellular Biology

    Article Title: Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis

    doi: 10.1128/MCB.24.24.10894-10904.2004

    Figure Lengend Snippet: FRET analysis reveals SBM-dependent interaction of La with nucleolin in the nucleolus. (A) Constructs used for the interaction analysis below were first examined for expression in HeLa cells prior to FRET analysis. Column I shows the yellow channel fluorescence of the YFP-hLa constructs, column II shows the cyan channel fluorescence of the CFP-nucleolin constructs, and column III shows the overlaid views for the indicated constructs. Note that the last row shows CFP-fibrillarin. (B) Acceptor photobleaching was used to monitor FRET between the YFP-hLa (acceptor) constructs and the CFP-nucleolin or CFP-fibrillarin (donor) after specific bleaching of the acceptor with YFP-wavelength light. FRET efficiencies reflective of positive interactions were plotted as bars above the horizontal line (see Materials and Methods). Bars below the horizontal line reflect control measurements in the same cells in nucleolar areas in which there was no prebleaching of the acceptor. At least 30 measurements were collected and quantitated for each bar in the graph. CFP/YFP, CFP alone plus YFP alone; CFP-YFP, CFP-YFP fusion protein.

    Article Snippet: Four different La-YFP constructs were each cotransfected with nucleolin-CFP into HeLa cells and visualized for either YFP or CFP or both (Fig. , columns I, II, and III, respectively).

    Techniques: Construct, Expressing, Fluorescence

    Nucleolin interacts with epitope-tagged La-FLAG-HA or native endogenous La in HeLa cells. (A) SDS-PAGE followed by Coomassie blue staining after two epitope-directed affinity purifications of extract from cells transfected with empty plasmid (lane 1) or cells expressing hLa-FLAG-HA (lane 2). (B) The affinity-purified material derived from cells transfected with hLa-FLAG-HA was untreated (lane 1) or treated with RNase A (lane 2) and then subjected to SDS-PAGE analysis followed by silver staining. (C) Identity of the five peptide sequences identified from the La-FLAG-HA-associated band with an apparent molecular mass of ∼116 kDa (see text). (D to G) Immunoprecipitation of HeLa cell extract by anti-La and nonimmune antibodies and analysis by Western blotting to detect nucleolin(D), fibrillarin (E), La (F), and β-actin (G). Samples are numbered below the lanes. in, input; S, supernatant; IP+, immunoprecipitate after treatment with RNase A. For panel H, material was collected and labeled with 32 P-Cp and RNA ligase and analyzed by denaturing PAGE and autoradiography. The asterisk represents RNase A-resistant, protease-sensitive material that results from the labeling reaction (data not shown), which serves as a recovery-loading control for labeling. Molecular mass markers (lanes M) are expressed in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis

    doi: 10.1128/MCB.24.24.10894-10904.2004

    Figure Lengend Snippet: Nucleolin interacts with epitope-tagged La-FLAG-HA or native endogenous La in HeLa cells. (A) SDS-PAGE followed by Coomassie blue staining after two epitope-directed affinity purifications of extract from cells transfected with empty plasmid (lane 1) or cells expressing hLa-FLAG-HA (lane 2). (B) The affinity-purified material derived from cells transfected with hLa-FLAG-HA was untreated (lane 1) or treated with RNase A (lane 2) and then subjected to SDS-PAGE analysis followed by silver staining. (C) Identity of the five peptide sequences identified from the La-FLAG-HA-associated band with an apparent molecular mass of ∼116 kDa (see text). (D to G) Immunoprecipitation of HeLa cell extract by anti-La and nonimmune antibodies and analysis by Western blotting to detect nucleolin(D), fibrillarin (E), La (F), and β-actin (G). Samples are numbered below the lanes. in, input; S, supernatant; IP+, immunoprecipitate after treatment with RNase A. For panel H, material was collected and labeled with 32 P-Cp and RNA ligase and analyzed by denaturing PAGE and autoradiography. The asterisk represents RNase A-resistant, protease-sensitive material that results from the labeling reaction (data not shown), which serves as a recovery-loading control for labeling. Molecular mass markers (lanes M) are expressed in kilodaltons.

    Article Snippet: Four different La-YFP constructs were each cotransfected with nucleolin-CFP into HeLa cells and visualized for either YFP or CFP or both (Fig. , columns I, II, and III, respectively).

    Techniques: SDS Page, Staining, Transfection, Plasmid Preparation, Expressing, Affinity Purification, Derivative Assay, Silver Staining, Immunoprecipitation, Western Blot, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography

    Effect of substrate availability on maintenance of infectivity. P. amoebophila and C. trachomatis cells were harvested from infected amoeba and HeLa 229 cell cultures, respectively, and incubated for indicated periods of time in different host-free media. Subsequently, incubated bacteria were used to infect amoebae ( P. amoebophila ) or HeLa 229 cells ( C. trachomatis ), which were then fixed at 48 h or 24 h p.i., respectively. Bacteria were detected by FISH ( P. amoebophila ) or immunostaining ( C. trachomatis ). The observed infectivity, relative to that observed for 2 h incubation in DGM-D ( P. amoebophila ) or 30 min incubation in DGM-D6P ( C. trachomatis ) is depicted in (A) and (C), respectively. Data represent means and standard deviations from at least three replicate host-free incubations. For each sample a minimum of 600 amoebae (A) or 300 HeLa 229 cells (C) was counted. Statistically significant differences compared to the values obtained for DGM-D (A) or DGM-D6P (C) are indicated (ANOVA; ***, p≤0.001; **, p≤0.01; *, p≤0.05). In (B) representative fluorescence and DIC images of amoebae infected with P. amoebophila after 48 h host-free incubation in the indicated media are shown (FISH, red). The bar indicates 10 µm. In (D) representative confocal fluorescence images of HeLa 229 cells infected with C. trachomatis after 2 h host-free incubation in the indicated media are shown. Bacteria were detected by immunostaining (red), host cells and DNA were stained using HCS cytoplasmic stain (grey) and DAPI (blue), respectively. The bar indicates 25 µm.

    Journal: PLoS Pathogens

    Article Title: Metabolic Features of Protochlamydia amoebophila Elementary Bodies - A Link between Activity and Infectivity in Chlamydiae

    doi: 10.1371/journal.ppat.1003553

    Figure Lengend Snippet: Effect of substrate availability on maintenance of infectivity. P. amoebophila and C. trachomatis cells were harvested from infected amoeba and HeLa 229 cell cultures, respectively, and incubated for indicated periods of time in different host-free media. Subsequently, incubated bacteria were used to infect amoebae ( P. amoebophila ) or HeLa 229 cells ( C. trachomatis ), which were then fixed at 48 h or 24 h p.i., respectively. Bacteria were detected by FISH ( P. amoebophila ) or immunostaining ( C. trachomatis ). The observed infectivity, relative to that observed for 2 h incubation in DGM-D ( P. amoebophila ) or 30 min incubation in DGM-D6P ( C. trachomatis ) is depicted in (A) and (C), respectively. Data represent means and standard deviations from at least three replicate host-free incubations. For each sample a minimum of 600 amoebae (A) or 300 HeLa 229 cells (C) was counted. Statistically significant differences compared to the values obtained for DGM-D (A) or DGM-D6P (C) are indicated (ANOVA; ***, p≤0.001; **, p≤0.01; *, p≤0.05). In (B) representative fluorescence and DIC images of amoebae infected with P. amoebophila after 48 h host-free incubation in the indicated media are shown (FISH, red). The bar indicates 10 µm. In (D) representative confocal fluorescence images of HeLa 229 cells infected with C. trachomatis after 2 h host-free incubation in the indicated media are shown. Bacteria were detected by immunostaining (red), host cells and DNA were stained using HCS cytoplasmic stain (grey) and DAPI (blue), respectively. The bar indicates 25 µm.

    Article Snippet: HeLa 229 cells (ATCC, CCL-2.1) were cultivated at 37°C, 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (PAA).

    Techniques: Infection, Incubation, Fluorescence In Situ Hybridization, Immunostaining, Fluorescence, Staining

    The effect of Enhancer nanobody binding on the repeated photoswitching behavior of rsGreens in HeLa cells (500 cycles): ( A ) representative fluorescence photoswitching trace of rsGreen1, inset shows the initial five switching cycles; ( B ) signal of the maximally on-switched frame for each switching cycle scaled to the initial on-state fluorescence; and ( C ) baseline fluorescence (maximally off-switched frame) as fraction of the initial on-state fluorescence.

    Journal: International Journal of Molecular Sciences

    Article Title: Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

    doi: 10.3390/ijms18092015

    Figure Lengend Snippet: The effect of Enhancer nanobody binding on the repeated photoswitching behavior of rsGreens in HeLa cells (500 cycles): ( A ) representative fluorescence photoswitching trace of rsGreen1, inset shows the initial five switching cycles; ( B ) signal of the maximally on-switched frame for each switching cycle scaled to the initial on-state fluorescence; and ( C ) baseline fluorescence (maximally off-switched frame) as fraction of the initial on-state fluorescence.

    Article Snippet: High-power photoswitching in HeLa cells was measured on a setup comprising an Olympus IX 71 inverted microscope (Olympus, Berchem, Belgium) coupled to a Spectra X Light Engine (Lumencor, Beaverton, OR, USA), equipped with a 10 × objective (UplanSApo, Olympus, Berchem, Belgium), a dichroic turret wheel mounting a ZT488RDC (Chroma, Olching, Germany) dichroic filter with a 535/30 bandpass emission filter.

    Techniques: Binding Assay, Fluorescence

    Global fits describing a two-species model for photoswitching in HeLa cells. Contribution of the two switching species of ( A ) rsGreen1, ( B ) rsGreen1-Enhancer, ( C ) rsGreenF, ( D ) rsGreenF-Enhancer and their respective fits ( Appendix B ). Red curves represent the fast-switching species A, blue curves the slower-switching species B. Inset: single off-switching decays and their respective fits for switching cycles 1 (purple), 250 (light blue) and 500 (green).

    Journal: International Journal of Molecular Sciences

    Article Title: Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

    doi: 10.3390/ijms18092015

    Figure Lengend Snippet: Global fits describing a two-species model for photoswitching in HeLa cells. Contribution of the two switching species of ( A ) rsGreen1, ( B ) rsGreen1-Enhancer, ( C ) rsGreenF, ( D ) rsGreenF-Enhancer and their respective fits ( Appendix B ). Red curves represent the fast-switching species A, blue curves the slower-switching species B. Inset: single off-switching decays and their respective fits for switching cycles 1 (purple), 250 (light blue) and 500 (green).

    Article Snippet: High-power photoswitching in HeLa cells was measured on a setup comprising an Olympus IX 71 inverted microscope (Olympus, Berchem, Belgium) coupled to a Spectra X Light Engine (Lumencor, Beaverton, OR, USA), equipped with a 10 × objective (UplanSApo, Olympus, Berchem, Belgium), a dichroic turret wheel mounting a ZT488RDC (Chroma, Olching, Germany) dichroic filter with a 535/30 bandpass emission filter.

    Techniques:

    The effect of Enhancer nanobody binding on the photoswitching behavior of rsGreens. ( A ) Normalized average fluorescence of E. coli colonies expressing indicated FPs upon irradiation with violet, blue and violet light. ( B ) Normalized average fluorescence in HeLa cells upon irradiation with violet and cyan light with indicated preset powers. ( C ) Spontaneous, thermal recovery of the fluorescence in HeLa cells after off-switching with cyan light. a.u.: arbitrary units.

    Journal: International Journal of Molecular Sciences

    Article Title: Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

    doi: 10.3390/ijms18092015

    Figure Lengend Snippet: The effect of Enhancer nanobody binding on the photoswitching behavior of rsGreens. ( A ) Normalized average fluorescence of E. coli colonies expressing indicated FPs upon irradiation with violet, blue and violet light. ( B ) Normalized average fluorescence in HeLa cells upon irradiation with violet and cyan light with indicated preset powers. ( C ) Spontaneous, thermal recovery of the fluorescence in HeLa cells after off-switching with cyan light. a.u.: arbitrary units.

    Article Snippet: High-power photoswitching in HeLa cells was measured on a setup comprising an Olympus IX 71 inverted microscope (Olympus, Berchem, Belgium) coupled to a Spectra X Light Engine (Lumencor, Beaverton, OR, USA), equipped with a 10 × objective (UplanSApo, Olympus, Berchem, Belgium), a dichroic turret wheel mounting a ZT488RDC (Chroma, Olching, Germany) dichroic filter with a 535/30 bandpass emission filter.

    Techniques: Binding Assay, Fluorescence, Expressing, Irradiation

    Confocal microscopy of HeLa cells after transfection with DNA2aD1 – DNA3rD5 (row 1), DNA2rD1 – DNA3aD8 (row 2), DNA2aD2–DNA3aD8 (row 3) and DNA2rD4 – DNA3aD8 (row 4). The visualization was performed using a Leica TCS-SPE (DMi8) inverted microscope with an ACS APO 63×/1.30 oil objective. For DNA2aD1 – DNA3rD5 λ exc = 405 nm (UV laser), λ em = 435–470 nm (blue) and 575–750 nm (yellow), for DNA2rD1 – DNA3aD8 λ exc = 405 nm (UV laser), λ em = 415–550 nm (blue) and 575–750 nm (red), for DNA2aD2–DNA3aD8 λ exc = 488 nm (argon ion laser), λ em = 490–550 nm (green) and 550–675 nm (red), for DNA2rD4 – DNA3aD8 λ exc = 488 nm (argon ion laser), λ em = 490–550 nm (green) and 675–800 nm (red), scale bar = 20 µm.

    Journal: Beilstein Journal of Organic Chemistry

    Article Title: A postsynthetically 2’-“clickable” uridine with arabino configuration and its application for fluorescent labeling and imaging of DNA

    doi: 10.3762/bjoc.13.16

    Figure Lengend Snippet: Confocal microscopy of HeLa cells after transfection with DNA2aD1 – DNA3rD5 (row 1), DNA2rD1 – DNA3aD8 (row 2), DNA2aD2–DNA3aD8 (row 3) and DNA2rD4 – DNA3aD8 (row 4). The visualization was performed using a Leica TCS-SPE (DMi8) inverted microscope with an ACS APO 63×/1.30 oil objective. For DNA2aD1 – DNA3rD5 λ exc = 405 nm (UV laser), λ em = 435–470 nm (blue) and 575–750 nm (yellow), for DNA2rD1 – DNA3aD8 λ exc = 405 nm (UV laser), λ em = 415–550 nm (blue) and 575–750 nm (red), for DNA2aD2–DNA3aD8 λ exc = 488 nm (argon ion laser), λ em = 490–550 nm (green) and 550–675 nm (red), for DNA2rD4 – DNA3aD8 λ exc = 488 nm (argon ion laser), λ em = 490–550 nm (green) and 675–800 nm (red), scale bar = 20 µm.

    Article Snippet: 24 h before transfection 5 × 104 HeLa cells per well were seeded in an 8-well chamber slide (µ Slide 8 well ibiTreat, IBIDI, Martinsried, Germany) in 200 µL of media.

    Techniques: Confocal Microscopy, Transfection, Inverted Microscopy

    Species-specific differences in primate CMV TRS1 PKR antagonism. (A) HeLa PKR KO, HeLa (human), or BSC40 (Agm) cells were mock infected or infected (MOI 0.1) with WT VacV, VacVΔE3L, or VacVΔE3L recombinants containing HCMV TRS1 , AgmCMV TRS1 , RhCMV TRS1 , or SmCMV TRS1 . At 48 hpi, viral replication was quantified by measuring β-gal activity (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected HeLa PKR KO cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length.

    Journal: PLoS Pathogens

    Article Title: A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    doi: 10.1371/journal.ppat.1005966

    Figure Lengend Snippet: Species-specific differences in primate CMV TRS1 PKR antagonism. (A) HeLa PKR KO, HeLa (human), or BSC40 (Agm) cells were mock infected or infected (MOI 0.1) with WT VacV, VacVΔE3L, or VacVΔE3L recombinants containing HCMV TRS1 , AgmCMV TRS1 , RhCMV TRS1 , or SmCMV TRS1 . At 48 hpi, viral replication was quantified by measuring β-gal activity (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected HeLa PKR KO cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length.

    Article Snippet: HeLa PKR KO cell lines were generated by transfecting HeLa cells with plasmid vectors that express Cas9 (HCas9, a gift from George Church, Addgene #41815 [ ], two guide RNAs (pEQ 1451 and pEQ1452) that target genomic sequences upstream (5’-TCTCTTCCATTGTAGGATA-3’) and downstream (5’-CTTTTCTTCCACACAGTCA-3’) of PKR, and a homologous recombination vector containing mCherry and puromycin (pEQ1489).

    Techniques: Infection, Activity Assay, Construct, Western Blot

    Species-specific differences in primate CMV TRS1 PKR antagonism map to a single amino acid. (A) Schematic representation of the SEAP assay. Transfection of PKR leads to decreased activity of a co-transfected reporter construct expressing SEAP. This PKR-driven repression can be counteracted by co-transfection of a functional TRS1 antagonist, resulting in a rescue of SEAP activity. (B) The SEAP assay recapitulates species-specific differences in HuPKR antagonism by TRS1 alleles. HeLa PKR KO cells were co-transfected with a SEAP reporter plasmid along with either a control vector or HuPKR and the indicated TRS1 alleles or a vector control. At 48 h post-transfection, SEAP activity in the medium was measured (mean ± s.d., n = 2). Data are representative of three independent experiments. (C) A single amino acid change, F489S, confers resistance to HCMV TRS1 . Point mutants were generated in HuPKR to introduce the six AgmPKR-specific residues that differ between HuPKR and AgmPKR within the region spanning codons 475 to 520, shown in the alignment. The ability of the point mutants to antagonize HuPKR was evaluated as described in (B) (mean ± s.d., n = 2). Data are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    doi: 10.1371/journal.ppat.1005966

    Figure Lengend Snippet: Species-specific differences in primate CMV TRS1 PKR antagonism map to a single amino acid. (A) Schematic representation of the SEAP assay. Transfection of PKR leads to decreased activity of a co-transfected reporter construct expressing SEAP. This PKR-driven repression can be counteracted by co-transfection of a functional TRS1 antagonist, resulting in a rescue of SEAP activity. (B) The SEAP assay recapitulates species-specific differences in HuPKR antagonism by TRS1 alleles. HeLa PKR KO cells were co-transfected with a SEAP reporter plasmid along with either a control vector or HuPKR and the indicated TRS1 alleles or a vector control. At 48 h post-transfection, SEAP activity in the medium was measured (mean ± s.d., n = 2). Data are representative of three independent experiments. (C) A single amino acid change, F489S, confers resistance to HCMV TRS1 . Point mutants were generated in HuPKR to introduce the six AgmPKR-specific residues that differ between HuPKR and AgmPKR within the region spanning codons 475 to 520, shown in the alignment. The ability of the point mutants to antagonize HuPKR was evaluated as described in (B) (mean ± s.d., n = 2). Data are representative of three independent experiments.

    Article Snippet: HeLa PKR KO cell lines were generated by transfecting HeLa cells with plasmid vectors that express Cas9 (HCas9, a gift from George Church, Addgene #41815 [ ], two guide RNAs (pEQ 1451 and pEQ1452) that target genomic sequences upstream (5’-TCTCTTCCATTGTAGGATA-3’) and downstream (5’-CTTTTCTTCCACACAGTCA-3’) of PKR, and a homologous recombination vector containing mCherry and puromycin (pEQ1489).

    Techniques: SEAP Assay, Transfection, Activity Assay, Construct, Expressing, Cotransfection, Functional Assay, Plasmid Preparation, Generated, Introduce

    HuPKR F489S confers resistance to VacV K3L H47R. HeLa PKR KO cells inducibly expressing the indicated PKR variants were infected and evaluated as described in Fig 3A . (mean ± s.d., n = 3, ** p

    Journal: PLoS Pathogens

    Article Title: A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    doi: 10.1371/journal.ppat.1005966

    Figure Lengend Snippet: HuPKR F489S confers resistance to VacV K3L H47R. HeLa PKR KO cells inducibly expressing the indicated PKR variants were infected and evaluated as described in Fig 3A . (mean ± s.d., n = 3, ** p

    Article Snippet: HeLa PKR KO cell lines were generated by transfecting HeLa cells with plasmid vectors that express Cas9 (HCas9, a gift from George Church, Addgene #41815 [ ], two guide RNAs (pEQ 1451 and pEQ1452) that target genomic sequences upstream (5’-TCTCTTCCATTGTAGGATA-3’) and downstream (5’-CTTTTCTTCCACACAGTCA-3’) of PKR, and a homologous recombination vector containing mCherry and puromycin (pEQ1489).

    Techniques: Expressing, Infection

    The F489S mutation eliminates HCMV TRS1 binding to PKR. HeLa PKR KO cells were co-transfected with WT HuPKR or HuPKR F489S and either His-tagged HCMV TRS1 , AgmCMV TRS1 , SmCMV TRS1 or EGFP. At 48h post transfection, lysates were prepared and incubated with nickel-agarose beads. Cell lysates and bound proteins were analyzed by western blotting, probing for His-tagged TRS1 proteins and for PKR. Data are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    doi: 10.1371/journal.ppat.1005966

    Figure Lengend Snippet: The F489S mutation eliminates HCMV TRS1 binding to PKR. HeLa PKR KO cells were co-transfected with WT HuPKR or HuPKR F489S and either His-tagged HCMV TRS1 , AgmCMV TRS1 , SmCMV TRS1 or EGFP. At 48h post transfection, lysates were prepared and incubated with nickel-agarose beads. Cell lysates and bound proteins were analyzed by western blotting, probing for His-tagged TRS1 proteins and for PKR. Data are representative of three independent experiments.

    Article Snippet: HeLa PKR KO cell lines were generated by transfecting HeLa cells with plasmid vectors that express Cas9 (HCas9, a gift from George Church, Addgene #41815 [ ], two guide RNAs (pEQ 1451 and pEQ1452) that target genomic sequences upstream (5’-TCTCTTCCATTGTAGGATA-3’) and downstream (5’-CTTTTCTTCCACACAGTCA-3’) of PKR, and a homologous recombination vector containing mCherry and puromycin (pEQ1489).

    Techniques: Mutagenesis, Binding Assay, Transfection, Incubation, Western Blot

    The PKR pathway is activated in HuPKR F489S cells after infection with VacVΔE3L+HCMV TRS1 . HeLa PKR KO cells with stably integrated HuPKR or HuPKR F489S were induced with doxycycline to express PKR and 24 hours later mock-infected or infected at an MOI of 3 with the indicated viruses. At 6 hpi, cells were lysed and levels of PKR-P, total PKR, eIF2α-P, total eIF2α and actin were evaluated by western blotting.

    Journal: PLoS Pathogens

    Article Title: A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    doi: 10.1371/journal.ppat.1005966

    Figure Lengend Snippet: The PKR pathway is activated in HuPKR F489S cells after infection with VacVΔE3L+HCMV TRS1 . HeLa PKR KO cells with stably integrated HuPKR or HuPKR F489S were induced with doxycycline to express PKR and 24 hours later mock-infected or infected at an MOI of 3 with the indicated viruses. At 6 hpi, cells were lysed and levels of PKR-P, total PKR, eIF2α-P, total eIF2α and actin were evaluated by western blotting.

    Article Snippet: HeLa PKR KO cell lines were generated by transfecting HeLa cells with plasmid vectors that express Cas9 (HCas9, a gift from George Church, Addgene #41815 [ ], two guide RNAs (pEQ 1451 and pEQ1452) that target genomic sequences upstream (5’-TCTCTTCCATTGTAGGATA-3’) and downstream (5’-CTTTTCTTCCACACAGTCA-3’) of PKR, and a homologous recombination vector containing mCherry and puromycin (pEQ1489).

    Techniques: Infection, Stable Transfection, Western Blot

    HuPKR F489S confers resistance in the context of viral replication. (A) Triplicate wells of HeLa PKR KO cells inducibly expressing the indicated PKR variants were treated +/- doxycycline and infected (MOI 0.1) with a panel of VacVs. At 48 hpi, viral replication was quantified by measuring β-gal activity and is reported as percent replication in doxycycline treated cells relative to replication in the same cells without induction of PKR expression (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected empty vector cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length. (C) PKR expression in lysates of mock-infected cells from (A) was evaluated by western blotting. *AgmPKR does not cross react with the antibody used.

    Journal: PLoS Pathogens

    Article Title: A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    doi: 10.1371/journal.ppat.1005966

    Figure Lengend Snippet: HuPKR F489S confers resistance in the context of viral replication. (A) Triplicate wells of HeLa PKR KO cells inducibly expressing the indicated PKR variants were treated +/- doxycycline and infected (MOI 0.1) with a panel of VacVs. At 48 hpi, viral replication was quantified by measuring β-gal activity and is reported as percent replication in doxycycline treated cells relative to replication in the same cells without induction of PKR expression (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected empty vector cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length. (C) PKR expression in lysates of mock-infected cells from (A) was evaluated by western blotting. *AgmPKR does not cross react with the antibody used.

    Article Snippet: HeLa PKR KO cell lines were generated by transfecting HeLa cells with plasmid vectors that express Cas9 (HCas9, a gift from George Church, Addgene #41815 [ ], two guide RNAs (pEQ 1451 and pEQ1452) that target genomic sequences upstream (5’-TCTCTTCCATTGTAGGATA-3’) and downstream (5’-CTTTTCTTCCACACAGTCA-3’) of PKR, and a homologous recombination vector containing mCherry and puromycin (pEQ1489).

    Techniques: Expressing, Infection, Activity Assay, Construct, Plasmid Preparation, Western Blot

    Effect of DNA sequences susceptible or unsusceptible to AAV Rep-mediated nicking on the stable transfection levels with AAVS1 -targeting vectors. ( A ) Structures of the nicking-competent targeting plasmids pA1.p5.GFP.A2, pA1.RBE/trs.GFP.A2 and pA1.trs/RBE.GFP.A2 and that of the nicking-resistant donor constructs pA1.GFP.A2, pA1.RBE/Δtrs.GFP.A2 and pA1.RBEst/trs.GFP.A2. The nucleotide sequence corresponding to the wild-type RBE is shown in black uppercase letters whereas the trs (i.e. the position at which Rep78/68-mediated nicking takes place) is indicated by black lowercase letters and a vertical arrow. The DNA sequence between the RBE and the trs is called the spacer. Targeting plasmid pA1.RBE/Δtrs.GFP.A2 has the dinucleotide at which Rep endonuclease-mediated nicking occurs mutated from TT to AA while in pA1.RBE.st/trs.GFP.A2 a 21-bp stuffer positions the trs at a bigger distance from RBE-bound AAV Rep molecules. For an explanation of the other symbols and elements see the legend of Figure 3 A. ( B ) Representative dot plots of flow cytometric analysis of the frequency of GFP -modified HeLa cells at 37 days post-transfection in cultures initially exposed to pA1.p5.GFP.A2 and ‘empty’ plasmid (p5 + +empty), pA1.p5.GFP.A2 and pGAPDH.Rep68(Y156F) (p5 + +Y156F), pA1.GFP.A2 and pGAPDH.Rep68 (p5 − +Rep68), pA1.RBE/Δtrs.GFP.A2 and pGAPDH.Rep68 (RBE/Δtrs+Rep68), pA1.RBEst/trs.GFP.A2 and pGAPDH.Rep68 (RBEst/trs+Rep68), pA1.p5.GFP.A2 and pGAPDH.Rep68 (p5 + +Rep68), pA1.RBE/trs.GFP.A2 and pGAPDH.Rep68 (RBE/trs+Rep68) or to pA1.trs/RBE.GFP.A2 and pGAPDH.Rep68 (trs/RBE+Rep68). Untransfected HeLa cells were used to set the background of the assay at 0.00% GFP-positive cells (HeLa). For each sample, 10 5 viable single cells were analyzed. ( C ) PCR analysis using primer set #649/#651 on chromosomal DNA extracted from untransfected HeLa cells (HeLa) and from HeLa cells co-transfected with pGAPDH.Rep68 (Rep68) and targeting constructs pA1.GFP.A2 (p5 − ), pA1.RBE/Δtrs.GFP.A2 (RBE/Δtrs), pA1.RBEst/trs.GFP.A2 (RBEst/trs), pA1.p5.GFP.A2 (p5 + ), pA1.RBE/trs.GFP.A2 (RBE/trs) or pA1.trs/RBE.GFP.A2 (trs/RBE). HeLa cells were also co-transfected with pA1.p5.GFP.A2 (p5 + ) and pGAPDH.Rep68(Y156F) or with an ‘empty’ control plasmid (empty). The genomic DNA was isolated at 43 days post-transfection. HPRT1 -specific PCRs served as control for the integrity of the input DNA. ( D ) In vivo nicking assay based on Southern blot analysis of DpnI-resistant extrachromosomal DNA. Episomal DNA was isolated at 4 days post-transfection from 911 cells co-transfected with pGAPDH.Rep68 and pA1.p5.GFP.A2 (lanes 1 and 2), pGAPDH.Rep68 and pA1.RBE/trs.GFP.A2 (lane 4) or pGAPDH.Rep68 and pA1.RBE/Δtrs.GFP.A2 (lane 5). Episomal DNA isolated from 911 cells co-transfected with pGAPDH.Rep68(Y156F) and pA1.p5.GFP.A2 (lane 3) served as a negative control. All cell cultures except for the one represented by lane 2 were exposed to Ad.floxedΨ.F50 at a multiplicity of infection of 25 infectious units per cell. Lane M, Gene Ruler DNA Ladder Mix. Prior to Southern blot analysis, the DNA was digested with DpnI (to fragment the input prokaryotic DNA) and with NcoI. Southern blots were exposed to a radiolabeled GFP -specific probe. The position of the 4.9-kb GFP -containing NcoI fragments derived from de novo synthesized DNA is indicated by an arrow at the right of the autoradiogram.

    Journal: Nucleic Acids Research

    Article Title: Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

    doi: 10.1093/nar/gkr1234

    Figure Lengend Snippet: Effect of DNA sequences susceptible or unsusceptible to AAV Rep-mediated nicking on the stable transfection levels with AAVS1 -targeting vectors. ( A ) Structures of the nicking-competent targeting plasmids pA1.p5.GFP.A2, pA1.RBE/trs.GFP.A2 and pA1.trs/RBE.GFP.A2 and that of the nicking-resistant donor constructs pA1.GFP.A2, pA1.RBE/Δtrs.GFP.A2 and pA1.RBEst/trs.GFP.A2. The nucleotide sequence corresponding to the wild-type RBE is shown in black uppercase letters whereas the trs (i.e. the position at which Rep78/68-mediated nicking takes place) is indicated by black lowercase letters and a vertical arrow. The DNA sequence between the RBE and the trs is called the spacer. Targeting plasmid pA1.RBE/Δtrs.GFP.A2 has the dinucleotide at which Rep endonuclease-mediated nicking occurs mutated from TT to AA while in pA1.RBE.st/trs.GFP.A2 a 21-bp stuffer positions the trs at a bigger distance from RBE-bound AAV Rep molecules. For an explanation of the other symbols and elements see the legend of Figure 3 A. ( B ) Representative dot plots of flow cytometric analysis of the frequency of GFP -modified HeLa cells at 37 days post-transfection in cultures initially exposed to pA1.p5.GFP.A2 and ‘empty’ plasmid (p5 + +empty), pA1.p5.GFP.A2 and pGAPDH.Rep68(Y156F) (p5 + +Y156F), pA1.GFP.A2 and pGAPDH.Rep68 (p5 − +Rep68), pA1.RBE/Δtrs.GFP.A2 and pGAPDH.Rep68 (RBE/Δtrs+Rep68), pA1.RBEst/trs.GFP.A2 and pGAPDH.Rep68 (RBEst/trs+Rep68), pA1.p5.GFP.A2 and pGAPDH.Rep68 (p5 + +Rep68), pA1.RBE/trs.GFP.A2 and pGAPDH.Rep68 (RBE/trs+Rep68) or to pA1.trs/RBE.GFP.A2 and pGAPDH.Rep68 (trs/RBE+Rep68). Untransfected HeLa cells were used to set the background of the assay at 0.00% GFP-positive cells (HeLa). For each sample, 10 5 viable single cells were analyzed. ( C ) PCR analysis using primer set #649/#651 on chromosomal DNA extracted from untransfected HeLa cells (HeLa) and from HeLa cells co-transfected with pGAPDH.Rep68 (Rep68) and targeting constructs pA1.GFP.A2 (p5 − ), pA1.RBE/Δtrs.GFP.A2 (RBE/Δtrs), pA1.RBEst/trs.GFP.A2 (RBEst/trs), pA1.p5.GFP.A2 (p5 + ), pA1.RBE/trs.GFP.A2 (RBE/trs) or pA1.trs/RBE.GFP.A2 (trs/RBE). HeLa cells were also co-transfected with pA1.p5.GFP.A2 (p5 + ) and pGAPDH.Rep68(Y156F) or with an ‘empty’ control plasmid (empty). The genomic DNA was isolated at 43 days post-transfection. HPRT1 -specific PCRs served as control for the integrity of the input DNA. ( D ) In vivo nicking assay based on Southern blot analysis of DpnI-resistant extrachromosomal DNA. Episomal DNA was isolated at 4 days post-transfection from 911 cells co-transfected with pGAPDH.Rep68 and pA1.p5.GFP.A2 (lanes 1 and 2), pGAPDH.Rep68 and pA1.RBE/trs.GFP.A2 (lane 4) or pGAPDH.Rep68 and pA1.RBE/Δtrs.GFP.A2 (lane 5). Episomal DNA isolated from 911 cells co-transfected with pGAPDH.Rep68(Y156F) and pA1.p5.GFP.A2 (lane 3) served as a negative control. All cell cultures except for the one represented by lane 2 were exposed to Ad.floxedΨ.F50 at a multiplicity of infection of 25 infectious units per cell. Lane M, Gene Ruler DNA Ladder Mix. Prior to Southern blot analysis, the DNA was digested with DpnI (to fragment the input prokaryotic DNA) and with NcoI. Southern blots were exposed to a radiolabeled GFP -specific probe. The position of the 4.9-kb GFP -containing NcoI fragments derived from de novo synthesized DNA is indicated by an arrow at the right of the autoradiogram.

    Article Snippet: DNA transfections DNA transfections of 8 × 104 HeLa cells seeded in wells of 24-well plates (Greiner Bio-One) were performed by using ExGen 500 (Fermentas) as detailed before ( ).

    Techniques: Stable Transfection, Construct, Sequencing, Plasmid Preparation, Flow Cytometry, Modification, Transfection, Polymerase Chain Reaction, Isolation, In Vivo, Southern Blot, Negative Control, Infection, Derivative Assay, Synthesized

    Stable genetic modification of human cells with AAVS1 -targeting vectors containing minimal AAV Rep endonuclease recognition sequences. ( A ) Structures of the p5-negative pA1.GFP.A2 and the p5-positive pA1.p5.GFP.A2 donor constructs and of the targeting plasmids pA1.RBE.GFP.A2, pA1.mRBE.GFP.A2 and pA1.RBE/trs.GFP.A2 harboring the minimal AAV Rep endonuclease recognition sites RBE, mutant RBE (mRBE) and RBE/ trs , respectively. The nucleotide sequence corresponding to the wild-type RBE is shown in black uppercase letters whereas the trs (i.e. the position at which Rep78/68-mediated nicking takes place) is indicated by black lowercase letters and a vertical arrow. The DNA sequence between the RBE and the trs is dubbed the spacer. The nucleotide sequence of the mRBE contains guanines (shown in lowercase and marked with asterisks) in place of cytosines. R6K, prokaryotic origin of DNA replication; KanR, transposon Tn5 neomycin phosphotransferase II gene conferring resistance to kanamycin. For an explanation of the other symbols and elements see the legend of Figure 2 A. ( B ) Stable transfection levels in cultures of Hela cells initially co-transfected with the targeting vector pA1.GFP.A2 (p5 − ), pA1.mRBE.GFP.A2 (mRBE), pA1.RBE.GFP.A2 (RBE), pA1.RBE/trs.GFP.A2 (RBE/trs) or pA1.p5.GFP.A2 (p5 + ) and either pGAPDH.Rep68(Y156F) (white bars) or pGAPDH.Rep68 (black bars). Flow cytometric analysis of 10 4 viable cells per sample was performed at 37 days post-transfection. Results shown correspond to means ± SD from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

    doi: 10.1093/nar/gkr1234

    Figure Lengend Snippet: Stable genetic modification of human cells with AAVS1 -targeting vectors containing minimal AAV Rep endonuclease recognition sequences. ( A ) Structures of the p5-negative pA1.GFP.A2 and the p5-positive pA1.p5.GFP.A2 donor constructs and of the targeting plasmids pA1.RBE.GFP.A2, pA1.mRBE.GFP.A2 and pA1.RBE/trs.GFP.A2 harboring the minimal AAV Rep endonuclease recognition sites RBE, mutant RBE (mRBE) and RBE/ trs , respectively. The nucleotide sequence corresponding to the wild-type RBE is shown in black uppercase letters whereas the trs (i.e. the position at which Rep78/68-mediated nicking takes place) is indicated by black lowercase letters and a vertical arrow. The DNA sequence between the RBE and the trs is dubbed the spacer. The nucleotide sequence of the mRBE contains guanines (shown in lowercase and marked with asterisks) in place of cytosines. R6K, prokaryotic origin of DNA replication; KanR, transposon Tn5 neomycin phosphotransferase II gene conferring resistance to kanamycin. For an explanation of the other symbols and elements see the legend of Figure 2 A. ( B ) Stable transfection levels in cultures of Hela cells initially co-transfected with the targeting vector pA1.GFP.A2 (p5 − ), pA1.mRBE.GFP.A2 (mRBE), pA1.RBE.GFP.A2 (RBE), pA1.RBE/trs.GFP.A2 (RBE/trs) or pA1.p5.GFP.A2 (p5 + ) and either pGAPDH.Rep68(Y156F) (white bars) or pGAPDH.Rep68 (black bars). Flow cytometric analysis of 10 4 viable cells per sample was performed at 37 days post-transfection. Results shown correspond to means ± SD from three independent experiments.

    Article Snippet: DNA transfections DNA transfections of 8 × 104 HeLa cells seeded in wells of 24-well plates (Greiner Bio-One) were performed by using ExGen 500 (Fermentas) as detailed before ( ).

    Techniques: Modification, Construct, Mutagenesis, Sequencing, Stable Transfection, Transfection, Plasmid Preparation, Flow Cytometry

    Detection of homology-directed gene targeting events. ( A ) Diagram of the PCR assay deployed to identify cells genetically modified through HR-mediated GFP gene addition. The primer pairs #649/#651 and #650/#635 allow the detection of HR events at the AAVS1 of human cells transfected with the targeting construct pA1.p5.GFP.A2 by yielding diagnostic 2868-bp and 5361-bp PCR amplicons, respectively (horizontal black bars). Half arrows, primers #649, #651, #650 and #635 drawn in relation to their respective target sequences; thin black line, AAVS1 chromosomal region; horizontal thick grey lines, sequences shared by target and donor DNA; grey bar and vertical black line, RBE and trs , respectively; black box, nicking-prone p5 element; open box with broken arrow, EF1α promoter; large grey box, GFP ORF; open box, SV40 pA signal; open circle, prokaryotic origin of DNA replication. ( B ) PCR screening of clones derived from stably transfected HeLa cell populations initially co-transfected with pA1.p5.GFP.A2 and pGAPDH.Rep78/68. The panels labeled GT display the results of amplification reactions carried out with primers #649 and #651. PCR amplification of a 1.9-kb segment of the hypoxanthine phosphoribosyltransferase 1 gene ( HPRT1 ) was performed in parallel to ascertain the integrity of the various genomic DNA templates (panels marked HPRT1). Marker, Gene Ruler DNA Ladder Mix (Fermentas); H 2 0, PCR performed with nuclease-free water instead of chromosomal DNA. The positions (arrowheads) and sizes (in kb) of the PCR products are indicated at the left. ( C ) PCR screening of clones derived from stably transfected HeLa cell populations originally co-transfected with pA1.p5.GFP.A2 and pGAPDH.Rep78/68. The panels marked GT correspond to the PCR assay performed with primers #650 and #635 whereas those labeled HPRT1 are for the purpose specified in the legend of Figure 2 B. ( D ) Summary of the data presented in Figure 2 B and 2 C, which resulted from the PCR assays depicted in Figure 2 A. ( E ) Schematic representation of the Southern blot assay with ApaLI-digested genomic DNA from randomly selected clones of pA1.p5.GFP.A2-transfected HeLa cells. Unmodified target loci should yield a 7.1-kb AAVS1 -specific restriction fragment while ‘two-sided’ HR should give rise to a DNA species of 10.1 kb hybridizing to the AAVS1 - as well as the GFP -specific probe (black horizontal bars). Both probes are drawn in relation to their respective target DNA sequences. For an explanation of the other elements and symbols see the legend of Figure 2 A. ( F ) Southern blots of ApaLI-treated genomic DNA of untransfected HeLa cells (HeLa) and of HeLa cell clones derived from cultures co-transfected with pA1.p5.GFP.A2 and pGAPDH.Rep78/68 (+Rep) or with pA1.p5.GFP.A2 and ‘empty’ plasmid (−Rep). The 12.9-kb ApaLI-linearized pA1.p5.GFP.A2 DNA (donor) served as an internal reference. Marker, Gene Ruler DNA Ladder Mix (Fermentas). ( G ) Diagram of the GFP expression unit (yellow and green boxes) inserted at 19q13.42-qter (horizontal yellow lines) upon homology-directed gene targeting deploying pA1.p5.GFP.A2 as donor template. Primers used to amplify the left- and right-hand junctions (dark and light blue half arrows, respectively) are drawn in relation to their recognition sequences. The 2.9- and 5.4-kb PCR amplicons specific for ‘telomeric’ and ‘centromeric’ DNA junctions are indicated by dark and light blue bars, respectively. ( H ) Nucleotide sequence analysis of ‘telomeric’ and ‘centromeric’ junctions between endogenous and exogenous DNA resulting from Rep78/68-induced HR events. The nucleotide sequences of the transition regions between pA1.p5.GFP.A2 sequences and flanking genomic or transgene DNA for three different clones that underwent HR are shown.

    Journal: Nucleic Acids Research

    Article Title: Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

    doi: 10.1093/nar/gkr1234

    Figure Lengend Snippet: Detection of homology-directed gene targeting events. ( A ) Diagram of the PCR assay deployed to identify cells genetically modified through HR-mediated GFP gene addition. The primer pairs #649/#651 and #650/#635 allow the detection of HR events at the AAVS1 of human cells transfected with the targeting construct pA1.p5.GFP.A2 by yielding diagnostic 2868-bp and 5361-bp PCR amplicons, respectively (horizontal black bars). Half arrows, primers #649, #651, #650 and #635 drawn in relation to their respective target sequences; thin black line, AAVS1 chromosomal region; horizontal thick grey lines, sequences shared by target and donor DNA; grey bar and vertical black line, RBE and trs , respectively; black box, nicking-prone p5 element; open box with broken arrow, EF1α promoter; large grey box, GFP ORF; open box, SV40 pA signal; open circle, prokaryotic origin of DNA replication. ( B ) PCR screening of clones derived from stably transfected HeLa cell populations initially co-transfected with pA1.p5.GFP.A2 and pGAPDH.Rep78/68. The panels labeled GT display the results of amplification reactions carried out with primers #649 and #651. PCR amplification of a 1.9-kb segment of the hypoxanthine phosphoribosyltransferase 1 gene ( HPRT1 ) was performed in parallel to ascertain the integrity of the various genomic DNA templates (panels marked HPRT1). Marker, Gene Ruler DNA Ladder Mix (Fermentas); H 2 0, PCR performed with nuclease-free water instead of chromosomal DNA. The positions (arrowheads) and sizes (in kb) of the PCR products are indicated at the left. ( C ) PCR screening of clones derived from stably transfected HeLa cell populations originally co-transfected with pA1.p5.GFP.A2 and pGAPDH.Rep78/68. The panels marked GT correspond to the PCR assay performed with primers #650 and #635 whereas those labeled HPRT1 are for the purpose specified in the legend of Figure 2 B. ( D ) Summary of the data presented in Figure 2 B and 2 C, which resulted from the PCR assays depicted in Figure 2 A. ( E ) Schematic representation of the Southern blot assay with ApaLI-digested genomic DNA from randomly selected clones of pA1.p5.GFP.A2-transfected HeLa cells. Unmodified target loci should yield a 7.1-kb AAVS1 -specific restriction fragment while ‘two-sided’ HR should give rise to a DNA species of 10.1 kb hybridizing to the AAVS1 - as well as the GFP -specific probe (black horizontal bars). Both probes are drawn in relation to their respective target DNA sequences. For an explanation of the other elements and symbols see the legend of Figure 2 A. ( F ) Southern blots of ApaLI-treated genomic DNA of untransfected HeLa cells (HeLa) and of HeLa cell clones derived from cultures co-transfected with pA1.p5.GFP.A2 and pGAPDH.Rep78/68 (+Rep) or with pA1.p5.GFP.A2 and ‘empty’ plasmid (−Rep). The 12.9-kb ApaLI-linearized pA1.p5.GFP.A2 DNA (donor) served as an internal reference. Marker, Gene Ruler DNA Ladder Mix (Fermentas). ( G ) Diagram of the GFP expression unit (yellow and green boxes) inserted at 19q13.42-qter (horizontal yellow lines) upon homology-directed gene targeting deploying pA1.p5.GFP.A2 as donor template. Primers used to amplify the left- and right-hand junctions (dark and light blue half arrows, respectively) are drawn in relation to their recognition sequences. The 2.9- and 5.4-kb PCR amplicons specific for ‘telomeric’ and ‘centromeric’ DNA junctions are indicated by dark and light blue bars, respectively. ( H ) Nucleotide sequence analysis of ‘telomeric’ and ‘centromeric’ junctions between endogenous and exogenous DNA resulting from Rep78/68-induced HR events. The nucleotide sequences of the transition regions between pA1.p5.GFP.A2 sequences and flanking genomic or transgene DNA for three different clones that underwent HR are shown.

    Article Snippet: DNA transfections DNA transfections of 8 × 104 HeLa cells seeded in wells of 24-well plates (Greiner Bio-One) were performed by using ExGen 500 (Fermentas) as detailed before ( ).

    Techniques: Polymerase Chain Reaction, Genetically Modified, Transfection, Construct, Diagnostic Assay, Clone Assay, Derivative Assay, Stable Transfection, Labeling, Amplification, Marker, Southern Blot, Plasmid Preparation, Expressing, Sequencing

    Stable genetic modification of human cells with p5-negative or -positive targeting vectors containing DNA sequences homologous to the genomic region framing the RBE and trs at AAVS1 . ( A ) Experimental setup deployed to investigate the role of sequence- and strand-specific cleavage of donor and acceptor DNA molecules on mitotic HR at an endogenous human locus . The donor template pA1.GFP.A2 differs from pA1.p5.GFP.A2 by lacking p5. Both targeting constructs contain a 4.1-kb transcription unit consisting of the EF1α promoter (large yellow box), the GFP ORF (green box) and the SV40 pA signal (small yellow box). Immediately upstream of the EF1α promoter in pA1.p5.GFP.A2 lies the nicking-competent p5 element, whose RBE and trs are indicated by a red bar and vertical thin black line, respectively. The GFP gene in both donor plasmids is bracketed by DNA segments homologous to those framing the trs (vertical thin black line) and RBE (red box) at the chromosomal target site (i.e. the AAVS1 locus embedded in the PPP1R12C gene at 19q13.42-qter). The arbitrarily designated homology ‘arms’ 1 and 2 (thick yellow lines) are 2063 and 4381 bps in length, respectively. The AAV endonucleases Rep78 and Rep68 are represented by a cyan oval. ( B ) Flow cytometric quantification of the frequency of GFP-positive HeLa cells at different times after co-transfection with pA1.GFP.A2 or pA1.p5.GFP.A2 and either the AAV rep78/68 expression plasmid pGAPDH.Rep78/68 (+Rep) or an ‘empty’ control vector (−Rep). The frequencies of GFP-positive cells at the different time points in each of the experimental groups are plotted relative to those measured at 2 days post-transfection. Bars represent means ± SD of three independent experiments. ( C ) Representative flow cytometry dot plots corresponding to untransfected HeLa cells (control cells) and to HeLa cells initially co-transfected with pA1.GFP.A2 and pGAPDH.Rep78/68 (pA1.GFP.A2 + Rep) or with pA1.p5.GFP.A2 and pGAPDH.Rep78/68 (pA1.p5.GFP.A2 + Rep) at 44 days post-transfection. The frequency of stably transfected cells in each of the cell populations is indicated. The insets show direct fluorescence micrographs of each of the three types of HeLa cell populations. The GFP-specific signals (green) are overlaid with those of the DNA-binding dye Hoechst 33342 (blue).

    Journal: Nucleic Acids Research

    Article Title: Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

    doi: 10.1093/nar/gkr1234

    Figure Lengend Snippet: Stable genetic modification of human cells with p5-negative or -positive targeting vectors containing DNA sequences homologous to the genomic region framing the RBE and trs at AAVS1 . ( A ) Experimental setup deployed to investigate the role of sequence- and strand-specific cleavage of donor and acceptor DNA molecules on mitotic HR at an endogenous human locus . The donor template pA1.GFP.A2 differs from pA1.p5.GFP.A2 by lacking p5. Both targeting constructs contain a 4.1-kb transcription unit consisting of the EF1α promoter (large yellow box), the GFP ORF (green box) and the SV40 pA signal (small yellow box). Immediately upstream of the EF1α promoter in pA1.p5.GFP.A2 lies the nicking-competent p5 element, whose RBE and trs are indicated by a red bar and vertical thin black line, respectively. The GFP gene in both donor plasmids is bracketed by DNA segments homologous to those framing the trs (vertical thin black line) and RBE (red box) at the chromosomal target site (i.e. the AAVS1 locus embedded in the PPP1R12C gene at 19q13.42-qter). The arbitrarily designated homology ‘arms’ 1 and 2 (thick yellow lines) are 2063 and 4381 bps in length, respectively. The AAV endonucleases Rep78 and Rep68 are represented by a cyan oval. ( B ) Flow cytometric quantification of the frequency of GFP-positive HeLa cells at different times after co-transfection with pA1.GFP.A2 or pA1.p5.GFP.A2 and either the AAV rep78/68 expression plasmid pGAPDH.Rep78/68 (+Rep) or an ‘empty’ control vector (−Rep). The frequencies of GFP-positive cells at the different time points in each of the experimental groups are plotted relative to those measured at 2 days post-transfection. Bars represent means ± SD of three independent experiments. ( C ) Representative flow cytometry dot plots corresponding to untransfected HeLa cells (control cells) and to HeLa cells initially co-transfected with pA1.GFP.A2 and pGAPDH.Rep78/68 (pA1.GFP.A2 + Rep) or with pA1.p5.GFP.A2 and pGAPDH.Rep78/68 (pA1.p5.GFP.A2 + Rep) at 44 days post-transfection. The frequency of stably transfected cells in each of the cell populations is indicated. The insets show direct fluorescence micrographs of each of the three types of HeLa cell populations. The GFP-specific signals (green) are overlaid with those of the DNA-binding dye Hoechst 33342 (blue).

    Article Snippet: DNA transfections DNA transfections of 8 × 104 HeLa cells seeded in wells of 24-well plates (Greiner Bio-One) were performed by using ExGen 500 (Fermentas) as detailed before ( ).

    Techniques: Modification, Sequencing, Construct, Flow Cytometry, Cotransfection, Expressing, Plasmid Preparation, Transfection, Cytometry, Stable Transfection, Fluorescence, Binding Assay