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  • 99
    Thermo Fisher hela cells
    Experimental design. (A) Experimental setting for pharmaceutical treatment: <t>HeLa</t> cells were seeded and cultured for 24 h before infection with MOI = 1 of C. trachomatis Serovar E. wIRA/VIS irradiation was performed at 24, 36, and 40 hpi, for 30 min application time of irradiation, ranging between 2340 and 3400 W/m 2 . Cell densities were 5 × 10 4 for pharmaceutical inhibition of cytokines and corresponding controls and 3 × 10 5 for cycloheximide supplementation and 2 × 10 5 for controls, respectively. After three additional hours of incubation (43 hpi), sampling for further analyses (including direct IFA, titration by sub-passage, ELISA of supernatants) was performed. (B) Experimental setting for gene silencing: After seeding and culture of HeLa cells (density 2.5 × 10 4 cells/well), gene silencing (or negative control siRNA transfection) was performed, followed by an additional incubation time of 35 h before infection with C. trachomatis Serovar E at MOI = 1. Irradiation and sampling time points were the same as described in (A) .
    Hela Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 39520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hela cells hela cells
    A) <t>HeLa</t> cells were <t>transfected</t> with pcDNA3 (-) or CD74 (+), together with Notch . The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with 35 S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.
    Hela Cells Hela Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore treatments hela cells
    Over-expressed RelA peptide co-localizes with SETD6 and inhibits its catalytic activity in cells ( A ) The 15 amino acids (302-316) sequence of the RelA peptide. Methylated lysine residue (K310) is labeled in red. ( B ) MCF7 cells were transfected with increasing amounts of the Flag-RelA302-316 plasmid. 24 hours <t>post-transfection,</t> cells were harvested, RNA was extracted and used for a qPCR analysis of the indicated genes. mRNA levels were normalized to GAPDH and to control cells. Error bars represent triplicates. ( C ) <t>HeLa</t> or MDA-MB-231 cells were plated at 15,000 cells/well and transfected either with Flag-RelA302-316 or an empty plasmid. 48 hours after transfection viability was measured using the PrestoBlue reagent. Standard deviation represents quadruplicates of two independent experiments. HeLa cells were transfected with the indicated combinations of the GFP-RelA302-316, mCherry-H2B ( D ) and mCherry-SETD6 ( E ). 24 hours after transfection cells were visualized by fluorescent microscopy.
    Treatments Hela Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Horizon Discovery hela cells
    Effect of prolonged Cdk1 inhibition on Gwl dephosphorylation in <t>Fcp1-depleted</t> cells. Control (Cont.) or Fcp1-depleted (Fcp1) <t>HeLa</t> cells were arrested at prometaphase, split into two samples, one sample receiving vehicle (-), the other RO3306 (+), and further incubated for 30 min. Cell lysates were processed for mock (Mk) or Gwl IP that were then probed for indicated antigens. Data shown are representative of at least four independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.010
    Hela Cells, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 1566 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    National Centre for Cell Science hela cells
    Two dimensional gel electrophoresis of hTERT overexpressing (A) <t>U2OS</t> and (B) <t>HeLa</t> cell line. Total proteins were extracted from hTERT overexpressing U2OS and HeLa cells and separated non-linearly on IPG strip of PH 3–10, followed by electrophoresis through 12% polyacrylamide gels. The gels were further stained and analyzed by image master 2D platinum software.
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    93
    Santa Cruz Biotechnology hela cells
    Proximity Ligation Assay on <t>Scramble/Cas9</t> <t>HeLa</t> cells showing interactions between TMEM97 and PGRMC1, LDLR and PGRMC1, and LDLR and TMEM97. ( A ) standard, non-lipoprotein depleted conditions, ( B ) sterol-starved conditions, and ( C ) sterol-starved conditions followed with treatment with 50 μg/mL LDL for 3 hours. Inserts represent an enlarged region focusing on a single cell.
    Hela Cells, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    SignaGen hela cells
    SIRPα proteolysis modulates inflammatory signaling. a , <t>HeLa</t> cells were incubated with TNFα (10 ng/ml) for the indicated times. Cells were harvested and probed for pIKKα/β and total IKKα. b , HeLa cells were <t>transfected</t>
    Hela Cells, supplied by SignaGen, used in various techniques. Bioz Stars score: 97/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Active Motif hela cells
    Expression and localization of <t>NKAP.</t> ( A ) Presence of NKAP in mouse tissues. Homogenates of adult mouse tissues as indicated were separated by SDS-PAGE (12% acrylamide) and the corresponding blot probed with NKAP-specific mAb K85-80-5. ( B ) Localization of NKAP during mitosis. <t>HeLa</t> cells were synchronized using nocodazole to block progression of the cell cycle and then released and fixed using 4% paraformaldehyde (PFA). Cells were stained with mAb K85-80-5, and nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). The phases of the cell cycle are indicated. Bar, 10 µm.
    Hela Cells, supplied by Active Motif, used in various techniques. Bioz Stars score: 92/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Biovest hela cells
    Binding of proteins to AREs within the 3′-UTR of PTHrP 1-141 mRNA. A . EMSA using ARE-containing region of the 3′-UTR and <t>S100</t> extract from <t>HeLa</t> cells. 20 μg of HeLa S100 cytoplasmic extract were incubated with a 32 P-labeled RNA
    Hela Cells, supplied by Biovest, used in various techniques. Bioz Stars score: 91/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 393 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Enzo Biochem hela cells
    Ubc9 co-localizes with SUMO1-modified RanGAP1 and RanBP2 at both NPCs and <t>ALPCs.</t> (A) <t>HeLa</t> cells were analyzed by immunofluorescence microscopy using anti-Ubc9 antibody and mAb414. (B and C) HeLa cells were transfected with the construct encoding Myc-tagged Ubc9, double-stained with mouse anti-Myc mAb (9E10) and rabbit anti-RanGAP1 antibodies or with rabbit anti-Myc antibody and mouse anti-RanBP2 mAb, and then analyzed by immunofluorescence microscopy. Bar, 10 μm. The enlarged versions of inlets are shown at the top-right corner of each image.
    Hela Cells, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 94/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare hela cells
    SIRT1 dissociates with CHK2 for its activation upon oxidative stress. a SIRT1 bound CHK2 under control conditions but dissociated with CHK2 after treatment with 1 mM of H 2 O 2 in <t>HeLa</t> cells. b The acetylated CHK2 level was elevated in response to oxidative stress in HeLa cells. c DDR pathways were activated upon oxidative stress. The phosphorylation levels of CHK2 (p-CHK2) and p53 (p-p53) were highly increased in response to oxidative stress in <t>HCT116</t> cells. IP immunoprecipitation, WB Western blot. Western blots in figures are representative of more than three independent experiments
    Hela Cells, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega hela cells
    Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. ( A ) <t>U2OS</t> or <t>HeLa</t> cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. ( B ) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.
    Hela Cells, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Texcell SA hela cells
    Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. ( A ) <t>U2OS</t> or <t>HeLa</t> cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. ( B ) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.
    Hela Cells, supplied by Texcell SA, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Immunodiagnostic Systems hela cells
    Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. ( A ) <t>U2OS</t> or <t>HeLa</t> cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. ( B ) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.
    Hela Cells, supplied by Immunodiagnostic Systems, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    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: 98/100, based on 1541 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems hela cells
    In vitro cell invasion is stimulated by the SDF-1/CXCR4 pathway independently from HPV status. The modulation of <t>E6</t> expression was monitored using Western-blot in HPV-positive <t>HeLa</t> (A) and TC-1 (B) cells and in HPV-negative B16F10 (C) cells, after 3 and 6 days of incubation with Cidofovir (CDF). Modulation of P53 expression was assessed in HeLa cells after CDF incubation (A). Cell invasion was measured using a Matrigel assay in HeLa (A), TC-1 (B) and B16F10 (C) cells. Recombinant human CXCL12/SDF-1 (100 ng/mL; R D Systems) was used as chemoattractant and modulation of cell migration was recorded after treatment with CXCR4-blocking antibody or/and Cidofovir (CDF). The invasion rate was determined by counting crystal violet-stained cells. Invasion was stimulated by SDF-1α/CXCR4 independently from the HPV status of the cells but Cidofovir anti-invasive action was restricted to the two HPV-positive cell lines. Three independent experiments with three chambers each time were performed. ***P
    Hela Cells, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 336 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa hela cells
    In vitro MMR. ( A ) <t>HeLa</t> nuclear extracts from cells carrying empty vector (vector), wild type Polδ, PolδD316A;E318A or PolδD515V were analyzed by western blot. Western blot data are presented for MSH2, MLH1 and PCNA. ( B) Mutation frequency in HCT116 (MLH1-/-) cells <t>overexpressing</t> PolδD316A;E318A or PolδD515V. (C) In vitro MMR assay using 2 nt indel heteroduplex DNA substrates Δ2–3′ or Δ2–5′ (see Materials and Methods). (D) As in B except using chromosome 3 complemented HCT116 (HCT116+Chr3) cells. The data are the mean ± SD of three independent experiments. * P
    Hela Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1994 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Xenogen Corporation hela cells
    Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. <t>HeLa</t> cells were <t>transfected</t> with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. ( A ) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; ( B ) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; ( C ) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and ( D ) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.
    Hela Cells, supplied by Xenogen Corporation, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    EpiCypher hela cells
    Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. <t>HeLa</t> cells were <t>transfected</t> with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. ( A ) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; ( B ) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; ( C ) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and ( D ) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.
    Hela Cells, supplied by EpiCypher, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Eurofins hela cells
    A statistical analysis of the level of FITC fluorescence in <t>HeLa</t> cells transfected with nanoparticles, in the presence of Lipofectamine, GeneJuice or Matra, compared to untransfected HeLa cells. HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence or the presence of <t>transfection</t> reagents. The FITC fluorescence in the cells was quantified as described in the methodology, and the levels of fluorescence in nanoparticle-transfected cells compared to those in untransfected cells. This multi-level statistical analysis takes into account local variations within the cell population. The bars represent the region where there is a 95% probability that the mean fluorescence increase lies within it. The dot represents the mean calculated effect size. Bars not crossing the 1 line show significant evidence for an effect following transfection. The p-value indicates the probability that there was no difference in expression levels between the control and treatment samples. Hence, a lower p-value indicates a greater likelihood that there was a difference between the transfected and the untransfected cells.
    Hela Cells, supplied by Eurofins, used in various techniques. Bioz Stars score: 96/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    LGC Standards GmbH hela cells
    CUX1, YY1, EAP1, and <t>TTF1</t> are recruited to different regions of the human EAP1 promoter in <t>Hela</t> cells, as assessed by ChIP assays Putative binding sites for three transcription factors (TTF1, CUX1, and YY1) implicated in the control of puberty were identified in silico (TESS, Vector NTI) and are shown by horizontal wide arrows. The direction of each arrow indicates the presence of each binding site on either the sense or antisense DNA strand. Potential EAP1 binding sites are not depicted, because it is currently unknown if EAP1 binds directly to DNA. Endogenous CUX1, as well as transfected HA-tagged EAP1 and untagged TTF1, strongly associates with the proximal promoter region of the EAP1 gene (Amplicon II). No association of YY1 to this region was detected. In contrast to the proximal promoter, an upstream region (Amplicon I) showed CUX1 and YY1 binding, but no EAP1 or TTF1 association. The endogenous proteins (CUX1 and YY1) were immunoprecipitated using specific monoclonal antibodies (see Material and Methods) and mouse IgGs as a negative control for the immunoprecipitation. To immunoprecipitate proteins produced by transfected expression vectors (EAP1-HA and TTF1), we used a mouse monoclonal antibody against HA (for EAP) and rabbit polyclonal antibodies (against TTF1). Chromatin prepared from cells transfected with the empty expression vector was used as the negative control. Inp: input DNA, chromatin precleared with protein A beads before immunoprecipitation. Transf = transfected.
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    Abcam hela cells lysates
    Neither low protein high carbohydrate diet ( LPHC ) nor high protein low carbohydrate ( HPLC ) diet altered neither skeletal muscle glycogen content nor cellular signaling. (A) Tibialis anterior ( TA ) muscle glycogen content. The color legend in panel A is shared with panel B. (B) TA muscle cellular signaling determined by western blotting. The signal intensities are presented relative to chow (dashed line). Representative blots and a coomassie loading control are shown to the left. For p70S6K T389 and GCN 2 T899, 25 μ g of total protein were loaded, and the positive controls were HEK 293 and 100 <t>nmol/L</t> Calyculin A‐treated <t>HeLa</t> cell lysates, respectively. Data are expressed as mean ± SEM . N = 7 for chow and n = 8 for LPHC /HPLC.
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    Keygen Biotech hela cells
    Evaluation of NAMAs self-assembled on GONPs for target miR-21 imaging in single cells (HepG2, <t>MCF-7</t> cells, <t>Hela</t> and L-02 cells).
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    Malvern Panalytical hela cells
    Targeting labeling of cells using <t>CPV-VLPs-QDs.</t> DAPI-labeled nucleus (blue), QDs (red), and FITC-labeled goat anti-mouse secondary antibody (green). Anti-CPV mouse monoclonal antibody was used as the primary antibody. <t>Hela</t> and F81 cells show high uptake (red), whereas no obvious fluorescence signal was detected in BHK-21 cells.
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    Pasteur Institute hela cells
    DNA laddering: apoptotic activity was detected by agarose gel electrophoretic separation of DNA fragmentation after exposure to rStx, A and B subunits. Concentration of A and B subunits was two times higher than rStx. DNA was isolated and analyzed by ethidium bromide/agarose gel electrophoresis. DNA fragmentation in a dose-dependent manner was shown after 16 h treatment with rStx ( AB ), A and B subunits. a DNA laddering in <t>HeLa</t> cell. b DNA laddering in <t>Vero</t> cell
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    ScienCell hela cells
    <t>Girdin</t> regulates cell proliferation and apoptosis in <t>HeLa</t> cells. (A) Western blot analysis indicated that Girdin was suppressed in the Girdin shRNA group but not in the control after 48 h of transfection. (B) Cell proliferation detected by trypan blue staining indicated that Girdin shRNA caused significant attenuation of proliferation in HeLa cells at 72 h (P
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    Lonza hela cells
    Screening of candidate human PRE/TREs by dual luciferase reporter assay. ( A ) Overview of the screening strategy. Two plasmids were common for all samples: 2FRT-YY1pLuc and pGL4.74. Each of the candidate human PRE/TREs was inserted between the two FRT sequences in 2FRT-YY1pLuc to yield 2FRT-PRE/TRE-YY1pLuc. pGL4.74 contains a Renilla luciferase gene under the control of an upstream TK promoter. This allowed the signal of firefly luciferase to be normalized against that of Renilla luciferase from the same sample, yielding relative luciferase activity (RLU), to account for any variations in <t>transfection</t> and expression. In FLP(+) samples, an additional plasmid harboring the wild-type FLPe recombinase gene was co-transfected with the two luciferase vectors to allow for partial excision of candidate PRE/TRE at the two FRT sites. In FLP(m), a plasmid containing a C-terminally truncated, inactive FLPe (shown with an asterisk in the FLPe gene) was used in the transfection to serve as a negative control. The RLU values of the FLP(m) and FLP(+) samples were normalized against that of the FLP(-) for the same DNA insert to give rise to Normalized RLU. As PREs or TREs were supposed to repress or activate gene expression, respectively, the partial excision of inserted DNA fragments by FLPe in FLP(+) samples should increase the firefly luciferase signals for PREs, and decrease the firefly luciferase signals for TREs. ( B ) Results of dual luciferase assay in <t>HeLa</t> cells. The data were represented as mean ± SD (standard deviations) from at least three biological replicates that were performed on different samples on different days. The mean signal of the Gene Desert FLP(+) sample and its plus and minus 3*SD were shown as black dashed line or red or blue solid line, respectively. Candidate PRE/TREs were selected as PRE-like or TRE-like if the Normalized RLU values of their FLP(+) samples were above the red line or below the blue line, respectively.
    Hela Cells, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PAA Laboratories hela cells
    Immunoprecipitation of xDYRK1B . A , C-terminal sequences of human and Xenopus DYRK1A and DYRK1B. Amino acids identical with the immunogenic peptide used to raise the DYRK1B antibody are highlighted in bold. B , Immunoprecipitation of xDYRK1B. <t>HeLa</t> cells were transiently transfected with expression plasmids for xDYRK1B, GFP-DYRK1B or the empty vector ( control ). After immunoprecipitation with DYRK1B-specific antiserum ( αD1B ) or preimmune serum ( Pre ), bound proteins were subjected to Western blot analysis with a phosphotyrosine-specific antibody ( P-Tyr ) and the DYRK1B antiserum as indicated. Detection of IgG is shown as a loading control. The asterisk marks an unspecific band. C , Specificity of the DYRK1B antiserum. <t>COS7</t> cells were transiently transfected with expression plasmids for the GFP fusion proteins of mammalian DYRK1A or DYRK1B as indicated on top of the lanes. Aliquots of the whole cell lysates ( input ) or proteins eluted from the immunoprecipitates ( αD1B, Pre ) were subjected to Western blot analysis with anti GFP and anti DYRK1B antibodies as indicated. Detection of IgG is shown as a loading control.
    Hela Cells, supplied by PAA Laboratories, used in various techniques. Bioz Stars score: 91/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene hela cells
    NleC inhibits NFκB-dependent protein expression and IL-8 secretion. A , relative change in NFκB-dependent luciferase activity in pEGFP (empty vector control)-transfected <t>HeLa</t> cells following treatment with NFκB activation inhibitor (Calbiochem product no. 481406). B , fold increase in NFκB-dependent luciferase activity. C , representative immunoblot showing similar expression levels of eGFP and eGFP-NleC in transfected cells. D , IL-8 secretion levels in untreated or TNFα-treated HeLa cells transfected with pEGFP-NleC or pEGFP vectors. <t>Transfection</t> with pEGFP-NleC inhibits luciferase activity and IL-8 secretion. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. **, p ≤ 0.01; ***, p ≤ 0.005 as compared with empty vector control.
    Hela Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Experimental design. (A) Experimental setting for pharmaceutical treatment: HeLa cells were seeded and cultured for 24 h before infection with MOI = 1 of C. trachomatis Serovar E. wIRA/VIS irradiation was performed at 24, 36, and 40 hpi, for 30 min application time of irradiation, ranging between 2340 and 3400 W/m 2 . Cell densities were 5 × 10 4 for pharmaceutical inhibition of cytokines and corresponding controls and 3 × 10 5 for cycloheximide supplementation and 2 × 10 5 for controls, respectively. After three additional hours of incubation (43 hpi), sampling for further analyses (including direct IFA, titration by sub-passage, ELISA of supernatants) was performed. (B) Experimental setting for gene silencing: After seeding and culture of HeLa cells (density 2.5 × 10 4 cells/well), gene silencing (or negative control siRNA transfection) was performed, followed by an additional incubation time of 35 h before infection with C. trachomatis Serovar E at MOI = 1. Irradiation and sampling time points were the same as described in (A) .

    Journal: Frontiers in Microbiology

    Article Title: Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition

    doi: 10.3389/fmicb.2018.02757

    Figure Lengend Snippet: Experimental design. (A) Experimental setting for pharmaceutical treatment: HeLa cells were seeded and cultured for 24 h before infection with MOI = 1 of C. trachomatis Serovar E. wIRA/VIS irradiation was performed at 24, 36, and 40 hpi, for 30 min application time of irradiation, ranging between 2340 and 3400 W/m 2 . Cell densities were 5 × 10 4 for pharmaceutical inhibition of cytokines and corresponding controls and 3 × 10 5 for cycloheximide supplementation and 2 × 10 5 for controls, respectively. After three additional hours of incubation (43 hpi), sampling for further analyses (including direct IFA, titration by sub-passage, ELISA of supernatants) was performed. (B) Experimental setting for gene silencing: After seeding and culture of HeLa cells (density 2.5 × 10 4 cells/well), gene silencing (or negative control siRNA transfection) was performed, followed by an additional incubation time of 35 h before infection with C. trachomatis Serovar E at MOI = 1. Irradiation and sampling time points were the same as described in (A) .

    Article Snippet: Transfection Procedure Transfection of HeLa cells was performed according to Ambion manufacturer’s guidelines (Life technologies) in 24-well culture plates using 6.25 nM siRNA and 2 μl Lipofectamine® RNAiMAX reagent for each siRNA (Thermo Fisher Scientific) under normoxic conditions in OptiMEM I reduced serum medium (Thermo Fisher Scientific).

    Techniques: Cell Culture, Infection, Irradiation, Inhibition, Incubation, Sampling, Immunofluorescence, Titration, Enzyme-linked Immunosorbent Assay, Negative Control, Transfection

    A) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with Notch . The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with 35 S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.

    Journal: Molecular Neurodegeneration

    Article Title: CD74 interacts with APP and suppresses the production of A?

    doi: 10.1186/1750-1326-4-41

    Figure Lengend Snippet: A) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with Notch . The total cell lysates (TL) and FLAG IPs were analyzed as in Figure 1B. Notch was detected with αmyc antibody. B) HeLa cells were transfected with pcDNA3 (-) or CD74 (+), together with APLP2, and were analyzed as in A. C) Left panels: HEK293APP cells were transfected with the indicated plasmids. The transfected cells were further incubated for 24 hours with fresh media, and secreted sAPPα/β were measured by Westerm blot. The cell lysates were analyzed as in Figure 1B. Right panels: the quantification of sAPPα/β. The levels in pcDNA3 transfected cells were set to 100. D) HEK293APP cells were transfected with pcDNA3 or CD74 and treated as in C. Aβ40 and 42 secreted in the media, and Aβ40 in the lysates were measured with specific ELISA. The amount of Aβ was normalized to the amount of the protein in the total lysates. The asterisks (**) indicate that p values by Student's t test are less than 0.01. E) HeLa cells were transfected with pcDNA3 or CD74, and the media was replaced with fresh media containing synthetic Aβ40 and Aβ42. The media of pcDNA3 (open circles) and CD74 (closed circles) transfected cells were incubated for indicated hours, and the remaining Aβ40 and Aβ42 were measured as in D. F) HeLa cells were transfected with pcDNA3 or FLAG-CD74 together with APP. The cells were pulsed for 30 min with 35 S methionine+cysteine, and chased in fresh media for indicated hours. APP was immunoprecipitated with αAPPct antibody and analyzed by SDS-PAGE followed by autoradiography. G) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with wild type APP (wt APP) or the indicated mutants. The total lysates and FLAG IPs were processed as in Figure 1B. Note that APP Ncas lacks C-terminal 31 amino acids of APP and has therefore a smaller in size.

    Article Snippet: Staining of transfected HeLa cells HeLa cells were plated on coverslips coated with poly L-lysine (Sigma), and were transfected with indicated plasmids.

    Techniques: Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, SDS Page, Autoradiography

    A) A cartoon of various FLAG-tagged CD74 constructs: the yeast split-ubiquitin (TH) clone, full length CD74, two deletions constructs of CD74, and two splicing variants of mouse CD74 . The cytoplasmic and luminal parts, the locations of the FLAG tag, the endosomal-sorting signal in the cytoplasmic domain (LL), the CLIP domain (C), and the trimeric domain (T) are indicated. B) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with APP, and the total cell lysates (TL) and FLAG immunoprecipitants (IP) were analyzed by Westerm blot. Full length APP, APP CTF (C99 and C83), and CD74 were detected with 22C11, αAPPct, and αFLAG antibodies, respectively. The 33 and 14 kDa N-terminal fragments of CD74 were designated as CD74 NTF33 and NTF14. C) HeLa cells were transfected with FLAG-CD74 and APP, and the total lysates of the transfected cells were immunoprecipitated with either rabbit polyclonal antibody (RP) or αAPPct, and analyzed as in B. D) HeLa cells were transfected with various CD74 constructs and APP. The total lysates and FLAG IPs were analyzed as in B. Mature APP and immature APP (mAPP/imAPP) are indicated. The bands marked with asterisks (*) were attributed to the FLAG antibody used in the immunoprecipitation.

    Journal: Molecular Neurodegeneration

    Article Title: CD74 interacts with APP and suppresses the production of A?

    doi: 10.1186/1750-1326-4-41

    Figure Lengend Snippet: A) A cartoon of various FLAG-tagged CD74 constructs: the yeast split-ubiquitin (TH) clone, full length CD74, two deletions constructs of CD74, and two splicing variants of mouse CD74 . The cytoplasmic and luminal parts, the locations of the FLAG tag, the endosomal-sorting signal in the cytoplasmic domain (LL), the CLIP domain (C), and the trimeric domain (T) are indicated. B) HeLa cells were transfected with pcDNA3 (-) or FLAG-CD74 (+) together with APP, and the total cell lysates (TL) and FLAG immunoprecipitants (IP) were analyzed by Westerm blot. Full length APP, APP CTF (C99 and C83), and CD74 were detected with 22C11, αAPPct, and αFLAG antibodies, respectively. The 33 and 14 kDa N-terminal fragments of CD74 were designated as CD74 NTF33 and NTF14. C) HeLa cells were transfected with FLAG-CD74 and APP, and the total lysates of the transfected cells were immunoprecipitated with either rabbit polyclonal antibody (RP) or αAPPct, and analyzed as in B. D) HeLa cells were transfected with various CD74 constructs and APP. The total lysates and FLAG IPs were analyzed as in B. Mature APP and immature APP (mAPP/imAPP) are indicated. The bands marked with asterisks (*) were attributed to the FLAG antibody used in the immunoprecipitation.

    Article Snippet: Staining of transfected HeLa cells HeLa cells were plated on coverslips coated with poly L-lysine (Sigma), and were transfected with indicated plasmids.

    Techniques: Construct, FLAG-tag, Cross-linking Immunoprecipitation, Transfection, Immunoprecipitation

    A) HeLa cells were transfected with APP-YFP or FLAG-CD74 . APP and CD74 were detected by YFP fluorescence and αFLAG antibody staining with Alexa594 anti-mouse secondary antibody, respectively. Corresponding phase contrast views were shown. B) HeLa cells were transfected with APP-YFP or Notch-GFP together with FLAG-CD74. Notch was detected by GFP fluorescence. APP and CD74 were detected as in A. Digitally merged pictures of Green and Red channels and phase contrast view are also displayed. Colocalization Coefficient (CC) and Intensity Correlation Quotient (ICQ) were indicated. C) HeLa cells were transfected with APP, C99 or C83 together with FLAG-CD74. APP, C99, and C83 were detected with the αAPPct antibody, and FLAG-CD74 was detected as above. Merged pictures and phase contrast views were shown as in B. Bar = 10 μm. CC and ICQ are as in B.

    Journal: Molecular Neurodegeneration

    Article Title: CD74 interacts with APP and suppresses the production of A?

    doi: 10.1186/1750-1326-4-41

    Figure Lengend Snippet: A) HeLa cells were transfected with APP-YFP or FLAG-CD74 . APP and CD74 were detected by YFP fluorescence and αFLAG antibody staining with Alexa594 anti-mouse secondary antibody, respectively. Corresponding phase contrast views were shown. B) HeLa cells were transfected with APP-YFP or Notch-GFP together with FLAG-CD74. Notch was detected by GFP fluorescence. APP and CD74 were detected as in A. Digitally merged pictures of Green and Red channels and phase contrast view are also displayed. Colocalization Coefficient (CC) and Intensity Correlation Quotient (ICQ) were indicated. C) HeLa cells were transfected with APP, C99 or C83 together with FLAG-CD74. APP, C99, and C83 were detected with the αAPPct antibody, and FLAG-CD74 was detected as above. Merged pictures and phase contrast views were shown as in B. Bar = 10 μm. CC and ICQ are as in B.

    Article Snippet: Staining of transfected HeLa cells HeLa cells were plated on coverslips coated with poly L-lysine (Sigma), and were transfected with indicated plasmids.

    Techniques: Transfection, Fluorescence, Staining

    HERV-K CA-NTD and MHR are not required for reduction of HIV-1 infectivity. HeLa cells were cotransfected with pNL4-3 and indicated plasmids. a Virus in the supernatant from cotransfected HeLa cells were collected and fractionated in rate-zonal gradient analysis. The amount of HIV-1 Gag were measured by ELISA. b Virus stocks were prepared from each fraction after rate-zonal gradient analysis. The viruses were purified and normalized by p24 ELISA. TZM-bl cells, which harbor an HIV-1 LTR-driven luciferase-reporter gene, were infected with the purified viruses. At 2 days post-infection, luciferase activities were measured by luminometor. Data from three independent experiments are shown as means ± standard deviations. P values were determined using a Student’s t test. * P

    Journal: Retrovirology

    Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

    doi: 10.1186/s12977-017-0351-8

    Figure Lengend Snippet: HERV-K CA-NTD and MHR are not required for reduction of HIV-1 infectivity. HeLa cells were cotransfected with pNL4-3 and indicated plasmids. a Virus in the supernatant from cotransfected HeLa cells were collected and fractionated in rate-zonal gradient analysis. The amount of HIV-1 Gag were measured by ELISA. b Virus stocks were prepared from each fraction after rate-zonal gradient analysis. The viruses were purified and normalized by p24 ELISA. TZM-bl cells, which harbor an HIV-1 LTR-driven luciferase-reporter gene, were infected with the purified viruses. At 2 days post-infection, luciferase activities were measured by luminometor. Data from three independent experiments are shown as means ± standard deviations. P values were determined using a Student’s t test. * P

    Article Snippet: Cells HeLa cells and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 5% FBS (DMEM-10).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Purification, Luciferase

    Influence of the protein corona on the interactions between RGD-targeted TMV nanoparticles (of three different RGD coverages) and different cell lines A, TMV–HeLa cell interactions in serum-free (sfMEM) and complete (cMEM) media as a function of surface chemistry studied by FACS. Statistical significant differences are indicated by p-values. B, Hypothesis for the promotion of VNP uptake in HeLa cells by coronal proteins. The energy of nonspecific interactions between coronal proteins and cell surface receptors is not sufficient for effective binding of the VNP. However, the RGD-targeted VNPs can “dock”, and uptake is enhanced by nonspecific interactions between coronal proteins and cell surface receptors. C, TMV–RAW264.7 cell interactions. a. D, TMV–SC cell interactions. E, Schematic diagram showing the specificity of RGD-targeted TMV VNPs. Red arrows indicate uptake in cMEM (a protein corona is present); blue arrows indicate uptake in serum-free medium (a protein corona is absent).

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: The protein corona of plant virus nanoparticles influences their dispersion properties, cellular interactions and in vivo fates

    doi: 10.1002/smll.201502458

    Figure Lengend Snippet: Influence of the protein corona on the interactions between RGD-targeted TMV nanoparticles (of three different RGD coverages) and different cell lines A, TMV–HeLa cell interactions in serum-free (sfMEM) and complete (cMEM) media as a function of surface chemistry studied by FACS. Statistical significant differences are indicated by p-values. B, Hypothesis for the promotion of VNP uptake in HeLa cells by coronal proteins. The energy of nonspecific interactions between coronal proteins and cell surface receptors is not sufficient for effective binding of the VNP. However, the RGD-targeted VNPs can “dock”, and uptake is enhanced by nonspecific interactions between coronal proteins and cell surface receptors. C, TMV–RAW264.7 cell interactions. a. D, TMV–SC cell interactions. E, Schematic diagram showing the specificity of RGD-targeted TMV VNPs. Red arrows indicate uptake in cMEM (a protein corona is present); blue arrows indicate uptake in serum-free medium (a protein corona is absent).

    Article Snippet: For additional experiments on HeLa cells ( ) cells were resuspended in either (A) cMEM and heat inactivated cMEM (30 minutes incubation in 56°C), or (B) sfMEM and sfMEM supplemented with 1.5 mg/ml total human IgG (I4506 Sigma Aldrich).

    Techniques: FACS, Binding Assay

    (A) Schematic representation of the interaction of the luminal domain of GPx8 with the loop region between Cys 208 and Cys 241 in Ero1α (left panel) and the Bi-molecular fluorescence complementation (BiFC) of two YFP half-sites fused to either Ero1α or GPx8 (right panel). (B) 18 h after transfection with the indicated constructs, HeLa cells were trypsinized and analyzed by flow cytometry for BiFC fluorescence ( n ≥5; mean±SD). a.u. arbitrary unit, ** p

    Journal: Redox Biology

    Article Title: Cysteines 208 and 241 in Ero1α are required for maximal catalytic turnover

    doi: 10.1016/j.redox.2015.11.004

    Figure Lengend Snippet: (A) Schematic representation of the interaction of the luminal domain of GPx8 with the loop region between Cys 208 and Cys 241 in Ero1α (left panel) and the Bi-molecular fluorescence complementation (BiFC) of two YFP half-sites fused to either Ero1α or GPx8 (right panel). (B) 18 h after transfection with the indicated constructs, HeLa cells were trypsinized and analyzed by flow cytometry for BiFC fluorescence ( n ≥5; mean±SD). a.u. arbitrary unit, ** p

    Article Snippet: 2.4 Cell culture and transient transfections The culturing of HeLa cells and FlipIn TRex293 cells for doxycycline (1 μg/ml, Sigma)-inducible expression of Ero1 variants has been described.

    Techniques: Fluorescence, Bimolecular Fluorescence Complementation Assay, Transfection, Construct, Flow Cytometry, Cytometry

    Cep152 recruits Plk4 to the centrosome. (A) Centriole overduplication in HeLa Tet-on cells was forced by induction of Plk4 expression through addition of doxycycline. Simultaneously, cells were transfected with either GL2 or one of two different Cep152 siRNAs (O1 or O2). Centrosomes were stained with γ-tubulin (green) antibodies or Cep152 (red). 72 h after transfection, cells with less than two centrosomes were counted. (B) Hela Tet-on cells were transfected with GL2 or Cep152 siRNAs (O1 and O2) treated with aphidicolin for 24 h. Plk4 expression was induced in the last 20 h of aphidicolin treatment. Cells were scored for centrosomal HA-Plk4 signal. Induced HA-Plk4 signal (green) locates to the centrosome (red, γ-tubulin). Blue, DNA. (right) Insets display enlargements of the selected regions in the indicated channels (arrowheads). The last set of insets includes a triple merge. Corresponding immunoblots from siRNA-treated and induced cells were analyzed for Plk4 levels using antibodies against HA and Plk4 in comparison with uninduced, control siRNA-treated samples. (A and B) Error bars indicate SDs ( n = 3). (C) Dynamics of Plk4 at centrosomes in response to Cep152 RNAi. FRAP was performed on U2OS cells treated with either GL2 or Cep152 siRNAs (O2) for 60 h followed by GFP-Plk4 transfections for 14 h. GFP-Plk4–positive, unsplit centrosomes in the same plane of focus were selected for photobleaching and subsequent imaging. 80 × 80–pixel squares surrounding the centrosome were bleached (bleach time 2.5 s), and the recovery of GFP fluorescence on centrosomes was imaged over time. (left) Arrows mark photobleached regions on the centrosome. (right) Relative expression levels of GFP-Plk4 in GL2- and O2-transfected cells were determined. (bottom) Mean fluorescence recovery profiles of GFP-Plk4 on the centrosome were depicted in GL2- and O2-treated cells ( n = 12). Mean t 1/2 of experiments is shown ± SD. Student’s t test was performed with GL2 or Cep152 siRNA-treated cells for recovery of GFP-Plk4 on the centrosome with P

    Journal: The Journal of Cell Biology

    Article Title: Cep152 acts as a scaffold for recruitment of Plk4 and CPAP to the centrosome

    doi: 10.1083/jcb.201007107

    Figure Lengend Snippet: Cep152 recruits Plk4 to the centrosome. (A) Centriole overduplication in HeLa Tet-on cells was forced by induction of Plk4 expression through addition of doxycycline. Simultaneously, cells were transfected with either GL2 or one of two different Cep152 siRNAs (O1 or O2). Centrosomes were stained with γ-tubulin (green) antibodies or Cep152 (red). 72 h after transfection, cells with less than two centrosomes were counted. (B) Hela Tet-on cells were transfected with GL2 or Cep152 siRNAs (O1 and O2) treated with aphidicolin for 24 h. Plk4 expression was induced in the last 20 h of aphidicolin treatment. Cells were scored for centrosomal HA-Plk4 signal. Induced HA-Plk4 signal (green) locates to the centrosome (red, γ-tubulin). Blue, DNA. (right) Insets display enlargements of the selected regions in the indicated channels (arrowheads). The last set of insets includes a triple merge. Corresponding immunoblots from siRNA-treated and induced cells were analyzed for Plk4 levels using antibodies against HA and Plk4 in comparison with uninduced, control siRNA-treated samples. (A and B) Error bars indicate SDs ( n = 3). (C) Dynamics of Plk4 at centrosomes in response to Cep152 RNAi. FRAP was performed on U2OS cells treated with either GL2 or Cep152 siRNAs (O2) for 60 h followed by GFP-Plk4 transfections for 14 h. GFP-Plk4–positive, unsplit centrosomes in the same plane of focus were selected for photobleaching and subsequent imaging. 80 × 80–pixel squares surrounding the centrosome were bleached (bleach time 2.5 s), and the recovery of GFP fluorescence on centrosomes was imaged over time. (left) Arrows mark photobleached regions on the centrosome. (right) Relative expression levels of GFP-Plk4 in GL2- and O2-transfected cells were determined. (bottom) Mean fluorescence recovery profiles of GFP-Plk4 on the centrosome were depicted in GL2- and O2-treated cells ( n = 12). Mean t 1/2 of experiments is shown ± SD. Student’s t test was performed with GL2 or Cep152 siRNA-treated cells for recovery of GFP-Plk4 on the centrosome with P

    Article Snippet: Culturing of cells, generation of stable cell lines, transfections, and cell synchronization HeLa cells, HeLa Tet-on cells, and U2OS cells were grown in DME (Sigma-Aldrich) containing 1 g/liter glucose, 10% fetal calf serum, and 2 mM glutamine.

    Techniques: Expressing, Transfection, Staining, Western Blot, Imaging, Fluorescence

    Importin α accumulates in the nucleus in response to either the oxidative stress or the heat shock stress. After HeLa cells were treated with hydrogen peroxide or incubated at 42°C at the indicated time, the cells were stained by specific antibodies for importin α (mRch1) and Alexa488-conjugated secondary antibodies. (A) HeLa cells were cultured with a medium containing 200 μM H 2 O 2 . (B) After HeLa cells were treated with 200 μM H 2 O 2 for 1 h, the cells were incubated with fresh medium in the absence of H 2 O 2 . (C) HeLa cells were incubated at 42°C. (D) After HeLa cells were incubated for 1 h at 42°C, they were incubated at 37°C for the indicated times.

    Journal: The Journal of Cell Biology

    Article Title: Cellular stresses induce the nuclear accumulation of importin ? and cause a conventional nuclear import block

    doi: 10.1083/jcb.200312008

    Figure Lengend Snippet: Importin α accumulates in the nucleus in response to either the oxidative stress or the heat shock stress. After HeLa cells were treated with hydrogen peroxide or incubated at 42°C at the indicated time, the cells were stained by specific antibodies for importin α (mRch1) and Alexa488-conjugated secondary antibodies. (A) HeLa cells were cultured with a medium containing 200 μM H 2 O 2 . (B) After HeLa cells were treated with 200 μM H 2 O 2 for 1 h, the cells were incubated with fresh medium in the absence of H 2 O 2 . (C) HeLa cells were incubated at 42°C. (D) After HeLa cells were incubated for 1 h at 42°C, they were incubated at 37°C for the indicated times.

    Article Snippet: Cell culture HeLa cells were incubated in Dulbecco's modified MEM (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS at 37°C in a 5% CO2 atmosphere.

    Techniques: Incubation, Staining, Cell Culture

    Clonal variations in CRISPRi and their associated off-target transcriptional effects. ( A ) Expression in counts-per-million (CPM) of Cas9 in CRISPRi clonal, CRISPRi non-clonal and untransduced HeLa cells. Clone 2 was used for showing Cas9 expression in CRISPRi clonal cells. ( B) Comparison of the transcriptional differences between parental HeLa untreated cells, CRISPRi clones expressing only dCas9–KRAB (clone 2) and clones treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled and shown as connecting lines of proportional thickness. ( C ) Comparison of the transcriptional differences between parental HeLa untreated cells, non-clonal CRISPRi cells expressing dCas9–KRAB and non-clonal cells treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled as described in B. ( D ) Expression of dCas9–KRAB in three different CRISPRi clones derived from single cell cloning, confirmed by immunoblot using a Cas9 antibody. β-tubulin was used as a loading control. A Venn diagram of DEGs detected in the three different clones against untransduced cells in the absence of any guide RNAs identified 37 genes as a common transcriptional signature of cloning. The total number of genes in this analysis was 18224 and DEGs were detected at a FDR of 5% with a log 2 -fold change threshold of 0.5. ( E ) Heat map of DEGs from three different CRISPRi clones compared to non-clonal cells and parental untransduced HeLa cells in the absence of any guide RNAs. 33 out of 37 genes were downregulated in clonal cells compared to the parental population. ( F ) Downregulation of two randomly selected DEGs from E ( SCIN and CDH2 ) was validated by qPCR in clonal cells (clone 2) and in non-clonal populations. Expression levels were normalized to the geometric mean of GAPDH and RPS18 . Error bars, s.e.m. ( n = 4 biological replicates). Statistical significance by two-tailed Student's t -test: * P

    Journal: Nucleic Acids Research

    Article Title: Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

    doi: 10.1093/nar/gky437

    Figure Lengend Snippet: Clonal variations in CRISPRi and their associated off-target transcriptional effects. ( A ) Expression in counts-per-million (CPM) of Cas9 in CRISPRi clonal, CRISPRi non-clonal and untransduced HeLa cells. Clone 2 was used for showing Cas9 expression in CRISPRi clonal cells. ( B) Comparison of the transcriptional differences between parental HeLa untreated cells, CRISPRi clones expressing only dCas9–KRAB (clone 2) and clones treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled and shown as connecting lines of proportional thickness. ( C ) Comparison of the transcriptional differences between parental HeLa untreated cells, non-clonal CRISPRi cells expressing dCas9–KRAB and non-clonal cells treated with two negative guide RNAs (negative guide 1 and 2). The number of genes differing between each pair of treatments is labelled as described in B. ( D ) Expression of dCas9–KRAB in three different CRISPRi clones derived from single cell cloning, confirmed by immunoblot using a Cas9 antibody. β-tubulin was used as a loading control. A Venn diagram of DEGs detected in the three different clones against untransduced cells in the absence of any guide RNAs identified 37 genes as a common transcriptional signature of cloning. The total number of genes in this analysis was 18224 and DEGs were detected at a FDR of 5% with a log 2 -fold change threshold of 0.5. ( E ) Heat map of DEGs from three different CRISPRi clones compared to non-clonal cells and parental untransduced HeLa cells in the absence of any guide RNAs. 33 out of 37 genes were downregulated in clonal cells compared to the parental population. ( F ) Downregulation of two randomly selected DEGs from E ( SCIN and CDH2 ) was validated by qPCR in clonal cells (clone 2) and in non-clonal populations. Expression levels were normalized to the geometric mean of GAPDH and RPS18 . Error bars, s.e.m. ( n = 4 biological replicates). Statistical significance by two-tailed Student's t -test: * P

    Article Snippet: FACS analysis and cell sorting HeLa cells were transduced with lentivirus containing the pHR-SFFV-dCAS9-BFP-KRAB vector together with polybrene (5 μg/ml, Sigma).

    Techniques: Expressing, Clone Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Two Tailed Test

    Endosomal tubulation in spastin-depleted cells requires intact MTs. (a) HeLa cells depleted of spastin by transfection with an siRNA pool were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). In this and subsequent color images, the color of the lettering in the black and white images indicates the color of that image in the corresponding merged image. Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (b–d) Mock-transfected cells or cells depleted of spastin by transfection with an siRNA pool were treated with vehicle (DMSO) or the MT-depolymerizing agent nocodazole (b), the MT stabilizing agent taxol (c), or the actin-depolymerizing agent latrunculin (d), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three (or four for latrunculin treatment) independent experiments were plotted. Representative immunofluorescence images from these experiments are shown in Fig. S2 . KD, knockdown. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Endosomal tubulation in spastin-depleted cells requires intact MTs. (a) HeLa cells depleted of spastin by transfection with an siRNA pool were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). In this and subsequent color images, the color of the lettering in the black and white images indicates the color of that image in the corresponding merged image. Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (b–d) Mock-transfected cells or cells depleted of spastin by transfection with an siRNA pool were treated with vehicle (DMSO) or the MT-depolymerizing agent nocodazole (b), the MT stabilizing agent taxol (c), or the actin-depolymerizing agent latrunculin (d), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three (or four for latrunculin treatment) independent experiments were plotted. Representative immunofluorescence images from these experiments are shown in Fig. S2 . KD, knockdown. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Immunofluorescence

    Ist1 is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 (IST1 knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. GAPDH immunoblotting serves as a loading control. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in d. (e) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were labeled for TfnR. Increased TfnR tubulation is seen in the IST1-depleted cells (see arrowheads in inset magnified region of the boxed area). The exposure settings in the IST1 knockdown image have been increased to compensate for the reduced TfnR levels in these cells. (f–h) Confocal micrographs of HeLa cells subjected to mock transfection (f) or transfected with IST1 siRNA1 (g and h) and then labeled with the markers shown. Confocal micrograph gain settings were identical in f and g, but in h, which shows higher magnification images of the dashed areas indicated in g, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Insets are magnified areas of the boxed regions. (i–k) Uptake of Alexa Fluor Tfn 647 was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with spastin siRNA1 (as a positive control), and IST1 siRNA1 (i) or IST1 siRNA3 (j). Mean Tfn uptake at the 20-min time point is shown ( n = 3 experiments plotted for each histogram). A representative time course experiment is shown in k. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Ist1 is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 (IST1 knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. GAPDH immunoblotting serves as a loading control. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in d. (e) Mock-transfected HeLa cells or HeLa cells transfected with IST1 siRNA1 were labeled for TfnR. Increased TfnR tubulation is seen in the IST1-depleted cells (see arrowheads in inset magnified region of the boxed area). The exposure settings in the IST1 knockdown image have been increased to compensate for the reduced TfnR levels in these cells. (f–h) Confocal micrographs of HeLa cells subjected to mock transfection (f) or transfected with IST1 siRNA1 (g and h) and then labeled with the markers shown. Confocal micrograph gain settings were identical in f and g, but in h, which shows higher magnification images of the dashed areas indicated in g, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Insets are magnified areas of the boxed regions. (i–k) Uptake of Alexa Fluor Tfn 647 was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with spastin siRNA1 (as a positive control), and IST1 siRNA1 (i) or IST1 siRNA3 (j). Mean Tfn uptake at the 20-min time point is shown ( n = 3 experiments plotted for each histogram). A representative time course experiment is shown in k. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, FACS, Positive Control

    IST1 regulates endosomal tubulation. (a and b) Wild-type HeLa cells were subjected to mock transfection or transfected with either of the two IST1 siRNA oligonucleotides indicated and then labeled with SNX1. The number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted in b. (c) Depletion of IST1 was verified by immunoblotting. (d–g) Wild-type HeLa cells (d) or HeLa cells stably expressing myc-tagged siRNA-resistant IST1 (e) were subjected to mock transfection or were transfected with an siRNA targeting endogenous IST1. The number of SNX1 tubules per cell was counted as in a. To control for any variation in the baseline number of tubules per cell in the two cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. wt, wild type. (g) Depletion of endogenous IST1 was verified by immunoblotting. (h) HeLa cells depleted of IST1 by transfection with IST1 siRNA1 were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (i–k) Mock-transfected cells or cells depleted of IST1 by transfection with siRNA1 were treated with vehicle (DMSO), nocodazole (i), taxol (j), or latrunculin A (k), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted. KD, knockdown. Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: IST1 regulates endosomal tubulation. (a and b) Wild-type HeLa cells were subjected to mock transfection or transfected with either of the two IST1 siRNA oligonucleotides indicated and then labeled with SNX1. The number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted in b. (c) Depletion of IST1 was verified by immunoblotting. (d–g) Wild-type HeLa cells (d) or HeLa cells stably expressing myc-tagged siRNA-resistant IST1 (e) were subjected to mock transfection or were transfected with an siRNA targeting endogenous IST1. The number of SNX1 tubules per cell was counted as in a. To control for any variation in the baseline number of tubules per cell in the two cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. wt, wild type. (g) Depletion of endogenous IST1 was verified by immunoblotting. (h) HeLa cells depleted of IST1 by transfection with IST1 siRNA1 were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (i–k) Mock-transfected cells or cells depleted of IST1 by transfection with siRNA1 were treated with vehicle (DMSO), nocodazole (i), taxol (j), or latrunculin A (k), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted. KD, knockdown. Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Stable Transfection, Expressing

    M1 and M87 spastin are recruited to endosomes and regulate endosomal tubulation. (a and b) GFP-VPS4-E235Q was transiently transfected into cell lines stably expressing myc-tagged M87 spastin (a) or myc-tagged M1 spastin M87A (b), and then, the cells were labeled with an anti-myc antibody. (c–f) HeLa cells (c), HeLa cells stably expressing myc-tagged M87 spastin (d), or HeLa cells expressing myc-tagged M1 spastinM87A (e) were subjected to mock transfection, transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant), or with a combination of two siRNA oligonucleotides that together targeted endogenous and transfected spastin (spastin 1 and 6). The cells were labeled with an antibody to endogenous SNX1, and the number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: M1 and M87 spastin are recruited to endosomes and regulate endosomal tubulation. (a and b) GFP-VPS4-E235Q was transiently transfected into cell lines stably expressing myc-tagged M87 spastin (a) or myc-tagged M1 spastin M87A (b), and then, the cells were labeled with an anti-myc antibody. (c–f) HeLa cells (c), HeLa cells stably expressing myc-tagged M87 spastin (d), or HeLa cells expressing myc-tagged M1 spastinM87A (e) were subjected to mock transfection, transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant), or with a combination of two siRNA oligonucleotides that together targeted endogenous and transfected spastin (spastin 1 and 6). The cells were labeled with an antibody to endogenous SNX1, and the number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Stable Transfection, Expressing, Labeling, Immunofluorescence

    ATPase activity and interaction with ESCRT-III are required for spastin to regulate endosomal tubulation. (a–d) Wild-type HeLa cells (a), HeLa cell lines stably expressing myc-tagged M87 spastin (b), myc-tagged M87 spastinK388R (c), or myc-tagged M87 spastinF124D (d) were subjected to mock transfection or were transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant) or with siRNA oligonucleotides directed against endogenous and exogenous spastin (spastin 1 and 6). Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). The number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in e; n = 3 independent experiments. (f–h) GFP-tagged VPS4-E235Q was transiently transfected into HeLa cells stably expressing myc-tagged wild-type (wt) M87 spastin (f) or myc-tagged M87 spastinF124D (g), which has dramatically reduced binding to CHMP1B and IST1. The cells were labeled with anti-myc antibodies. The extent of colocalization between GFP-VPS4-E235Q and the spastin proteins was estimated by calculating the Pearson’s correlation coefficient for red and green pixels in each cell, using Volocity software (h; n = 3 experiments, 20 cells per condition quantified in each experiment). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: ATPase activity and interaction with ESCRT-III are required for spastin to regulate endosomal tubulation. (a–d) Wild-type HeLa cells (a), HeLa cell lines stably expressing myc-tagged M87 spastin (b), myc-tagged M87 spastinK388R (c), or myc-tagged M87 spastinF124D (d) were subjected to mock transfection or were transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant) or with siRNA oligonucleotides directed against endogenous and exogenous spastin (spastin 1 and 6). Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting ( Fig. S3 ). The number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in e; n = 3 independent experiments. (f–h) GFP-tagged VPS4-E235Q was transiently transfected into HeLa cells stably expressing myc-tagged wild-type (wt) M87 spastin (f) or myc-tagged M87 spastinF124D (g), which has dramatically reduced binding to CHMP1B and IST1. The cells were labeled with anti-myc antibodies. The extent of colocalization between GFP-VPS4-E235Q and the spastin proteins was estimated by calculating the Pearson’s correlation coefficient for red and green pixels in each cell, using Volocity software (h; n = 3 experiments, 20 cells per condition quantified in each experiment). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Immunofluorescence, Binding Assay, Labeling, Software

    Spastin regulates endosomal tubulation. (a and b) HeLa cells were subject to mock transfection or transfection with a spastin siRNA pool (spastin knockdown [KD]) and labeled versus endogenous SNX1 (a) or transfected SNX4-mCherry (b). The insets in a and b are magnified images of the boxed regions indicated. (c) The mean number of SNX1 and SNX4 tubules per cell was quantified ( n = 5 independent experiments for SNX1, and n = 3 for SNX4, 30 cells counted per experimental condition in each experiment). (d) The length of the longest tubule per cell was measured in 30 cells, and the mean values were plotted. (e) Depletion of spastin was confirmed by immunoblotting. Note that M1 spastin is not routinely seen in immunoblots such as this, in which the exposure is optimized to show the much stronger M87 band. Actin labeling is shown to verify equal sample loading. (f) Complex structures, defined as having at least three tubules emanating from a central punctum (arrowheads), were commonly seen in spastin-depleted cells. (g) The mean number of complex structures per cell was quantified ( n = 3, 30 cells counted per condition in each experiment). Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Spastin regulates endosomal tubulation. (a and b) HeLa cells were subject to mock transfection or transfection with a spastin siRNA pool (spastin knockdown [KD]) and labeled versus endogenous SNX1 (a) or transfected SNX4-mCherry (b). The insets in a and b are magnified images of the boxed regions indicated. (c) The mean number of SNX1 and SNX4 tubules per cell was quantified ( n = 5 independent experiments for SNX1, and n = 3 for SNX4, 30 cells counted per experimental condition in each experiment). (d) The length of the longest tubule per cell was measured in 30 cells, and the mean values were plotted. (e) Depletion of spastin was confirmed by immunoblotting. Note that M1 spastin is not routinely seen in immunoblots such as this, in which the exposure is optimized to show the much stronger M87 band. Actin labeling is shown to verify equal sample loading. (f) Complex structures, defined as having at least three tubules emanating from a central punctum (arrowheads), were commonly seen in spastin-depleted cells. (g) The mean number of complex structures per cell was quantified ( n = 3, 30 cells counted per condition in each experiment). Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Western Blot

    Spastin is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA (spastin knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in eight independent experiments was plotted in d. (e–g) Confocal micrographs of HeLa cells subjected to mock transfection (e) or transfected with a spastin siRNA pool (f and g) and then labeled with the markers shown. Confocal micrograph gain settings were identical in e and f, but in g, which shows a higher magnification image of the dashed area indicated in f, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Note that tubular TfnR structures are not readily seen under confocal microscopy, as the tubules tend to leave the plane of section. Insets are magnified regions of the boxed areas. (h) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were fixed and labeled (without permeabilization) with a FITC-conjugated antibody against TfnR, and then, the cell-associated fluorescent signal was quantified by FACS analysis. The mean fluorescence values for three independent experiments were plotted. (i–k) Uptake of Alexa Fluor 647–conjugated Tfn was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with siRNA targeting clathrin heavy chain (CHC), or cells transfected with one of two siRNAs directed against spastin (spas1, spastin 1; spas3, spastin 3). Mean Tfn uptake at the 20-min time point ( n = 3 experiments) is shown in i, and a representative time course experiment is shown in j. Depletion of the relevant proteins targeted by siRNA was confirmed by immunoblotting, and a representative example is shown in k. (l and m) Recycling of internalized fluorescently labeled Tfn was measured over a 20-min time course, and the mean cell-associated Tfn at the 20-min time point ( n = 3 experiments) is shown in l, with a representative time course experiment shown in m. Bars, 10 µm. Error bars show SEMs.

    Journal: The Journal of Cell Biology

    Article Title: An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome

    doi: 10.1083/jcb.201211045

    Figure Lengend Snippet: Spastin is required for sorting TfnR away from degradation. (a and b) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA (spastin knockdown [KD]) were immunoblotted for the antibodies indicated. TfnR band density was quantified, and the mean density in three independent experiments was plotted in b. (c and d) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were treated with vehicle (DMSO) or DMSO + bafilomycin and then immunoblotted versus the antibodies indicated. TfnR band density was quantified, and the mean density in eight independent experiments was plotted in d. (e–g) Confocal micrographs of HeLa cells subjected to mock transfection (e) or transfected with a spastin siRNA pool (f and g) and then labeled with the markers shown. Confocal micrograph gain settings were identical in e and f, but in g, which shows a higher magnification image of the dashed area indicated in f, gain settings were increased to reveal that the remaining TfnR signal strongly colocalized with M6PR. Note that tubular TfnR structures are not readily seen under confocal microscopy, as the tubules tend to leave the plane of section. Insets are magnified regions of the boxed areas. (h) Mock-transfected HeLa cells or HeLa cells transfected with a pool of spastin siRNA were fixed and labeled (without permeabilization) with a FITC-conjugated antibody against TfnR, and then, the cell-associated fluorescent signal was quantified by FACS analysis. The mean fluorescence values for three independent experiments were plotted. (i–k) Uptake of Alexa Fluor 647–conjugated Tfn was measured by FACS over a 20-min time course in mock-transfected HeLa cells, cells transfected with siRNA targeting clathrin heavy chain (CHC), or cells transfected with one of two siRNAs directed against spastin (spas1, spastin 1; spas3, spastin 3). Mean Tfn uptake at the 20-min time point ( n = 3 experiments) is shown in i, and a representative time course experiment is shown in j. Depletion of the relevant proteins targeted by siRNA was confirmed by immunoblotting, and a representative example is shown in k. (l and m) Recycling of internalized fluorescently labeled Tfn was measured over a 20-min time course, and the mean cell-associated Tfn at the 20-min time point ( n = 3 experiments) is shown in l, with a representative time course experiment shown in m. Bars, 10 µm. Error bars show SEMs.

    Article Snippet: Adherent HeLa cells were transfected with siRNA for 72 h. For the final 18 h, cells were incubated with 100 µM bafilomycin A1 (in DMSO diluted to a final concentration of 1 µM; Sigma-Aldrich) or DMSO vehicle and then washed and harvested in cell lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, pH 8.0, and 1% [vol/vol] Triton X-100).

    Techniques: Transfection, Labeling, Confocal Microscopy, FACS, Fluorescence

    HeLa cells transfected with IKCal siRNAs induces apoptosis. HeLa cells were assayed by flow cytometry for annexin V binding and PI staining 72 h after infection. The logarithm of annexin V-fluorescein isothiocyanate fluorescence and the logarithm of PI fluorescence were plotted on the x and y axes of the cytogram, respectively. The lower right quadrant shows the percentage of viable cells in the early stages of apoptosis (i.e., annexin V-positive, PI-negative cells), the upper right quadrant shows the percentage of cells in a later stage of apoptosis or necrosis (annexin V-positive, PI-positive cells), the lower left shows the percentage of cells in the proliferating-senescence state, and the upper left quadrant shows the percentage of dead cells. ( A ) and ( B ) represent blank control and negative control cells infected with expressing control siRNA but not target IKCal, respectively. ( C ) Represents cells infected with IKCa1 siRNA. ( D ) Percentages of cells in apoptosis. The results are presented as the mean of 3 similar experiments. * P

    Journal: Medical Science Monitor Basic Research

    Article Title: Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    doi: 10.12659/MSMBR.901462

    Figure Lengend Snippet: HeLa cells transfected with IKCal siRNAs induces apoptosis. HeLa cells were assayed by flow cytometry for annexin V binding and PI staining 72 h after infection. The logarithm of annexin V-fluorescein isothiocyanate fluorescence and the logarithm of PI fluorescence were plotted on the x and y axes of the cytogram, respectively. The lower right quadrant shows the percentage of viable cells in the early stages of apoptosis (i.e., annexin V-positive, PI-negative cells), the upper right quadrant shows the percentage of cells in a later stage of apoptosis or necrosis (annexin V-positive, PI-positive cells), the lower left shows the percentage of cells in the proliferating-senescence state, and the upper left quadrant shows the percentage of dead cells. ( A ) and ( B ) represent blank control and negative control cells infected with expressing control siRNA but not target IKCal, respectively. ( C ) Represents cells infected with IKCa1 siRNA. ( D ) Percentages of cells in apoptosis. The results are presented as the mean of 3 similar experiments. * P

    Article Snippet: Medicine intervention on HeLa cells assay An IKCa1 channel blocker, clotrimazole (Sigma) was used to test the effect on HeLa cell proliferation.

    Techniques: Transfection, Flow Cytometry, Cytometry, Binding Assay, Staining, Infection, Fluorescence, Negative Control, Expressing

    Expression of IKCa1 mRNA and channel current in HeLa cells are decreased by treatment with clotrimazole. ( A ) The average relative expression level of IKCa1 mRNA in HeLa cells treated by 0, 5, 10, 20, and 40 μM clotrimazole for 24, 48, and 72 h was detected by qRT-PCR. ( B ) IKCa1 channel current in HeLa cells treated with 0 or 20 μM clotrimazole was detected by patch clamp technique. All experiments were repeated 3 times with similar results. * Indicated significant difference (P

    Journal: Medical Science Monitor Basic Research

    Article Title: Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    doi: 10.12659/MSMBR.901462

    Figure Lengend Snippet: Expression of IKCa1 mRNA and channel current in HeLa cells are decreased by treatment with clotrimazole. ( A ) The average relative expression level of IKCa1 mRNA in HeLa cells treated by 0, 5, 10, 20, and 40 μM clotrimazole for 24, 48, and 72 h was detected by qRT-PCR. ( B ) IKCa1 channel current in HeLa cells treated with 0 or 20 μM clotrimazole was detected by patch clamp technique. All experiments were repeated 3 times with similar results. * Indicated significant difference (P

    Article Snippet: Medicine intervention on HeLa cells assay An IKCa1 channel blocker, clotrimazole (Sigma) was used to test the effect on HeLa cell proliferation.

    Techniques: Expressing, Quantitative RT-PCR, Patch Clamp

    The IKCa1 blocker clotrimazole reduces HeLa cell number and inhibits cellular proliferation compared to control. HeLa cell culture media was replaced with serum-containing media including 0, 5, 10, 20, and 40 μM clotrimazole inhibitors and grown for 24, 48, and 72 h. The number of cells were quantitatively assessed using a Coulter Counter ( A ). The rate of inhibition on cell proliferation was assessed using MTT assay (see methods) * P

    Journal: Medical Science Monitor Basic Research

    Article Title: Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    doi: 10.12659/MSMBR.901462

    Figure Lengend Snippet: The IKCa1 blocker clotrimazole reduces HeLa cell number and inhibits cellular proliferation compared to control. HeLa cell culture media was replaced with serum-containing media including 0, 5, 10, 20, and 40 μM clotrimazole inhibitors and grown for 24, 48, and 72 h. The number of cells were quantitatively assessed using a Coulter Counter ( A ). The rate of inhibition on cell proliferation was assessed using MTT assay (see methods) * P

    Article Snippet: Medicine intervention on HeLa cells assay An IKCa1 channel blocker, clotrimazole (Sigma) was used to test the effect on HeLa cell proliferation.

    Techniques: Cell Culture, Inhibition, MTT Assay

    IKCa1 siRNAs downregulates IKCa1 mRNA and protein expression in HeLa cells. ( A, B ) Effect of the IKCa1-specific siRNAs on IKCa1 mRNA and protein expression levels, respectively. “nc” represents cells transfected with pGensil-HK+Lipo. “sr1” represents that HeLa cells were transfected with pGenesil-IKCa1siRNA+ Lipo and “sr2” represents a repeat of this experiment. The letter b (blank) represents cells were transfected with RPMI-1640+Lipo. At 48 h post-transfection, mRNA and protein levels were quantified and normalized to housekeeping genes GAPDH and β-actin, respectively. Data are the mean values ±SE of 3–5 independent transfections. * p

    Journal: Medical Science Monitor Basic Research

    Article Title: Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    doi: 10.12659/MSMBR.901462

    Figure Lengend Snippet: IKCa1 siRNAs downregulates IKCa1 mRNA and protein expression in HeLa cells. ( A, B ) Effect of the IKCa1-specific siRNAs on IKCa1 mRNA and protein expression levels, respectively. “nc” represents cells transfected with pGensil-HK+Lipo. “sr1” represents that HeLa cells were transfected with pGenesil-IKCa1siRNA+ Lipo and “sr2” represents a repeat of this experiment. The letter b (blank) represents cells were transfected with RPMI-1640+Lipo. At 48 h post-transfection, mRNA and protein levels were quantified and normalized to housekeeping genes GAPDH and β-actin, respectively. Data are the mean values ±SE of 3–5 independent transfections. * p

    Article Snippet: Medicine intervention on HeLa cells assay An IKCa1 channel blocker, clotrimazole (Sigma) was used to test the effect on HeLa cell proliferation.

    Techniques: Expressing, Transfection

    The expression of IKCa1 mRNA and protein are elevated in HeLa cells. ( A ) RT-PCR was used to detect IKCa1 mRNA expression with GAPDH as a loading control. ( B ) IKCa1 protein expression was detected using Western blot in HeLa cells with β-actin used as a loading control. In both A and B ( right ), mRNA or protein levels were quantified by measurement of the optical density of the samples compared to the optical density of the loading controls. Mean values ±SE of gene or protein expression from 3 independent cell preparations for HeLa and H8. * P

    Journal: Medical Science Monitor Basic Research

    Article Title: Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    doi: 10.12659/MSMBR.901462

    Figure Lengend Snippet: The expression of IKCa1 mRNA and protein are elevated in HeLa cells. ( A ) RT-PCR was used to detect IKCa1 mRNA expression with GAPDH as a loading control. ( B ) IKCa1 protein expression was detected using Western blot in HeLa cells with β-actin used as a loading control. In both A and B ( right ), mRNA or protein levels were quantified by measurement of the optical density of the samples compared to the optical density of the loading controls. Mean values ±SE of gene or protein expression from 3 independent cell preparations for HeLa and H8. * P

    Article Snippet: Medicine intervention on HeLa cells assay An IKCa1 channel blocker, clotrimazole (Sigma) was used to test the effect on HeLa cell proliferation.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    IKCa1 gene silencing decreases K + current densities and proliferation rates in HeLa cells. (A) Average IKCa1 currents were measured at 48 h after transfection with negative control siRNA (NC) or siRNA targeting IKCa1. Data are the mean values ±SE from 14–15 cells per experimental group (n=6. * p

    Journal: Medical Science Monitor Basic Research

    Article Title: Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    doi: 10.12659/MSMBR.901462

    Figure Lengend Snippet: IKCa1 gene silencing decreases K + current densities and proliferation rates in HeLa cells. (A) Average IKCa1 currents were measured at 48 h after transfection with negative control siRNA (NC) or siRNA targeting IKCa1. Data are the mean values ±SE from 14–15 cells per experimental group (n=6. * p

    Article Snippet: Medicine intervention on HeLa cells assay An IKCa1 channel blocker, clotrimazole (Sigma) was used to test the effect on HeLa cell proliferation.

    Techniques: Transfection, Negative Control

    Cytopathic effect induced by infection with mouse-adapted RV1A variants At different times after infection, cell viability was measured by trypan blue exclusion. Graphs are labeled as follows: HeLa cells (solid lines, solid symbols), LA4 cells (dashed lines, empty symbols), mock-infected cells (diamonds), wild-type RV1A (squares), RV1A/M2M (triangles), and RV1A/M2H (inverted triangles). A. Cell viability in HeLa cells infected with RV1A. B. Cell viability in LA4 cells infected with RV1A.

    Journal: Virology

    Article Title: Selection of Rhinovirus 1A Variants Adapted for Growth in Mouse Lung Epithelial Cells

    doi: 10.1016/j.virol.2011.08.021

    Figure Lengend Snippet: Cytopathic effect induced by infection with mouse-adapted RV1A variants At different times after infection, cell viability was measured by trypan blue exclusion. Graphs are labeled as follows: HeLa cells (solid lines, solid symbols), LA4 cells (dashed lines, empty symbols), mock-infected cells (diamonds), wild-type RV1A (squares), RV1A/M2M (triangles), and RV1A/M2H (inverted triangles). A. Cell viability in HeLa cells infected with RV1A. B. Cell viability in LA4 cells infected with RV1A.

    Article Snippet: Virus titer was determined on confluent HeLa cell monolayers by standard plaque assay in RV1A plaquing medium composed of DMEM supplemented with 2% bovine calf serum, 40 mM MgCl2 , 0.01% sodium bicarbonate, 1% penicillin/streptomycin, and 1% low-gelling type VII agarose (Sigma).

    Techniques: Infection, Labeling

    Effect of changes in RV1A capsid proteins on host range At different times after infection of LA4 or HeLa cells, viral yields were determined by plaque assay. RV1A (square) was compared to RV1A-VP3/H5Y-VP1/N85D (triangle) in HeLa (solid line, solid symbols) and LA4 cells (dashed line, empty symbols) at MOI=1.

    Journal: Virology

    Article Title: Selection of Rhinovirus 1A Variants Adapted for Growth in Mouse Lung Epithelial Cells

    doi: 10.1016/j.virol.2011.08.021

    Figure Lengend Snippet: Effect of changes in RV1A capsid proteins on host range At different times after infection of LA4 or HeLa cells, viral yields were determined by plaque assay. RV1A (square) was compared to RV1A-VP3/H5Y-VP1/N85D (triangle) in HeLa (solid line, solid symbols) and LA4 cells (dashed line, empty symbols) at MOI=1.

    Article Snippet: Virus titer was determined on confluent HeLa cell monolayers by standard plaque assay in RV1A plaquing medium composed of DMEM supplemented with 2% bovine calf serum, 40 mM MgCl2 , 0.01% sodium bicarbonate, 1% penicillin/streptomycin, and 1% low-gelling type VII agarose (Sigma).

    Techniques: Infection, Plaque Assay

    Growth of mouse-adapted RV1A in LA4 cells Yields of infected cells were determined at different times post-infection by plaque assay. Graphs are labeled as follows: HeLa cells (solid lines, filled symbols), LA4 cells (dashed lines, empty symbols), wild-type RV1A (square), and RV1A/M2M or RV1A/M2H (inverted triangle). A. Replication of RV1A/M2M at MOI=1 in HeLa and LA4 cells. B. Replication of RV1A/M2H at MOI=1 in HeLa and LA4 cells. C. Replication of RV1A/M2M at MOI=0.1. D. Replication of RV1A/M2H at MOI=0.1.

    Journal: Virology

    Article Title: Selection of Rhinovirus 1A Variants Adapted for Growth in Mouse Lung Epithelial Cells

    doi: 10.1016/j.virol.2011.08.021

    Figure Lengend Snippet: Growth of mouse-adapted RV1A in LA4 cells Yields of infected cells were determined at different times post-infection by plaque assay. Graphs are labeled as follows: HeLa cells (solid lines, filled symbols), LA4 cells (dashed lines, empty symbols), wild-type RV1A (square), and RV1A/M2M or RV1A/M2H (inverted triangle). A. Replication of RV1A/M2M at MOI=1 in HeLa and LA4 cells. B. Replication of RV1A/M2H at MOI=1 in HeLa and LA4 cells. C. Replication of RV1A/M2M at MOI=0.1. D. Replication of RV1A/M2H at MOI=0.1.

    Article Snippet: Virus titer was determined on confluent HeLa cell monolayers by standard plaque assay in RV1A plaquing medium composed of DMEM supplemented with 2% bovine calf serum, 40 mM MgCl2 , 0.01% sodium bicarbonate, 1% penicillin/streptomycin, and 1% low-gelling type VII agarose (Sigma).

    Techniques: Infection, Plaque Assay, Labeling

    NDP52 is required for p62-mediated autophagic activity and vice versa. HeLa cells were treated with control ( CTRL ), p62, or NDP52 siRNA, treated or not with bafilomycin, and immunoblotted for GAPDH, p62, or NDP52. Representative blots from three independent

    Journal: The Journal of Biological Chemistry

    Article Title: p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria

    doi: 10.1074/jbc.M111.223610

    Figure Lengend Snippet: NDP52 is required for p62-mediated autophagic activity and vice versa. HeLa cells were treated with control ( CTRL ), p62, or NDP52 siRNA, treated or not with bafilomycin, and immunoblotted for GAPDH, p62, or NDP52. Representative blots from three independent

    Article Snippet: HeLa cells (uninfected) treated or not with bafilomycin, or HeLa cells infected with Shigella or the Listeria ActA mutant in the absence and presence of TNF-α, were lysed in Igepal buffer (20 m m Tris, pH 8.0, 1% (v/v) Igepal CA-630 (Sigma), 150 m m NaCl, 10% (v/v) glycerol, and protease inhibitors mixture). p62, NDP52, SEPT2, or SEPT9 was immunoprecipitated from 800 μg of the total protein extracts by the addition of 1 μg of anti-p62, anti-NDP52, anti-SEPT2, or anti-SEPT9 antibody and 40 μl of a 50% slurry suspension of protein A-Sepharose beads (Amersham Biosciences).

    Techniques: Activity Assay

    Interaction of IMP1 and UNR with RNA. ( A ) Presence of IMP1, PABP and UNR in the ARS RNA–protein complex. REMSA was performed using [ 32 P]-labeled ARS RNA and HeLa cell extract as described. Cell extract (≈20 µg) was incubated with ≈1 µg of either non-immunized serum (lane 3), IMP1 (lane 4), PABP (lane 5) or UNR (lane 6) antibody for 10 min on ice prior to the addition of the labeled ARS RNA (≈1 ng, 1 × 10 5 c.p.m.). Samples were subjected to 5% PAGE under non-denaturing conditions. ( B ) Binding of IMP1 to the ARS RNA. [ 35 S]methionine labeled 6× His-tagged IMP1, PABP, UNR and β-galactosidase were expressed in E.coli and purified by Ni-NTA agarose as described in Materials and Methods. The purified polypeptides were incubated with ARS or poly(A) 50 –agarose beads and the bound proteins were eluted by boiling the beads in protein sample loading buffer and analyzed by 10% SDS–PAGE. In some binding reactions the indicated amount of purified non-radiolabeled PABP was added. Lane 1, [ 35 S]methionine labeled total cell extract from non-transformed E.coli DH5α; Lane 2, [ 35 S]methionine labeled PABP; Lanes 3–5, [ 35 S]methionine labeled IMP1 at different NaCl concentration; Lanes 6–8, [ 35 S]methionine labeled IMP1 and indicated amount of unlabeled PABP. Lane 9, [ 35 S]methionine labeled Luciferase. Lane 10, [ 35 S]methionine labeled His-tagged β-galactosidase. Lane 11, binding of IMP1 to poly(A) 50 –agarose. ( C ) Binding of UNR to the ARS RNA. Experiment was performed under similar conditions as in (B). Lanes 1, 2, 9 and 10 are same as in (A); Lanes 3–5, [ 35 S]methionine labeled UNR at different NaCl concentration; Lane 6–8, [ 35 S]methionine labeled UNR and indicated amount of unlabeled PABP. Lane 11, binding of UNR to poly(A) 50 –agarose.

    Journal: Nucleic Acids Research

    Article Title: The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex

    doi: 10.1093/nar/gki1014

    Figure Lengend Snippet: Interaction of IMP1 and UNR with RNA. ( A ) Presence of IMP1, PABP and UNR in the ARS RNA–protein complex. REMSA was performed using [ 32 P]-labeled ARS RNA and HeLa cell extract as described. Cell extract (≈20 µg) was incubated with ≈1 µg of either non-immunized serum (lane 3), IMP1 (lane 4), PABP (lane 5) or UNR (lane 6) antibody for 10 min on ice prior to the addition of the labeled ARS RNA (≈1 ng, 1 × 10 5 c.p.m.). Samples were subjected to 5% PAGE under non-denaturing conditions. ( B ) Binding of IMP1 to the ARS RNA. [ 35 S]methionine labeled 6× His-tagged IMP1, PABP, UNR and β-galactosidase were expressed in E.coli and purified by Ni-NTA agarose as described in Materials and Methods. The purified polypeptides were incubated with ARS or poly(A) 50 –agarose beads and the bound proteins were eluted by boiling the beads in protein sample loading buffer and analyzed by 10% SDS–PAGE. In some binding reactions the indicated amount of purified non-radiolabeled PABP was added. Lane 1, [ 35 S]methionine labeled total cell extract from non-transformed E.coli DH5α; Lane 2, [ 35 S]methionine labeled PABP; Lanes 3–5, [ 35 S]methionine labeled IMP1 at different NaCl concentration; Lanes 6–8, [ 35 S]methionine labeled IMP1 and indicated amount of unlabeled PABP. Lane 9, [ 35 S]methionine labeled Luciferase. Lane 10, [ 35 S]methionine labeled His-tagged β-galactosidase. Lane 11, binding of IMP1 to poly(A) 50 –agarose. ( C ) Binding of UNR to the ARS RNA. Experiment was performed under similar conditions as in (B). Lanes 1, 2, 9 and 10 are same as in (A); Lanes 3–5, [ 35 S]methionine labeled UNR at different NaCl concentration; Lane 6–8, [ 35 S]methionine labeled UNR and indicated amount of unlabeled PABP. Lane 11, binding of UNR to poly(A) 50 –agarose.

    Article Snippet: A PABP deficient cell extract was prepared by incubating 5 µl of the human PABP antibody with 25 µl of HeLa cell extract at 4°C for 2 h in CEB buffer followed by clearing the extract through a protein A–Sepharose column (Sigma).

    Techniques: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay, Purification, SDS Page, Transformation Assay, Concentration Assay, Luciferase

    Formation of ARS RNA–protein complex. ( A ) RNA–protein crosslinking by UV. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈3 ng, 3 × 10 5 c.p.m.) was incubated with different cell extract (≈60 µg protein) in the flat cap of a 0.2 ml PCR tube. Following the UV treatment, the samples were treated with RNaseA/RNase T1, analyzed by 10% SDS–PAGE and autoradiographed as described under experimental procedures. Cell extracts from mouse NIH3T3 fibroblasts (lane 2) and C2 myoblasts (lane 3), and human HEK293 (lane 4) and HeLa cells (lane 5) were used for these studies. One sample containing HeLa cell extract (lane 1) was analyzed without UV treatment as a control. Approximately 300 ng of the non-radioactive pGEM-T (lane 6), ARS (lane 7) or poly(A) 50 RNA (lane 8) was used for competition studies. ( B ) Analysis of RNP complex by REMSA. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈1 ng, 1 × 10 5 c.p.m.) was incubated with different cell extracts (20 µg) as indicated above each lane. The unbound RNA was digested with RNaseT1 and subjected to electrophoresis in a 2% agarose as described in the experimental procedures. Lane 1, radioactive ARS RNA incubated without the cell extract; lanes 2–5, radioactive ARS RNA incubated with NIH3T3, C2, HEK293 and HeLa cell extracts, respectively. Lanes 6–8, competition with 100 ng of non-radioactive pGEM-T, ARS, or poly(A) 50 RNA, respectively. Lane 9, cell extract was pre-incubated with the PABP antibody and precleared with protein A-sepharose beads before being used for REMSA. Approximately 20 µg of PABP deficient cell extract was used for REMSA. Lane 10, cell extract was similarly treated with the GFP antibody (BD Biosciences) before being used for REMSA. Lane 11, radiolabeled ARS RNA (≈ 1 ng, 1 × 10 5 c.p.m.) incubated with ≈2 ng of purified 6×His-PABP. ( C ) Analysis of RNP complexes by SDS–PAGE. RNP complex formation was initiated as described above and the samples were irradiated by UV before being resolved in a 2% agarose gel. The RNP bands (as shown in Figure 1B ) were excised from the gel, treated with RNaseA/RNase T1, and analyzed by 10% SDS–PAGE as described in the Materials and Methods. Lane 1, ARS RNA and HeLa extracts treated with UV and analyzed before gel purification. Lane 2, polypeptides from the gel purified slower migrating ARC. Lane 3, polypeptides from the faster migrating minor complex.

    Journal: Nucleic Acids Research

    Article Title: The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex

    doi: 10.1093/nar/gki1014

    Figure Lengend Snippet: Formation of ARS RNA–protein complex. ( A ) RNA–protein crosslinking by UV. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈3 ng, 3 × 10 5 c.p.m.) was incubated with different cell extract (≈60 µg protein) in the flat cap of a 0.2 ml PCR tube. Following the UV treatment, the samples were treated with RNaseA/RNase T1, analyzed by 10% SDS–PAGE and autoradiographed as described under experimental procedures. Cell extracts from mouse NIH3T3 fibroblasts (lane 2) and C2 myoblasts (lane 3), and human HEK293 (lane 4) and HeLa cells (lane 5) were used for these studies. One sample containing HeLa cell extract (lane 1) was analyzed without UV treatment as a control. Approximately 300 ng of the non-radioactive pGEM-T (lane 6), ARS (lane 7) or poly(A) 50 RNA (lane 8) was used for competition studies. ( B ) Analysis of RNP complex by REMSA. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈1 ng, 1 × 10 5 c.p.m.) was incubated with different cell extracts (20 µg) as indicated above each lane. The unbound RNA was digested with RNaseT1 and subjected to electrophoresis in a 2% agarose as described in the experimental procedures. Lane 1, radioactive ARS RNA incubated without the cell extract; lanes 2–5, radioactive ARS RNA incubated with NIH3T3, C2, HEK293 and HeLa cell extracts, respectively. Lanes 6–8, competition with 100 ng of non-radioactive pGEM-T, ARS, or poly(A) 50 RNA, respectively. Lane 9, cell extract was pre-incubated with the PABP antibody and precleared with protein A-sepharose beads before being used for REMSA. Approximately 20 µg of PABP deficient cell extract was used for REMSA. Lane 10, cell extract was similarly treated with the GFP antibody (BD Biosciences) before being used for REMSA. Lane 11, radiolabeled ARS RNA (≈ 1 ng, 1 × 10 5 c.p.m.) incubated with ≈2 ng of purified 6×His-PABP. ( C ) Analysis of RNP complexes by SDS–PAGE. RNP complex formation was initiated as described above and the samples were irradiated by UV before being resolved in a 2% agarose gel. The RNP bands (as shown in Figure 1B ) were excised from the gel, treated with RNaseA/RNase T1, and analyzed by 10% SDS–PAGE as described in the Materials and Methods. Lane 1, ARS RNA and HeLa extracts treated with UV and analyzed before gel purification. Lane 2, polypeptides from the gel purified slower migrating ARC. Lane 3, polypeptides from the faster migrating minor complex.

    Article Snippet: A PABP deficient cell extract was prepared by incubating 5 µl of the human PABP antibody with 25 µl of HeLa cell extract at 4°C for 2 h in CEB buffer followed by clearing the extract through a protein A–Sepharose column (Sigma).

    Techniques: In Vitro, Synthesized, Labeling, Incubation, Polymerase Chain Reaction, SDS Page, Electrophoresis, Purification, Irradiation, Agarose Gel Electrophoresis, Gel Purification

    Glycosylation profile of CD8 lumenal . HeLa cells expressing CD8 lumenal were incubated with the disaggregating drug at 16°C for 2 hr prior to being shifted at 37°C for 2 hr in the presence (1) or absence (2) of the drug. As negative control, cells were incubated at the same temperatures but without drug at any time (3). ( A ) Immunoblot showing that Jacalin, a lectin that binds Galatose residues, precipitates disaggregated CD8 lumenal from cells incubated at 37°C (accordingly to Figure 1D ). Helix-Pomatia, a lectin that binds N-Acetyl-Galactosamine, precipitates disaggregated CD8 lumenal from cells incubated at 16°C. DOI: http://dx.doi.org/10.7554/eLife.00558.008

    Journal: eLife

    Article Title: Stapled Golgi cisternae remain in place as cargo passes through the stack

    doi: 10.7554/eLife.00558

    Figure Lengend Snippet: Glycosylation profile of CD8 lumenal . HeLa cells expressing CD8 lumenal were incubated with the disaggregating drug at 16°C for 2 hr prior to being shifted at 37°C for 2 hr in the presence (1) or absence (2) of the drug. As negative control, cells were incubated at the same temperatures but without drug at any time (3). ( A ) Immunoblot showing that Jacalin, a lectin that binds Galatose residues, precipitates disaggregated CD8 lumenal from cells incubated at 37°C (accordingly to Figure 1D ). Helix-Pomatia, a lectin that binds N-Acetyl-Galactosamine, precipitates disaggregated CD8 lumenal from cells incubated at 16°C. DOI: http://dx.doi.org/10.7554/eLife.00558.008

    Article Snippet: For lectin precipitation, HeLa cells extracts containing CD8lumenal were incubated at 4°C with Jacalin-immobilized lectin (Sigma-Aldrich) or Helix pomatia Gel-HPA-immobilized lectin (EY Laboratories, San Mateo, CA, United States) prior to being processed for immunoblotting as described above.

    Techniques: Expressing, Incubation, Negative Control

    Figure 5. NuMA1 phosphorylations affecting spindle assembly and PARylation. ( A ) HeLa cells were treated as described in ; mock (Mk) or NuMA1 IPs probed for phospho-S/TQ sites (P-S/TQ; top panel), NuMA1 (bottom panel). ( B ) HeLa

    Journal: Cell Cycle

    Article Title: ATM controls proper mitotic spindle structure

    doi: 10.4161/cc.27945

    Figure Lengend Snippet: Figure 5. NuMA1 phosphorylations affecting spindle assembly and PARylation. ( A ) HeLa cells were treated as described in ; mock (Mk) or NuMA1 IPs probed for phospho-S/TQ sites (P-S/TQ; top panel), NuMA1 (bottom panel). ( B ) HeLa

    Article Snippet: For in vitro NuMA1 phosphorylation experiments, NuMA1 IPs from mitotic HeLa cells were incubated with Flag Ips from either mock- or Flag-ATM expression vector-transfected, mitotic, HeLa cells in a kinase reaction mixture (10 mM HEPES, pH 7.4; 10 mM MgCl2 ; 50 mM NaCl; 10 mM MnCl2 ), including Flag peptide (Sigma), 10 μCi γ-32 P-ATP, and KU-55933 where indicated, for 15 min at 30 °C.

    Techniques:

    Figure 4. ( A ) Mitotic localization of ATM, BRCA1, NuMA1, and TNKS1. HeLa cells were fixed and processed for immunofluorescence and confocal microscopy analysis. BRCA1, NuMA1, and TNKS1 were stained in red, ATM in green, and DNA in blue. Mitotic

    Journal: Cell Cycle

    Article Title: ATM controls proper mitotic spindle structure

    doi: 10.4161/cc.27945

    Figure Lengend Snippet: Figure 4. ( A ) Mitotic localization of ATM, BRCA1, NuMA1, and TNKS1. HeLa cells were fixed and processed for immunofluorescence and confocal microscopy analysis. BRCA1, NuMA1, and TNKS1 were stained in red, ATM in green, and DNA in blue. Mitotic

    Article Snippet: For in vitro NuMA1 phosphorylation experiments, NuMA1 IPs from mitotic HeLa cells were incubated with Flag Ips from either mock- or Flag-ATM expression vector-transfected, mitotic, HeLa cells in a kinase reaction mixture (10 mM HEPES, pH 7.4; 10 mM MgCl2 ; 50 mM NaCl; 10 mM MnCl2 ), including Flag peptide (Sigma), 10 μCi γ-32 P-ATP, and KU-55933 where indicated, for 15 min at 30 °C.

    Techniques: Immunofluorescence, Confocal Microscopy, Staining

    Figure 2. ATM requirements for NuMA1 PARylation. ( A ) Mitotic HeLa cells, detached after 14-h treatment with nocodazole, were treated with vehicle or KU-55933 (−/+) for 60 min in nocodazole. ( B ) Detached HeLa cells treated with control

    Journal: Cell Cycle

    Article Title: ATM controls proper mitotic spindle structure

    doi: 10.4161/cc.27945

    Figure Lengend Snippet: Figure 2. ATM requirements for NuMA1 PARylation. ( A ) Mitotic HeLa cells, detached after 14-h treatment with nocodazole, were treated with vehicle or KU-55933 (−/+) for 60 min in nocodazole. ( B ) Detached HeLa cells treated with control

    Article Snippet: For in vitro NuMA1 phosphorylation experiments, NuMA1 IPs from mitotic HeLa cells were incubated with Flag Ips from either mock- or Flag-ATM expression vector-transfected, mitotic, HeLa cells in a kinase reaction mixture (10 mM HEPES, pH 7.4; 10 mM MgCl2 ; 50 mM NaCl; 10 mM MnCl2 ), including Flag peptide (Sigma), 10 μCi γ-32 P-ATP, and KU-55933 where indicated, for 15 min at 30 °C.

    Techniques:

    Figure 3. ATM and BRCA1 interact with TNKS1 and control NuMA1 PARylation. ( A ) Total cytosolic extracts from asynchronous (Asy) and nocodazole-treated (Noco) HeLa cells and mock (Mk), ATM, and TNKS1 IPs from extracts of nocodazole-treated cells

    Journal: Cell Cycle

    Article Title: ATM controls proper mitotic spindle structure

    doi: 10.4161/cc.27945

    Figure Lengend Snippet: Figure 3. ATM and BRCA1 interact with TNKS1 and control NuMA1 PARylation. ( A ) Total cytosolic extracts from asynchronous (Asy) and nocodazole-treated (Noco) HeLa cells and mock (Mk), ATM, and TNKS1 IPs from extracts of nocodazole-treated cells

    Article Snippet: For in vitro NuMA1 phosphorylation experiments, NuMA1 IPs from mitotic HeLa cells were incubated with Flag Ips from either mock- or Flag-ATM expression vector-transfected, mitotic, HeLa cells in a kinase reaction mixture (10 mM HEPES, pH 7.4; 10 mM MgCl2 ; 50 mM NaCl; 10 mM MnCl2 ), including Flag peptide (Sigma), 10 μCi γ-32 P-ATP, and KU-55933 where indicated, for 15 min at 30 °C.

    Techniques:

    GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: GFP as a tag in immunoaffinity experiments. Although a commercial monoclonal anti-GFP antibody is capable of isolating significant amounts of free GFP from a stable HeLa cell line, the GFP binder is more efficient, as demonstrated both by Coomassie staining of protein eluted from the affinity matrices (A) and Western blotting using anti-GFP antibodies (B). Whether the mAb or GFP binder is used to purify GFP, there are very few proteins that bind nonspecifically to this tag (C). Four independent experiments were performed to identify proteins that may copurify with GFP, as indicated by SILAC ratios greater than 1 (IP1: whole cell extract, GFP binder; IP2: whole cell extract, monoclonal anti-GFP antibody; IP3: cytoplasmic extract, monoclonal anti-GFP antibody; IP4: nuclear extract, monoclonal anti-GFP antibody). No one protein was identified in every experiment, and most of them (in bold) have been identified as binding nonspecifically to the Sepharose bead matrix. This list was then screened against a set of 18 independent GFP protein immunoaffinity experiments performed using the GFP binder for purification and parental cells as the negative control. Proteins were scored for the percentage of experiments in which they were detected (yellow), and for the percentage of experiments in which they were detected and showed a SILAC ratio greater than 1 (green). Six proteins, representing three protein classes (heat shock/chaperone, cytokeratin, and ubiquitin), have been highlighted in green as the most frequently detected and potentially able to bind GFP.

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Staining, Western Blot, Binding Assay, Purification, Negative Control

    Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: Identification of proteins that interact with SMN and the SMN complex. The GFP binder was used to immunopurify GFP-SMN from a stable HeLa cell line as compared with the nonexpressing parental cell line. Like endogenous SMN, GFP-SMN is found in both cytoplasmic and nucleoplasmic pools and accumulates in gems within nuclei (A). Bar, 15 μM. Detailed biochemical and proteomic studies have revealed that the core SMN complex is composed of SMN itself and Gemins 2–8 (B). The stoichiometry is not known and, although not depicted here, the complex can oligomerize. Also listed are several other proteins that have been shown to interact with the SMN complex by similar experimental approaches. In the study presented here, separate experiments were performed for cytoplasmic and nuclear extracts to independently assess interacting partners and compare these two pools. The log SILAC (i.e., heavy/light arginine and/or lysine) ratio calculated for each protein identified in the cytoplasmic GFP-SMN immunoprecipitation experiment is plotted versus total peptide intensity in C. The nucleoplasmic GFP-SMN immunoprecipitation data are plotted in a similar fashion (D).

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Immunoprecipitation

    Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13 C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12 C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: Protocols used for SILAC-based analysis of protein interaction partners in pull-down experiments. (A) HeLa cells expressing a GFP-tagged protein are metabolically labeled by culturing in “heavy” media containing 13 C-isotopes of arginine and lysine, while the parental HeLa cells are grown in “light” media containing the 12 C-isotopes of arginine and lysine. Whole cell extracts can be prepared or, as shown here, cells can be fractionated for preparation of separate cytoplasmic and nuclear extracts. In this case, extracts are pre-cleared on Sepharose beads and then mixed in equal amounts before affinity purification of the GFP-tagged protein using the GFP binder (1 h incubation). Proteins are eluted from the beads and separated by 1D SDS-PAGE for digestion and LC-MS/MS analysis. (B) For SILAC analysis of an endogenous protein, two populations of HeLa cells are grown in light and heavy media, respectively, before harvesting and preparation of cellular extracts. Equal total protein amounts of each extract are subjected to separate immunoaffinity experiments, either using an antibody to the protein of interest or a control antibody covalently bound to beads at an equivalent concentration. The separate immunoprecipitates are mixed carefully to minimize variability and the proteins eluted and analyzed as described above.

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Expressing, Metabolic Labelling, Labeling, Affinity Purification, Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Concentration Assay

    Validation of mass spectrometric results. Cytoplasmic-specific copurification of the novel protein USP9X with GFP-SMN was confirmed by Western blotting (A). Two peptides, each with a SILAC ratio > 1, were found for USP9X in the SILAC analysis of a GFP-SMN pull-down from cytoplasmic extracts. The mass spectra of one of them is shown here for comparison (B). The quantifiable arginine is highlighted in red. This cytoplasmic enrichment of USP9X is consistent with immunostaining results using a monoclonal anti-USP9X antibody (C). Although predominantly cytoplasmic, there is a pool of USP9X in the nucleus (arrowhead), although it does not accumulate in gems (arrow). There is no difference in localization of USP9X in parental HeLa cells (top cell) versus HeLa cells stably expressing GFP-SMN (bottom cell). Bar, 5 μM. As a control, Western blotting was also used to confirm the enrichment of both endogenous SMN and GFP-SMN, and of the U1 snRNP protein U1A, from both cytoplasmic and nuclear extracts using the GFP binder, and the nuclear-specific enrichment of p80 coilin (D). For comparison, representative peptide spectra for these proteins from the SILAC analysis are shown (E). Quantifiable amino acids are highlighted in red, with the SILAC ratio in parentheses.

    Journal: The Journal of Cell Biology

    Article Title: Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    doi: 10.1083/jcb.200805092

    Figure Lengend Snippet: Validation of mass spectrometric results. Cytoplasmic-specific copurification of the novel protein USP9X with GFP-SMN was confirmed by Western blotting (A). Two peptides, each with a SILAC ratio > 1, were found for USP9X in the SILAC analysis of a GFP-SMN pull-down from cytoplasmic extracts. The mass spectra of one of them is shown here for comparison (B). The quantifiable arginine is highlighted in red. This cytoplasmic enrichment of USP9X is consistent with immunostaining results using a monoclonal anti-USP9X antibody (C). Although predominantly cytoplasmic, there is a pool of USP9X in the nucleus (arrowhead), although it does not accumulate in gems (arrow). There is no difference in localization of USP9X in parental HeLa cells (top cell) versus HeLa cells stably expressing GFP-SMN (bottom cell). Bar, 5 μM. As a control, Western blotting was also used to confirm the enrichment of both endogenous SMN and GFP-SMN, and of the U1 snRNP protein U1A, from both cytoplasmic and nuclear extracts using the GFP binder, and the nuclear-specific enrichment of p80 coilin (D). For comparison, representative peptide spectra for these proteins from the SILAC analysis are shown (E). Quantifiable amino acids are highlighted in red, with the SILAC ratio in parentheses.

    Article Snippet: For fixed cell imaging, a mix of parental HeLa cells and HeLa cells stably expressing GFP-SMN were paraformaldehyde fixed on glass coverslips, permeabilized with Triton X-100, stained with both anti-USP9X (detected by TRITC-anti–mouse secondary antibodies) and the DNA stain DAPI, and mounted in FluorSave mounting media (Calbiochem).

    Techniques: Copurification, Western Blot, Immunostaining, Stable Transfection, Expressing

    Evaluation of CDCA2 expression in OSCC-derived cell lines and the HeLa cell line. ( A ) Quantification of CDCA2 mRNA expression in OSCC-derived cell lines and the HeLa cell line by qRT-PCR analysis. Significant up-regulation of CDCA2 mRNA is seen in all cell lines compared with that in HNOKs (* P

    Journal: PLoS ONE

    Article Title: Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

    doi: 10.1371/journal.pone.0056381

    Figure Lengend Snippet: Evaluation of CDCA2 expression in OSCC-derived cell lines and the HeLa cell line. ( A ) Quantification of CDCA2 mRNA expression in OSCC-derived cell lines and the HeLa cell line by qRT-PCR analysis. Significant up-regulation of CDCA2 mRNA is seen in all cell lines compared with that in HNOKs (* P

    Article Snippet: The OSCC cell lines and HeLa cell line were grown in Dulbecco's modified Eagle medium (DMEM) F-12 HAM (Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 50 units/ml penicillin and streptomycin (Sigma).

    Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

    Paclitaxel (PTX)-induced cytotoxicity in neural stem cells (NSC). ( a ) Dose–response curve of cytotoxicity in mouse NSC (black dots), induced pluripotent stem cells (iPSC)-derived human neuronal progenitor cells (hNPC, open triangle) and post-mitotic mouse hippocampus neurons (HCN; clear dots) after 2 h exposure time with PTX. Data were fitted with a three-parameter logistic curve. ( b ) Representative histogram of cell cycle analysis in NSC. After both vehicle (VEH), respectively, PTX treatment, only small percentages of NSC were in G2/M-phase (insert). ( c ) Treatment of NSC with 30 n m PTX led to a significant increase in resting Ca 2+ levels, evidenced by a higher Fura-2 excitation ratio. ( d ) Caspase-3/7 activity was significantly increased after PTX treatment (30 n m ) and was inhibited by co-incubation with the calpain inhibitor MDL28170 (Calp-Inh; 10 μ m ), lithium chloride (Li + ; 1 m m ), and the caspase inhibitor AC-DEVD-CHO (C3/7-Inh; 1 μ m ). Caspase-3/7 activity remained high after co-treatment with the GSK3β inhibitor A1070722 (GSK3-Inh; 100 n m ). ( e ) Dose–response curve of caspase 3/7 (closed circles) and caspase 9 (open squares) activity in NSC 12 h after 2 h exposure with different PTX concentrations. ( f ) Protein expression of NCS-1 is higher in NSC compared with HCN and MCF-7 breast cancer cells. (Loading control of whole cell lysates: HDAC-1). ( g ) PTX induces an increase in calpain activity which could be prevented with the calpain inhibitor MDL28170 (Calp-Inh; 10 μ m ). ( h ) Impaired cell viability of NSC after exposure to 30 n m PTX was significantly improved by co-incubation with MDL28170 (Calp-Inh; 10 μ m ), Li + (1 m m ) or AC-DEVD-CHO (C3/7-Inh; 1 μ m ), respectively, but not with the GSK3β inhibitor A1070722. ( i ) Exposure of MCF-7 and HeLa cells to different PTX concentrations for 2 h with and without 1 m m Li + did not show a protective effect for the lithium co-medication in these cancer cell lines. Statistical analysis: data were checked for normal distribution using D’Agostino–Pearson normality test and then analyzed using ( b ) Mann–Whitney U test, ( c ) unpaired two-sided t -test and ( d-e and g – i ) ordinary one-way ANOVA and Holm–Sidak post hoc test (data are obtained from n =3 individual experiments with 4–6 technical replicates for each condition). * P

    Journal: Translational Psychiatry

    Article Title: A novel preventive therapy for paclitaxel-induced cognitive deficits: preclinical evidence from C57BL/6 mice

    doi: 10.1038/tp.2017.149

    Figure Lengend Snippet: Paclitaxel (PTX)-induced cytotoxicity in neural stem cells (NSC). ( a ) Dose–response curve of cytotoxicity in mouse NSC (black dots), induced pluripotent stem cells (iPSC)-derived human neuronal progenitor cells (hNPC, open triangle) and post-mitotic mouse hippocampus neurons (HCN; clear dots) after 2 h exposure time with PTX. Data were fitted with a three-parameter logistic curve. ( b ) Representative histogram of cell cycle analysis in NSC. After both vehicle (VEH), respectively, PTX treatment, only small percentages of NSC were in G2/M-phase (insert). ( c ) Treatment of NSC with 30 n m PTX led to a significant increase in resting Ca 2+ levels, evidenced by a higher Fura-2 excitation ratio. ( d ) Caspase-3/7 activity was significantly increased after PTX treatment (30 n m ) and was inhibited by co-incubation with the calpain inhibitor MDL28170 (Calp-Inh; 10 μ m ), lithium chloride (Li + ; 1 m m ), and the caspase inhibitor AC-DEVD-CHO (C3/7-Inh; 1 μ m ). Caspase-3/7 activity remained high after co-treatment with the GSK3β inhibitor A1070722 (GSK3-Inh; 100 n m ). ( e ) Dose–response curve of caspase 3/7 (closed circles) and caspase 9 (open squares) activity in NSC 12 h after 2 h exposure with different PTX concentrations. ( f ) Protein expression of NCS-1 is higher in NSC compared with HCN and MCF-7 breast cancer cells. (Loading control of whole cell lysates: HDAC-1). ( g ) PTX induces an increase in calpain activity which could be prevented with the calpain inhibitor MDL28170 (Calp-Inh; 10 μ m ). ( h ) Impaired cell viability of NSC after exposure to 30 n m PTX was significantly improved by co-incubation with MDL28170 (Calp-Inh; 10 μ m ), Li + (1 m m ) or AC-DEVD-CHO (C3/7-Inh; 1 μ m ), respectively, but not with the GSK3β inhibitor A1070722. ( i ) Exposure of MCF-7 and HeLa cells to different PTX concentrations for 2 h with and without 1 m m Li + did not show a protective effect for the lithium co-medication in these cancer cell lines. Statistical analysis: data were checked for normal distribution using D’Agostino–Pearson normality test and then analyzed using ( b ) Mann–Whitney U test, ( c ) unpaired two-sided t -test and ( d-e and g – i ) ordinary one-way ANOVA and Holm–Sidak post hoc test (data are obtained from n =3 individual experiments with 4–6 technical replicates for each condition). * P

    Article Snippet: MCF-7 and HeLa cell lines were obtained from Sigma-Aldrich (Taufkirchen, Germany; Authentication by STR profiling).

    Techniques: Derivative Assay, Cell Cycle Assay, Activity Assay, Incubation, Expressing, MANN-WHITNEY

    Modest expression of Ptc-ICD7 fragment leads to its nuclear accumulation. C-terminally-FLAG-tagged Ptc-ICD7 cDNA in pCI-neo expression vector was transfected into HeLa cells (Ptc-ICD7-FLAG TF) and immunostained with anti-FLAG antibody. At the time of cDNA transfection, protein synthesis inhibitor cyclohexamide (CHX) was added at indicated concentrations. “No CHX” indicates cell culture without CHX addition (A, B). Cells were harvested 24 hr after transfections. Addition of 12.5 µg/mL CHX completely suppressed protein expression from transfected expression plasmid (E, I), while the addition of 0.4 µg/mL CHX reduced expression of FLAG-tagged Ptc-ICD7 to less than half compared with “No CHX” (I). “Mock-TF” indicates empty vector transfection without CHX addition as a negative control (G, H). Anti-FLAG immunostain in green (A, C, E, G), and DAPI DNA stain in blue (B, D, F, H). Scale bar, 20 µm.

    Journal: PLoS ONE

    Article Title: A Novel Signaling Pathway Mediated by the Nuclear Targeting of C-Terminal Fragments of Mammalian Patched 1

    doi: 10.1371/journal.pone.0018638

    Figure Lengend Snippet: Modest expression of Ptc-ICD7 fragment leads to its nuclear accumulation. C-terminally-FLAG-tagged Ptc-ICD7 cDNA in pCI-neo expression vector was transfected into HeLa cells (Ptc-ICD7-FLAG TF) and immunostained with anti-FLAG antibody. At the time of cDNA transfection, protein synthesis inhibitor cyclohexamide (CHX) was added at indicated concentrations. “No CHX” indicates cell culture without CHX addition (A, B). Cells were harvested 24 hr after transfections. Addition of 12.5 µg/mL CHX completely suppressed protein expression from transfected expression plasmid (E, I), while the addition of 0.4 µg/mL CHX reduced expression of FLAG-tagged Ptc-ICD7 to less than half compared with “No CHX” (I). “Mock-TF” indicates empty vector transfection without CHX addition as a negative control (G, H). Anti-FLAG immunostain in green (A, C, E, G), and DAPI DNA stain in blue (B, D, F, H). Scale bar, 20 µm.

    Article Snippet: Mammalian cell culture and transfection HeLa cell was cultured in Dulbecco's modified Eagle's medium (Sigma Chemical Co., St Louis, MO) supplemented with 10% heat-inactivated Calf Serum at 37°C under a 5% CO2 atmosphere.

    Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Negative Control, Staining

    Gli1-based luciferase-reporter assay reveals Shh response in Ptc1 transfected cells. (A) Dual-luciferase reporter assay performed with Gli1 expression vector (Gli1) and a reporter construct consisting of eight copies of the Gli-binding site (8×3′Gli-BS) or its mutated version (8×m3′Gli-BS) as a negative control. Normalization of transfection efficiencies was carried out using Renilla luciferase activities as an internal control. Relative luciferase activity was monitored in cells stably expressing full-length Ptc1 (Ptc1 FL Stable exp.) and its negative control (Mock exp.) with (+) or without (−) Shh-N-conditioned media. This medium contains 19 kDa form of Shh-N fragment of Myc-tagged-Shh-N that was processed and secreted from in HeLa cells expressing full-length Shh. Addition of the Shh-N fragment stimulates transcriptional activity of Gli1 in cells stably expressing full-length Ptc1. All reporter assay experiments were repeated at least three times, and transfection was done in duplicate. (B) Stably expressed full-length Ptc1 binds with secreted Shh-N. HeLa cells that were stably expressed N-terminally T7-tagged full-length Ptc1 was exposed to Myc-Shh-N-conditioned medium. After cells were harvested, full-length Ptc1 was immunoprecipitated with N-terminal T7-tag from solubilized cell lysates (IP:anti-T7) and precipitates were probed with anti-Myc antibody (IB: anti-Myc). (C) Shh-N stimulates the production of Ptc-ICD7 fragments. Cells stably expressing full-length Ptc1 (with C-terminal FLAG-tag) were cultured with (+) or without (−) Shh-N-conditioned media. Solubilized cell lysates were subjected to FLAG-immunoprecipitation and subsequently probed with anti-FLAG antibody. (D) Relative luciferase activity as in (A) monitored in cells over-expressing Ptc-ICD7 fragment (pCI-Ptc1-ICD7), full-length Ptc1 (pCI-Ptc1 FL), and mock control (pCI-empty).

    Journal: PLoS ONE

    Article Title: A Novel Signaling Pathway Mediated by the Nuclear Targeting of C-Terminal Fragments of Mammalian Patched 1

    doi: 10.1371/journal.pone.0018638

    Figure Lengend Snippet: Gli1-based luciferase-reporter assay reveals Shh response in Ptc1 transfected cells. (A) Dual-luciferase reporter assay performed with Gli1 expression vector (Gli1) and a reporter construct consisting of eight copies of the Gli-binding site (8×3′Gli-BS) or its mutated version (8×m3′Gli-BS) as a negative control. Normalization of transfection efficiencies was carried out using Renilla luciferase activities as an internal control. Relative luciferase activity was monitored in cells stably expressing full-length Ptc1 (Ptc1 FL Stable exp.) and its negative control (Mock exp.) with (+) or without (−) Shh-N-conditioned media. This medium contains 19 kDa form of Shh-N fragment of Myc-tagged-Shh-N that was processed and secreted from in HeLa cells expressing full-length Shh. Addition of the Shh-N fragment stimulates transcriptional activity of Gli1 in cells stably expressing full-length Ptc1. All reporter assay experiments were repeated at least three times, and transfection was done in duplicate. (B) Stably expressed full-length Ptc1 binds with secreted Shh-N. HeLa cells that were stably expressed N-terminally T7-tagged full-length Ptc1 was exposed to Myc-Shh-N-conditioned medium. After cells were harvested, full-length Ptc1 was immunoprecipitated with N-terminal T7-tag from solubilized cell lysates (IP:anti-T7) and precipitates were probed with anti-Myc antibody (IB: anti-Myc). (C) Shh-N stimulates the production of Ptc-ICD7 fragments. Cells stably expressing full-length Ptc1 (with C-terminal FLAG-tag) were cultured with (+) or without (−) Shh-N-conditioned media. Solubilized cell lysates were subjected to FLAG-immunoprecipitation and subsequently probed with anti-FLAG antibody. (D) Relative luciferase activity as in (A) monitored in cells over-expressing Ptc-ICD7 fragment (pCI-Ptc1-ICD7), full-length Ptc1 (pCI-Ptc1 FL), and mock control (pCI-empty).

    Article Snippet: Mammalian cell culture and transfection HeLa cell was cultured in Dulbecco's modified Eagle's medium (Sigma Chemical Co., St Louis, MO) supplemented with 10% heat-inactivated Calf Serum at 37°C under a 5% CO2 atmosphere.

    Techniques: Luciferase, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Construct, Binding Assay, Negative Control, Activity Assay, Stable Transfection, Immunoprecipitation, FLAG-tag, Cell Culture

    p34 associates with AP-1 and AP-2 in the cytosol (a) Native immunoprecipitation (IP) of HeLa cytosolic extracts was performed with antibodies against p34 (p34-1 and p34-2), followed by Western blotting (WB) with antibodies against p34, AP-1 or AP-2. Note the association of both AP-1 and AP-2 adaptor complexes with p34. (b) HeLa cells were transiently transfected with either C-terminally (p34GFP) or N-terminally (GFPp34) GFP-tagged p34 constructs. Both constructs show a mainly cytosolic localization and this is consistent with the staining pattern (albeit faint) observed using an antibody against p34 in either transfected or untransfected cells (#). (c) HeLa lysates were subjected to fractionation into cytosol, membrane, and clathrin-coated vesicle (CCV) fractions, and then Western blotted for clathrin heavy chain (CHC), AP-1 and p34. Note that p34 is found principally in the cytosolic fraction and is virtually undetectable in membrane or CCV fractions in which AP-1 and CHC are enriched. (* = cross reacting band) (d) HeLa cells were treated with siRNA to knock down expression of p34 (kd), and western blotted for clathrin heavy chain (CHC) and p34 (* = cross reacting band). Note the efficient knockdown of p34 ( > 95%). (e) Localization of AP-1 and AP-2 visualized by immunofluorescence following knock down of p34 (kd). Note that the AP-2-containing membrane-associated vesicles or AP-1-trans-Golgi network appear unperturbed. Bar = 20 μm.

    Journal: Nature genetics

    Article Title: Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma

    doi: 10.1038/ng.2444

    Figure Lengend Snippet: p34 associates with AP-1 and AP-2 in the cytosol (a) Native immunoprecipitation (IP) of HeLa cytosolic extracts was performed with antibodies against p34 (p34-1 and p34-2), followed by Western blotting (WB) with antibodies against p34, AP-1 or AP-2. Note the association of both AP-1 and AP-2 adaptor complexes with p34. (b) HeLa cells were transiently transfected with either C-terminally (p34GFP) or N-terminally (GFPp34) GFP-tagged p34 constructs. Both constructs show a mainly cytosolic localization and this is consistent with the staining pattern (albeit faint) observed using an antibody against p34 in either transfected or untransfected cells (#). (c) HeLa lysates were subjected to fractionation into cytosol, membrane, and clathrin-coated vesicle (CCV) fractions, and then Western blotted for clathrin heavy chain (CHC), AP-1 and p34. Note that p34 is found principally in the cytosolic fraction and is virtually undetectable in membrane or CCV fractions in which AP-1 and CHC are enriched. (* = cross reacting band) (d) HeLa cells were treated with siRNA to knock down expression of p34 (kd), and western blotted for clathrin heavy chain (CHC) and p34 (* = cross reacting band). Note the efficient knockdown of p34 ( > 95%). (e) Localization of AP-1 and AP-2 visualized by immunofluorescence following knock down of p34 (kd). Note that the AP-2-containing membrane-associated vesicles or AP-1-trans-Golgi network appear unperturbed. Bar = 20 μm.

    Article Snippet: Immunoprecipitation and clathrin-coated vesicle isolation in HeLa cell extracts For immunoprecipitation, cells were lyzed in PBS containing 1% IGEPAL (Sigma-Aldrich), cleared of debris by centrifugation and antibody-protein complexes captured using protein-A Sepharose (Pharmacia, Milton Keynes, UK).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Construct, Staining, Fractionation, Expressing, Immunofluorescence

    F-box protein FBXW11 binds to NSs and regulates RVFV. (A) siRNA screening assay targeting 70 out of 72 human F-box genes to determine RVFV infection inhibitors. HeLa cells were transfected with non-targeting control siRNA or with a pool of four different siRNA targeting each of the 70 F-box genes for 48h followed by incubation with ZH501 (MOI = 1) for 24h. Percentage of infected cells was determined by HCA of N expressing cells. Each data point in panels A to D is an average of three replicates ±SD and is representative of 2 independent experiments. (B) Validation of FBXW11 as a true hit from the siRNA screening: Each siRNA from the pool of 4 siRNAs was individually tested for its ability to inhibit RVFV (ZH501 or MP-12) infection and reduce FBXW11 mRNA levels. Infection was quantified by HCA of viral antigen expressing cells as described in A, while mRNA levels were determined by real time PCR. The relative infection or mRNA levels were calculated by normalizing with the values derived from controls cells that were transfected with non-targeting siRNA and infected with the corresponding viruses. The infection rates of control siRNA treated cells are indicated in the brackets next to the virus names. (C) FBXW11 is the major homologue of βTrCP gene regulating RVFV infection, but its activity is enhanced by BTRC. Same as in B except siRNA targeting BTRC was used either singly or combined with FBXW11 siRNA. (D) Real-Time PCR analysis of BTRC and FBXW11 mRNA levels in HeLa cells that were transfected with the siRNAs are indicated on the X-axis. Relative mRNA levels were derived by normalizing mRNA levels in cells transfected with non-targeting siRNA. (E) Western blot analysis shows that βTrCP siRNA knockdown induces PKR activation and inhibition of viral gene expression similar to MLN4924 treated cells. HeLa cells were transfected with different siRNAs as indicated for 48h and then either mock infected or infected with rMP-12-NSs-V5 (MOI = 10, 8h), and were either treated with DMSO or MLN4924 at 2h, PI. Cells lysates were harvested at 8h, PI for Western blot analysis. (F) Co-immunoprecipitation (co-IP) assay is consistent with the WD40 domain regulating βTrCP binding to NSs: Top panel, depicts the domain structure of the BTRC and FBXW11 protein which contains D or the Dimerization domain, F or F-box motif, and the seven WD40 repeats. In the bottom panel is data from co-IP assay showing the binding of NSs or CUL1 to the various deletion mutants of BTRC and FBXW11. 293T cell lysates overexpressing myc-CUL1 and the vector alone or flag tagged wildtype βTrCP or deletion mutants of βTrCP were combined with rMP-12-NSs-V5 (MOI = 10, 8h) infected cell lysates and immunoprecipitated with anti-Flag antibody. The bound proteins were detected by Western blot analysis as described in the figure. Lysate controls represent 5% of extract used for co-IP.

    Journal: PLoS Pathogens

    Article Title: Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

    doi: 10.1371/journal.ppat.1005437

    Figure Lengend Snippet: F-box protein FBXW11 binds to NSs and regulates RVFV. (A) siRNA screening assay targeting 70 out of 72 human F-box genes to determine RVFV infection inhibitors. HeLa cells were transfected with non-targeting control siRNA or with a pool of four different siRNA targeting each of the 70 F-box genes for 48h followed by incubation with ZH501 (MOI = 1) for 24h. Percentage of infected cells was determined by HCA of N expressing cells. Each data point in panels A to D is an average of three replicates ±SD and is representative of 2 independent experiments. (B) Validation of FBXW11 as a true hit from the siRNA screening: Each siRNA from the pool of 4 siRNAs was individually tested for its ability to inhibit RVFV (ZH501 or MP-12) infection and reduce FBXW11 mRNA levels. Infection was quantified by HCA of viral antigen expressing cells as described in A, while mRNA levels were determined by real time PCR. The relative infection or mRNA levels were calculated by normalizing with the values derived from controls cells that were transfected with non-targeting siRNA and infected with the corresponding viruses. The infection rates of control siRNA treated cells are indicated in the brackets next to the virus names. (C) FBXW11 is the major homologue of βTrCP gene regulating RVFV infection, but its activity is enhanced by BTRC. Same as in B except siRNA targeting BTRC was used either singly or combined with FBXW11 siRNA. (D) Real-Time PCR analysis of BTRC and FBXW11 mRNA levels in HeLa cells that were transfected with the siRNAs are indicated on the X-axis. Relative mRNA levels were derived by normalizing mRNA levels in cells transfected with non-targeting siRNA. (E) Western blot analysis shows that βTrCP siRNA knockdown induces PKR activation and inhibition of viral gene expression similar to MLN4924 treated cells. HeLa cells were transfected with different siRNAs as indicated for 48h and then either mock infected or infected with rMP-12-NSs-V5 (MOI = 10, 8h), and were either treated with DMSO or MLN4924 at 2h, PI. Cells lysates were harvested at 8h, PI for Western blot analysis. (F) Co-immunoprecipitation (co-IP) assay is consistent with the WD40 domain regulating βTrCP binding to NSs: Top panel, depicts the domain structure of the BTRC and FBXW11 protein which contains D or the Dimerization domain, F or F-box motif, and the seven WD40 repeats. In the bottom panel is data from co-IP assay showing the binding of NSs or CUL1 to the various deletion mutants of BTRC and FBXW11. 293T cell lysates overexpressing myc-CUL1 and the vector alone or flag tagged wildtype βTrCP or deletion mutants of βTrCP were combined with rMP-12-NSs-V5 (MOI = 10, 8h) infected cell lysates and immunoprecipitated with anti-Flag antibody. The bound proteins were detected by Western blot analysis as described in the figure. Lysate controls represent 5% of extract used for co-IP.

    Article Snippet: Generation of doxycycline inducible PKR shRNA expressing stable cell line HeLa cells were transduced with lentivirus expressing doxycycline (Sigma) inducible human PKR shRNA (cat # V2THS_170555, GE Healthcare) encoding the antisense sequence 5’-TTTATCTCTGATGTATCTG-3’ and selected in Puromycin (1 μg/ml, from Sigma) containing media.

    Techniques: Screening Assay, Infection, Transfection, Incubation, High Content Screening, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Activity Assay, Western Blot, Activation Assay, Inhibition, Co-Immunoprecipitation Assay, Binding Assay, Plasmid Preparation, Immunoprecipitation

    MLN4924 regulates NSs-dependent early PKR activation in RVFV-infected cells. (A) Western blot analysis demonstrating MLN4924 regulation of PKR activation and inhibition of viral gene expression during the course of a single cycle of viral replication (~ 12h). HeLa cells treated with control (DMSO) or MLN4924 (1 μM) were either mock infected or infected with rMP12-NSs-V5. At the indicated time points, cell lysates were harvested and analyzed for gene expression by Western blot analysis. (B) NSs regulates MLN4924 mediated PKR activation during RVFV infection. HeLa cells expressing control non-targeting siRNA or PKR siRNA were treated with DMSO (vehicle control) or MLN4924 (1 μM) and infected with either wildtype viruses including RVFV ZH501 or rMP-12 or NSs deficient viruses including clone 13 or rMP-12ΔNSs::Luci at an MOI = 10 for 12h. The infection rates were determined by enumerating G expressing cells (rMP-12 and rMP-12ΔNSs::Luci) or N expressing cells (RVFV ZH501 and clone13) by HCA. The data derived from MLN4924 treated cells was normalized with the corresponding DMSO treated controls (average infection rates are indicated under the bar graph), which were considered as 100% and expressed as the mean ± SD. The inset shows a decrease in PKR expression levels in PKR siRNA transfected cells by Western blot analysis. (C) Western blot analysis demonstrating the regulation of the PKR activation pathway and viral gene expression kinetics by NSs deficient virus (rMP-12ΔNSs::Luci virus) during single cycle of virus infection. ( D ) Western blot analysis which shows that MLN4924 treatment of HeLa cells that were transiently transfected with plasmid DNA expressing the NSs gene was sufficient to activate PKR.

    Journal: PLoS Pathogens

    Article Title: Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

    doi: 10.1371/journal.ppat.1005437

    Figure Lengend Snippet: MLN4924 regulates NSs-dependent early PKR activation in RVFV-infected cells. (A) Western blot analysis demonstrating MLN4924 regulation of PKR activation and inhibition of viral gene expression during the course of a single cycle of viral replication (~ 12h). HeLa cells treated with control (DMSO) or MLN4924 (1 μM) were either mock infected or infected with rMP12-NSs-V5. At the indicated time points, cell lysates were harvested and analyzed for gene expression by Western blot analysis. (B) NSs regulates MLN4924 mediated PKR activation during RVFV infection. HeLa cells expressing control non-targeting siRNA or PKR siRNA were treated with DMSO (vehicle control) or MLN4924 (1 μM) and infected with either wildtype viruses including RVFV ZH501 or rMP-12 or NSs deficient viruses including clone 13 or rMP-12ΔNSs::Luci at an MOI = 10 for 12h. The infection rates were determined by enumerating G expressing cells (rMP-12 and rMP-12ΔNSs::Luci) or N expressing cells (RVFV ZH501 and clone13) by HCA. The data derived from MLN4924 treated cells was normalized with the corresponding DMSO treated controls (average infection rates are indicated under the bar graph), which were considered as 100% and expressed as the mean ± SD. The inset shows a decrease in PKR expression levels in PKR siRNA transfected cells by Western blot analysis. (C) Western blot analysis demonstrating the regulation of the PKR activation pathway and viral gene expression kinetics by NSs deficient virus (rMP-12ΔNSs::Luci virus) during single cycle of virus infection. ( D ) Western blot analysis which shows that MLN4924 treatment of HeLa cells that were transiently transfected with plasmid DNA expressing the NSs gene was sufficient to activate PKR.

    Article Snippet: Generation of doxycycline inducible PKR shRNA expressing stable cell line HeLa cells were transduced with lentivirus expressing doxycycline (Sigma) inducible human PKR shRNA (cat # V2THS_170555, GE Healthcare) encoding the antisense sequence 5’-TTTATCTCTGATGTATCTG-3’ and selected in Puromycin (1 μg/ml, from Sigma) containing media.

    Techniques: Activation Assay, Infection, Western Blot, Inhibition, Expressing, High Content Screening, Derivative Assay, Transfection, Plasmid Preparation

    SCF FBXW11-NSs is the E3 ligase that targets PKR degradation during RVFV infection. (A) Western blot analysis to determine the PKR activation and viral gene expression inhibition of rMP-12-NSs-V5 (MOI = 10 for 8h) infected HeLa cells in which the expression of the individual components of the SCF FBXW11 E3 ligases were either knocked down singly or in combination with siRNA. (B) Evaluating SCF FBXW11 regulation of RVFV infection under normal or PKR depleted conditions. The inset on the right is a Western blot showing PKR levels in the HeLa cell line that stably expresses doxycycline inducible PKR shRNA under untreated or doxycyline treated conditions. HeLa cells that were either treated with control DMSO or doxycycline were transfected with siRNA targeting the individual components of SCF FBXW11 , singly or in combination as indicated in the figure for 48h followed by infection with RVFV ZH501 (MOI = 1 for 24h). The percentage of infected cells was determined by HCA analysis of RVFV N expressing cells. Each data point is an average of 3 replicates ±SD. (C) Same as in B except that RVFV Clone 13 (MOI = 3, 24h) was used in infection. (D) Co-immunoprecipitation assay showing that PKR is recruited to the SCF FBXW11 complex by NSs. 293T cell lysates transfected with empty vector or expression plasmids of myc -CUL1 or HA-FBXW11 or myc -CUL1 and HA-FBXW11 (as indicated in the figure) were combined with mock infected or rMP-12-NSs-V5 (MOI = 10 for 8h) infected cell lysates and immunoprecipitated with anti- myc or anti-HA, or a combination of both. Western blot of lysates represents 5% of the total cellular extracts. (E) Model of NSs recruiting PKR to the SCF via NSs binding to FBXW11. The symbols R, S, and U represent RBX1, SKP1 and Ubiquitin respectively.

    Journal: PLoS Pathogens

    Article Title: Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

    doi: 10.1371/journal.ppat.1005437

    Figure Lengend Snippet: SCF FBXW11-NSs is the E3 ligase that targets PKR degradation during RVFV infection. (A) Western blot analysis to determine the PKR activation and viral gene expression inhibition of rMP-12-NSs-V5 (MOI = 10 for 8h) infected HeLa cells in which the expression of the individual components of the SCF FBXW11 E3 ligases were either knocked down singly or in combination with siRNA. (B) Evaluating SCF FBXW11 regulation of RVFV infection under normal or PKR depleted conditions. The inset on the right is a Western blot showing PKR levels in the HeLa cell line that stably expresses doxycycline inducible PKR shRNA under untreated or doxycyline treated conditions. HeLa cells that were either treated with control DMSO or doxycycline were transfected with siRNA targeting the individual components of SCF FBXW11 , singly or in combination as indicated in the figure for 48h followed by infection with RVFV ZH501 (MOI = 1 for 24h). The percentage of infected cells was determined by HCA analysis of RVFV N expressing cells. Each data point is an average of 3 replicates ±SD. (C) Same as in B except that RVFV Clone 13 (MOI = 3, 24h) was used in infection. (D) Co-immunoprecipitation assay showing that PKR is recruited to the SCF FBXW11 complex by NSs. 293T cell lysates transfected with empty vector or expression plasmids of myc -CUL1 or HA-FBXW11 or myc -CUL1 and HA-FBXW11 (as indicated in the figure) were combined with mock infected or rMP-12-NSs-V5 (MOI = 10 for 8h) infected cell lysates and immunoprecipitated with anti- myc or anti-HA, or a combination of both. Western blot of lysates represents 5% of the total cellular extracts. (E) Model of NSs recruiting PKR to the SCF via NSs binding to FBXW11. The symbols R, S, and U represent RBX1, SKP1 and Ubiquitin respectively.

    Article Snippet: Generation of doxycycline inducible PKR shRNA expressing stable cell line HeLa cells were transduced with lentivirus expressing doxycycline (Sigma) inducible human PKR shRNA (cat # V2THS_170555, GE Healthcare) encoding the antisense sequence 5’-TTTATCTCTGATGTATCTG-3’ and selected in Puromycin (1 μg/ml, from Sigma) containing media.

    Techniques: Infection, Western Blot, Activation Assay, Expressing, Inhibition, Stable Transfection, shRNA, Transfection, High Content Screening, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation, Binding Assay

    Characterization of the degron/destruction sequence that is essential for NSs-FBXW11 interaction to promote PKR degradation. (A) Description of the NSs degron mutants. The six linear amino acid sequence predicted as the degron sequence of the NSs gene is underlined and the alanine substitution mutations are marked in red. The number in the subscript denotes the amino acid residue number of the NSs protein. (B) Western blot analysis to determine the PKR activation and viral gene expression status in HeLa cells that were infected with rMP-12 encoding wildtype NSs gene or the NSs mutants as indicated in the figure at MOI = 10 for 8h. (C) IFA of NSs (green) filament formation in the nucleus of HeLa cells that were infected with rMP-12 virus expressing wildtype NSs gene or any of the various NSs mutants as indicated in the figure at MOI = 1 for 24h under normal or PKR depleted conditions. The inset shows the magnified image of white box marked in Doxycycline treated cells. Nuclei are stained blue with Hoechst (33342). The panel on the right side is the Western blot showing PKR levels in the HeLa cell line that stable express doxycycline inducible PKR shRNA under untreated or doxycycline treated conditions. (D) Same as C, but the percentage of infected cells was determined by HCA analysis of G expressing cells. Each data point is an average of 3 replicates ±SD. **** indicates a P

    Journal: PLoS Pathogens

    Article Title: Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

    doi: 10.1371/journal.ppat.1005437

    Figure Lengend Snippet: Characterization of the degron/destruction sequence that is essential for NSs-FBXW11 interaction to promote PKR degradation. (A) Description of the NSs degron mutants. The six linear amino acid sequence predicted as the degron sequence of the NSs gene is underlined and the alanine substitution mutations are marked in red. The number in the subscript denotes the amino acid residue number of the NSs protein. (B) Western blot analysis to determine the PKR activation and viral gene expression status in HeLa cells that were infected with rMP-12 encoding wildtype NSs gene or the NSs mutants as indicated in the figure at MOI = 10 for 8h. (C) IFA of NSs (green) filament formation in the nucleus of HeLa cells that were infected with rMP-12 virus expressing wildtype NSs gene or any of the various NSs mutants as indicated in the figure at MOI = 1 for 24h under normal or PKR depleted conditions. The inset shows the magnified image of white box marked in Doxycycline treated cells. Nuclei are stained blue with Hoechst (33342). The panel on the right side is the Western blot showing PKR levels in the HeLa cell line that stable express doxycycline inducible PKR shRNA under untreated or doxycycline treated conditions. (D) Same as C, but the percentage of infected cells was determined by HCA analysis of G expressing cells. Each data point is an average of 3 replicates ±SD. **** indicates a P

    Article Snippet: Generation of doxycycline inducible PKR shRNA expressing stable cell line HeLa cells were transduced with lentivirus expressing doxycycline (Sigma) inducible human PKR shRNA (cat # V2THS_170555, GE Healthcare) encoding the antisense sequence 5’-TTTATCTCTGATGTATCTG-3’ and selected in Puromycin (1 μg/ml, from Sigma) containing media.

    Techniques: Sequencing, Western Blot, Activation Assay, Expressing, Infection, Immunofluorescence, Staining, shRNA, High Content Screening

    TIN2 is phosphorylated on S295 and S330 during mitosis. ( A ) Detection of S295 and S330 phosphorylation of TIN2 during mitosis by the Phos-tag reagent after release from a double thymidine block. HeLa cells stably infected with a retrovirus encoding Flag-TIN2 in the WT, S330A, or S295A configuration were collected from asynchronous populations (A), populations arrested with a double thymidine block corresponding to the G1/S phase of the cell cycle, or populations at the points corresponding to S, G2, M and early or middle G1 (EG1 or MG1) after release from the double thymidine block. Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP) are denoted on the left of the upper panels. Left and right panels are different exposures. Representative of two experiments. ( B ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in cells arrested with nocodazole. HeLa cells stably infected with a retrovirus encoding no transgene (vector, V) or Flag-TIN2 in the WT, S330A, S295A, or AA configuration were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then subjected to either ( top ) immunoprecipitation (IP) with αFlag and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left of the upper panel. Representative of three experiments. ( C ) Detection of S295 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. Lysates from HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc), resolved by SDS-PAGE and immunoblotted (IB) with an anti-Phos-S295, anti-TIN2, anti-Phos-HH3, or anti-HH3 (loading control) antibody. Representative of two experiments. ( D ) Detection of S330 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-TIN2 antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with either an anti-Phos-S330 or anti-TIN2 antibody, or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with an anti-Phos-HH3 antibody, to monitor cell cycle progression, or an anti-Tubulin antibody as a loading control. Representative of one experiment.

    Journal: PLoS ONE

    Article Title: Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2

    doi: 10.1371/journal.pone.0071697

    Figure Lengend Snippet: TIN2 is phosphorylated on S295 and S330 during mitosis. ( A ) Detection of S295 and S330 phosphorylation of TIN2 during mitosis by the Phos-tag reagent after release from a double thymidine block. HeLa cells stably infected with a retrovirus encoding Flag-TIN2 in the WT, S330A, or S295A configuration were collected from asynchronous populations (A), populations arrested with a double thymidine block corresponding to the G1/S phase of the cell cycle, or populations at the points corresponding to S, G2, M and early or middle G1 (EG1 or MG1) after release from the double thymidine block. Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP) are denoted on the left of the upper panels. Left and right panels are different exposures. Representative of two experiments. ( B ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in cells arrested with nocodazole. HeLa cells stably infected with a retrovirus encoding no transgene (vector, V) or Flag-TIN2 in the WT, S330A, S295A, or AA configuration were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then subjected to either ( top ) immunoprecipitation (IP) with αFlag and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left of the upper panel. Representative of three experiments. ( C ) Detection of S295 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. Lysates from HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc), resolved by SDS-PAGE and immunoblotted (IB) with an anti-Phos-S295, anti-TIN2, anti-Phos-HH3, or anti-HH3 (loading control) antibody. Representative of two experiments. ( D ) Detection of S330 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then either subjected to ( top ) immunoprecipitation (IP) with an anti-TIN2 antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with either an anti-Phos-S330 or anti-TIN2 antibody, or ( bottom ) resolved by normal SDS-PAGE and immunoblotted with an anti-Phos-HH3 antibody, to monitor cell cycle progression, or an anti-Tubulin antibody as a loading control. Representative of one experiment.

    Article Snippet: Cell Synchronization by Nocodazole Arrest and Double Thymidine Block 106 of the described stably infected HeLa cells were either left untreated, arrested in G2/M by overnight treatment with 0.6 µg/ml nocodazole (catalogue # M1404, Sigma), or synchronized at G1/S by the double thymidine block, as outlined below.

    Techniques: Blocking Assay, Stable Transfection, Infection, Derivative Assay, Immunoprecipitation, SDS Page, Plasmid Preparation

    TIN2 is phosphorylated by the mitotic kinase RSK2. ( A ) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. ( B ) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. ( C ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. ( D ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2 Y707A ) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left ( top ). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control ( bottom ). Representative of two experiments. ( E ) Detection of TIN2 phosphorylation by RSK2 in vitro . Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP 32 , after which the reaction products were resolved by SDS-PAGE and either ( top ) exposed to autographic film or ( bottom ) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P 32 ) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.

    Journal: PLoS ONE

    Article Title: Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2

    doi: 10.1371/journal.pone.0071697

    Figure Lengend Snippet: TIN2 is phosphorylated by the mitotic kinase RSK2. ( A ) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. ( B ) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. ( C ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. ( D ) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2 Y707A ) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left ( top ). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control ( bottom ). Representative of two experiments. ( E ) Detection of TIN2 phosphorylation by RSK2 in vitro . Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP 32 , after which the reaction products were resolved by SDS-PAGE and either ( top ) exposed to autographic film or ( bottom ) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P 32 ) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.

    Article Snippet: Cell Synchronization by Nocodazole Arrest and Double Thymidine Block 106 of the described stably infected HeLa cells were either left untreated, arrested in G2/M by overnight treatment with 0.6 µg/ml nocodazole (catalogue # M1404, Sigma), or synchronized at G1/S by the double thymidine block, as outlined below.

    Techniques: Stable Transfection, Expressing, Derivative Assay, Immunoprecipitation, SDS Page, Staining, Fluorescence, FACS, Transfection, Mutagenesis, Activity Assay, In Vitro, Recombinant, Binding Assay, Purification, Incubation

    Characterization of HeLa cells cytosolic DUBs acting on human UBB. ( a ) Total cytosolic proteins (lane C) and the corresponding SEC fractions (lanes 1–16) were reacted with HA-UbVME and analyzed by SDS-PAGE/western blot with an anti-HA antibody. The asterisk and arrows a-c indicate HA-UbVME-reactive DUBs that co-elute with enzyme activities described in the main text. The elution positions of molecular mass protein standards, as well as the void volume (V 0 ), are indicated. ( b ) SDS-PAGE/Coomassie blue staining analysis of UBB processing by SEC fractions (upper panel). Lane C, total cytosolic proteins were also assayed. The elution profiles of USP5 (middle panel) and Otulin (lower panel) are also shown. ( c ) Fraction 8 from SEC was subjected to an immunoprecipitation/immunodepletion assay using control (lanes Ctrl) or anti-USP5 IgGs (lanes USP5). Fraction 8 (F8) and the corresponding immunoprecipitated (lanes IP) and immunodepleted fractions (lanes ID) were assayed for UBB processing activity (upper panel). The distribution of USP5 in the samples is shown (lower panel). ( d ) Otulin knockdown in HeLa cells. Western blot analysis of Otulin in HeLa cells transfected with Otulin-specific siRNA oligos #1 and/or #2, or with a scrambled (sc) control. The asterisk indicates a protein recognized by the anti-Otulin antibody in total homogenates, but not in cytosolic fractions. The corresponding Ponceau S-stained membrane is shown to assess protein loadings. ( e ) OTULIN knocked-down HeLa cell total extracts have decreased HA-UbVME-insensitive UBB processing activity. Inhib, HA-UbVME or HA-Ubal. In ( b , c , e ) the cleavage intermediate (Ub 2 ), and ubiquitin (Ub) are indicated. Lanes I, recombinant UBB used in the assays. In ( a – c , e ) numbers to the left indicate the molecular weights of protein standards in kDa.

    Journal: Scientific Reports

    Article Title: The de novo synthesis of ubiquitin: identification of deubiquitinases acting on ubiquitin precursors

    doi: 10.1038/srep12836

    Figure Lengend Snippet: Characterization of HeLa cells cytosolic DUBs acting on human UBB. ( a ) Total cytosolic proteins (lane C) and the corresponding SEC fractions (lanes 1–16) were reacted with HA-UbVME and analyzed by SDS-PAGE/western blot with an anti-HA antibody. The asterisk and arrows a-c indicate HA-UbVME-reactive DUBs that co-elute with enzyme activities described in the main text. The elution positions of molecular mass protein standards, as well as the void volume (V 0 ), are indicated. ( b ) SDS-PAGE/Coomassie blue staining analysis of UBB processing by SEC fractions (upper panel). Lane C, total cytosolic proteins were also assayed. The elution profiles of USP5 (middle panel) and Otulin (lower panel) are also shown. ( c ) Fraction 8 from SEC was subjected to an immunoprecipitation/immunodepletion assay using control (lanes Ctrl) or anti-USP5 IgGs (lanes USP5). Fraction 8 (F8) and the corresponding immunoprecipitated (lanes IP) and immunodepleted fractions (lanes ID) were assayed for UBB processing activity (upper panel). The distribution of USP5 in the samples is shown (lower panel). ( d ) Otulin knockdown in HeLa cells. Western blot analysis of Otulin in HeLa cells transfected with Otulin-specific siRNA oligos #1 and/or #2, or with a scrambled (sc) control. The asterisk indicates a protein recognized by the anti-Otulin antibody in total homogenates, but not in cytosolic fractions. The corresponding Ponceau S-stained membrane is shown to assess protein loadings. ( e ) OTULIN knocked-down HeLa cell total extracts have decreased HA-UbVME-insensitive UBB processing activity. Inhib, HA-UbVME or HA-Ubal. In ( b , c , e ) the cleavage intermediate (Ub 2 ), and ubiquitin (Ub) are indicated. Lanes I, recombinant UBB used in the assays. In ( a – c , e ) numbers to the left indicate the molecular weights of protein standards in kDa.

    Article Snippet: Subcellular fractionation and SEC HeLa cell total extracts were obtained by sonication in SEM buffer (0.25 M sucrose, 20 mM MOPS-KOH, pH 7.4, 1 mM EDTA-NaOH, pH 8.0, 1 mM DTT) containing 0.1% (w/v) Triton X-100, 0.1 mg/ml PMSF and 1:500 (v/v) mammalian protease inhibitor cocktail (Sigma).

    Techniques: Size-exclusion Chromatography, SDS Page, Western Blot, Staining, Immunoprecipitation, Activity Assay, Transfection, Inhibition, Recombinant

    Characterization of HeLa cells cytosolic DUBs acting on Ub-RPs. ( a ) SDS-PAGE/Coomassie blue staining analyses of UBA52 and UBA80 processing by HeLa cells cytosolic SEC fractions (first and second panels, respectively). Lane C, total cytosolic proteins were also assayed. Ubiquitin (Ub) and the ribosomal proteins L40 and S27A are indicated. The elution profiles of USP9X, USP7 and UCHL3 are also shown. The protein standards used to calibrate the column, as well as the void volume (V 0 ), are indicated. ( b ) Western blot analysis of UCHL3 in HeLa cells transfected with UCHL3-specific siRNA oligos #1 and/or #2, or with a scrambled (sc) control. The corresponding Ponceau S-stained membrane is shown to assess protein loadings. ( c ) SEC profiles of UBA52-cleavage activity in control (scrambled) and UCHL3 knocked-down (siUCHL3) HeLa cells cytosol. ( d ) Pooled SEC fractions 4 and 5 were subjected to an immunoprecipitation/immunodepletion assay using control (lanes Ctrl) or anti-USP9X IgGs (lanes USP9X). Pooled fractions (4 + 5) and the corresponding immunoprecipitated (lanes IP) and immunodepleted fractions (lanes ID) were assayed for UBA52 cleavage (upper panel). The distribution of USP9X in the samples is shown (lower panel). ( e ) Pooled fractions 6 and 7 (6 + 7) from SEC were incubated with HA-UbVME (lane +) or with HA-Ub (lane -) and subjected to immunoprecipitation using anti-HA-conjugated beads. A Coomassie blue-stained gel is shown. Numbers indicate protein bands of interest that were subjected to mass spectrometry (see Supplementary Table S1 ). ( f ) Immunoprecipitation/immunodepletion assay exactly as described in ( d ) but using pooled SEC fractions 6 and 7, and anti-USP7 IgGs. Lanes I in ( a , c , d , f ), recombinant UBA52 or UBA80, as indicated. In ( a , c – e ) numbers to the left indicate the molecular weights of protein standards in kDa.

    Journal: Scientific Reports

    Article Title: The de novo synthesis of ubiquitin: identification of deubiquitinases acting on ubiquitin precursors

    doi: 10.1038/srep12836

    Figure Lengend Snippet: Characterization of HeLa cells cytosolic DUBs acting on Ub-RPs. ( a ) SDS-PAGE/Coomassie blue staining analyses of UBA52 and UBA80 processing by HeLa cells cytosolic SEC fractions (first and second panels, respectively). Lane C, total cytosolic proteins were also assayed. Ubiquitin (Ub) and the ribosomal proteins L40 and S27A are indicated. The elution profiles of USP9X, USP7 and UCHL3 are also shown. The protein standards used to calibrate the column, as well as the void volume (V 0 ), are indicated. ( b ) Western blot analysis of UCHL3 in HeLa cells transfected with UCHL3-specific siRNA oligos #1 and/or #2, or with a scrambled (sc) control. The corresponding Ponceau S-stained membrane is shown to assess protein loadings. ( c ) SEC profiles of UBA52-cleavage activity in control (scrambled) and UCHL3 knocked-down (siUCHL3) HeLa cells cytosol. ( d ) Pooled SEC fractions 4 and 5 were subjected to an immunoprecipitation/immunodepletion assay using control (lanes Ctrl) or anti-USP9X IgGs (lanes USP9X). Pooled fractions (4 + 5) and the corresponding immunoprecipitated (lanes IP) and immunodepleted fractions (lanes ID) were assayed for UBA52 cleavage (upper panel). The distribution of USP9X in the samples is shown (lower panel). ( e ) Pooled fractions 6 and 7 (6 + 7) from SEC were incubated with HA-UbVME (lane +) or with HA-Ub (lane -) and subjected to immunoprecipitation using anti-HA-conjugated beads. A Coomassie blue-stained gel is shown. Numbers indicate protein bands of interest that were subjected to mass spectrometry (see Supplementary Table S1 ). ( f ) Immunoprecipitation/immunodepletion assay exactly as described in ( d ) but using pooled SEC fractions 6 and 7, and anti-USP7 IgGs. Lanes I in ( a , c , d , f ), recombinant UBA52 or UBA80, as indicated. In ( a , c – e ) numbers to the left indicate the molecular weights of protein standards in kDa.

    Article Snippet: Subcellular fractionation and SEC HeLa cell total extracts were obtained by sonication in SEM buffer (0.25 M sucrose, 20 mM MOPS-KOH, pH 7.4, 1 mM EDTA-NaOH, pH 8.0, 1 mM DTT) containing 0.1% (w/v) Triton X-100, 0.1 mg/ml PMSF and 1:500 (v/v) mammalian protease inhibitor cocktail (Sigma).

    Techniques: SDS Page, Staining, Size-exclusion Chromatography, Western Blot, Transfection, Activity Assay, Immunoprecipitation, Incubation, Mass Spectrometry, Recombinant

    Cx26 immunostaining performed in Hela DH transfectants overexpressing Cx26WT (top), and Cx26C169Y (bottom) proteins. Through focus confocal image sequence (z-stack) taken at 0.5 µm intervals of Hela DH cells expressing Cx26WT ( A – D ) and Cx26C169Y ( F – I ) proteins and their respective maximal projection rendering ( E and J ). Yellow arrow points to representative gap junction plaque, whereas red arrows indicate immunoreaction signals at the cell plasma membrane level, which are most likely due to unpaired connexons. Scale bar, 10 µm.

    Journal: Human Molecular Genetics

    Article Title: The p.Cys169Tyr variant of connexin 26 is not a polymorphism

    doi: 10.1093/hmg/ddv026

    Figure Lengend Snippet: Cx26 immunostaining performed in Hela DH transfectants overexpressing Cx26WT (top), and Cx26C169Y (bottom) proteins. Through focus confocal image sequence (z-stack) taken at 0.5 µm intervals of Hela DH cells expressing Cx26WT ( A – D ) and Cx26C169Y ( F – I ) proteins and their respective maximal projection rendering ( E and J ). Yellow arrow points to representative gap junction plaque, whereas red arrows indicate immunoreaction signals at the cell plasma membrane level, which are most likely due to unpaired connexons. Scale bar, 10 µm.

    Article Snippet: Cx26 immunoreactivity in Hela cells Hela DH cells (Sigma, cat. 96112022) were grown in DMEM (Gibco, catalog no. 41965-039) supplemented with 10% FBS (Gibco, catalog no. 10106169), plated on glass coverslips.

    Techniques: Immunostaining, Sequencing, Expressing

    GRK5 phosphorylates NPM1-Ser4 in HeLa cells. A , in vitro phosphorylation, detected by anti-pS4 NPM1 antibody, of purified 1 μ m NPM1 with 50 n m GRK5 or 50 n m PLK1 in the presence or absence of 60 n m BI 2536 at t = 30 min is shown. The blot is representative

    Journal: The Journal of Biological Chemistry

    Article Title: G Protein-coupled Receptor Kinase 5 Phosphorylates Nucleophosmin and Regulates Cell Sensitivity to Polo-like Kinase 1 Inhibition *

    doi: 10.1074/jbc.M112.353854

    Figure Lengend Snippet: GRK5 phosphorylates NPM1-Ser4 in HeLa cells. A , in vitro phosphorylation, detected by anti-pS4 NPM1 antibody, of purified 1 μ m NPM1 with 50 n m GRK5 or 50 n m PLK1 in the presence or absence of 60 n m BI 2536 at t = 30 min is shown. The blot is representative

    Article Snippet: To create stable HeLa cell lines, shRNA constructs specific for GRK5 along with a scrambled control shRNA were purchased from Sigma, and 10 μg of plasmid were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen).

    Techniques: In Vitro, Purification

    Characterization of NPM1 phosphorylation by GRK5. A , shown is purified NPM1 from a nucleolar preparation of HeLa cells compared with a crude nucleolar preparation on a Coomassie-stained polyacrylamide gel ( left panel ). Phosphorylation of nucleolar-derived

    Journal: The Journal of Biological Chemistry

    Article Title: G Protein-coupled Receptor Kinase 5 Phosphorylates Nucleophosmin and Regulates Cell Sensitivity to Polo-like Kinase 1 Inhibition *

    doi: 10.1074/jbc.M112.353854

    Figure Lengend Snippet: Characterization of NPM1 phosphorylation by GRK5. A , shown is purified NPM1 from a nucleolar preparation of HeLa cells compared with a crude nucleolar preparation on a Coomassie-stained polyacrylamide gel ( left panel ). Phosphorylation of nucleolar-derived

    Article Snippet: To create stable HeLa cell lines, shRNA constructs specific for GRK5 along with a scrambled control shRNA were purchased from Sigma, and 10 μg of plasmid were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen).

    Techniques: Purification, Staining, Derivative Assay

    NPM1 co-immunoprecipitates with GRK5 and their interaction is mediated by their respective N termini. A , shown is an immunoblot ( IB ) for GRK5 in HeLa cells stably transfected with control ( C ) or GRK5 ( 5 ) shRNA. B , detection of GRK5 in anti-NPM1 or anti-Ig2a

    Journal: The Journal of Biological Chemistry

    Article Title: G Protein-coupled Receptor Kinase 5 Phosphorylates Nucleophosmin and Regulates Cell Sensitivity to Polo-like Kinase 1 Inhibition *

    doi: 10.1074/jbc.M112.353854

    Figure Lengend Snippet: NPM1 co-immunoprecipitates with GRK5 and their interaction is mediated by their respective N termini. A , shown is an immunoblot ( IB ) for GRK5 in HeLa cells stably transfected with control ( C ) or GRK5 ( 5 ) shRNA. B , detection of GRK5 in anti-NPM1 or anti-Ig2a

    Article Snippet: To create stable HeLa cell lines, shRNA constructs specific for GRK5 along with a scrambled control shRNA were purchased from Sigma, and 10 μg of plasmid were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen).

    Techniques: Stable Transfection, Transfection, shRNA

    The activation of MAPKs following infection with ΔE3L vaccinia virus occurs through RLR signaling. Whole-cell extracts were prepared from parental PKR + HeLa cells, either uninfected (−) or infected with ΔE3L vaccinia virus

    Journal: Journal of Virology

    Article Title: Protein Kinase PKR-Dependent Activation of Mitogen-Activated Protein Kinases Occurs through Mitochondrial Adapter IPS-1 and Is Antagonized by Vaccinia Virus E3L ▿

    doi: 10.1128/JVI.00224-09

    Figure Lengend Snippet: The activation of MAPKs following infection with ΔE3L vaccinia virus occurs through RLR signaling. Whole-cell extracts were prepared from parental PKR + HeLa cells, either uninfected (−) or infected with ΔE3L vaccinia virus

    Article Snippet: HeLa cells with the stable knockdown of PKR (PKRkd cells) and PKR knockdown control HeLa cells with sufficient PKR (PKRkd-con cells) were described previously ( ); they were maintained in the above-described medium with the addition of 1 μg/ml puromycin (Sigma) ( ).

    Techniques: Activation Assay, Infection

    The expression of hPKR in PKR kd cells rescues MAPK activation by ΔE3L mutant infection. PKR kd HeLa cells were transfected with equimolar amounts of empty vector or the PKR WT (WT RSC ) or PKR catalytic mutant (K296R RSC ) expression construct. At

    Journal: Journal of Virology

    Article Title: Protein Kinase PKR-Dependent Activation of Mitogen-Activated Protein Kinases Occurs through Mitochondrial Adapter IPS-1 and Is Antagonized by Vaccinia Virus E3L ▿

    doi: 10.1128/JVI.00224-09

    Figure Lengend Snippet: The expression of hPKR in PKR kd cells rescues MAPK activation by ΔE3L mutant infection. PKR kd HeLa cells were transfected with equimolar amounts of empty vector or the PKR WT (WT RSC ) or PKR catalytic mutant (K296R RSC ) expression construct. At

    Article Snippet: HeLa cells with the stable knockdown of PKR (PKRkd cells) and PKR knockdown control HeLa cells with sufficient PKR (PKRkd-con cells) were described previously ( ); they were maintained in the above-described medium with the addition of 1 μg/ml puromycin (Sigma) ( ).

    Techniques: Expressing, Activation Assay, Mutagenesis, Infection, Transfection, Plasmid Preparation, Construct

    Ara C blocks PKR-dependent MAPK activation in ΔE3L mutant-infected cells. Whole-cell extracts were prepared from uninfected (−) or ΔE3L virus-infected (+) HeLa cells at 6 h after infection, either left untreated (−)

    Journal: Journal of Virology

    Article Title: Protein Kinase PKR-Dependent Activation of Mitogen-Activated Protein Kinases Occurs through Mitochondrial Adapter IPS-1 and Is Antagonized by Vaccinia Virus E3L ▿

    doi: 10.1128/JVI.00224-09

    Figure Lengend Snippet: Ara C blocks PKR-dependent MAPK activation in ΔE3L mutant-infected cells. Whole-cell extracts were prepared from uninfected (−) or ΔE3L virus-infected (+) HeLa cells at 6 h after infection, either left untreated (−)

    Article Snippet: HeLa cells with the stable knockdown of PKR (PKRkd cells) and PKR knockdown control HeLa cells with sufficient PKR (PKRkd-con cells) were described previously ( ); they were maintained in the above-described medium with the addition of 1 μg/ml puromycin (Sigma) ( ).

    Techniques: Acetylene Reduction Assay, Activation Assay, Mutagenesis, Infection

    PKR-dependent MAPK activation is antagonized by the vaccinia virus E3L protein. Whole-cell extracts were prepared from uninfected HeLa cells (mock) or HeLa cells infected with either WT vaccinia virus or one of the following E3L mutant viruses at 6 h

    Journal: Journal of Virology

    Article Title: Protein Kinase PKR-Dependent Activation of Mitogen-Activated Protein Kinases Occurs through Mitochondrial Adapter IPS-1 and Is Antagonized by Vaccinia Virus E3L ▿

    doi: 10.1128/JVI.00224-09

    Figure Lengend Snippet: PKR-dependent MAPK activation is antagonized by the vaccinia virus E3L protein. Whole-cell extracts were prepared from uninfected HeLa cells (mock) or HeLa cells infected with either WT vaccinia virus or one of the following E3L mutant viruses at 6 h

    Article Snippet: HeLa cells with the stable knockdown of PKR (PKRkd cells) and PKR knockdown control HeLa cells with sufficient PKR (PKRkd-con cells) were described previously ( ); they were maintained in the above-described medium with the addition of 1 μg/ml puromycin (Sigma) ( ).

    Techniques: Activation Assay, Infection, Mutagenesis

    dsRNA-mediated activation of MAPK phosphorylation is enhanced by PKR. Whole-cell extracts were prepared from cells with sufficient PKR (PKR + and PKR kd-con cells) and PKR kd HeLa cells that were untreated (mock), transfected with 3 μg/ml

    Journal: Journal of Virology

    Article Title: Protein Kinase PKR-Dependent Activation of Mitogen-Activated Protein Kinases Occurs through Mitochondrial Adapter IPS-1 and Is Antagonized by Vaccinia Virus E3L ▿

    doi: 10.1128/JVI.00224-09

    Figure Lengend Snippet: dsRNA-mediated activation of MAPK phosphorylation is enhanced by PKR. Whole-cell extracts were prepared from cells with sufficient PKR (PKR + and PKR kd-con cells) and PKR kd HeLa cells that were untreated (mock), transfected with 3 μg/ml

    Article Snippet: HeLa cells with the stable knockdown of PKR (PKRkd cells) and PKR knockdown control HeLa cells with sufficient PKR (PKRkd-con cells) were described previously ( ); they were maintained in the above-described medium with the addition of 1 μg/ml puromycin (Sigma) ( ).

    Techniques: Activation Assay, Transfection

    In Situ Analysis of Kinetics of p24 Loss from Cytoplasmic HIV HeLa cells were spinoculated with VSV-g pseudotyped, S15-mCherry, GFP-Vpr labeled HIV-1ΔEnv virions for 2 h at 17 °C. Infection was synchronized by washing off innocula and replaced with 37 °C media. HeLa cells were then fixed and immunostained for p24 CA (Cy-5) at the indicated time post infection and imaged. GFP (+) pucnta were then quantified and individually examined for the presence of mCherry and Cy-5 (p24 CA ) signal. The identity of the samples was blinded before the experiment. The percentage of the total number of virions that have stained for p24 CA over time following fusion is shown. The 0-h time point represents total number of GFP (+) virions that stained positive for p24 CA . HIV WT is represented by black lines/circles, the Q63/67A CA mutant by red line/diamonds, and the T54A/N57A CA mutant by blue line/triangles. The results shown are from three independently performed experiments and the standard deviation at each time point is shown.

    Journal: PLoS Pathogens

    Article Title: Evidence for Direct Involvement of the Capsid Protein in HIV Infection of Nondividing Cells

    doi: 10.1371/journal.ppat.0030156

    Figure Lengend Snippet: In Situ Analysis of Kinetics of p24 Loss from Cytoplasmic HIV HeLa cells were spinoculated with VSV-g pseudotyped, S15-mCherry, GFP-Vpr labeled HIV-1ΔEnv virions for 2 h at 17 °C. Infection was synchronized by washing off innocula and replaced with 37 °C media. HeLa cells were then fixed and immunostained for p24 CA (Cy-5) at the indicated time post infection and imaged. GFP (+) pucnta were then quantified and individually examined for the presence of mCherry and Cy-5 (p24 CA ) signal. The identity of the samples was blinded before the experiment. The percentage of the total number of virions that have stained for p24 CA over time following fusion is shown. The 0-h time point represents total number of GFP (+) virions that stained positive for p24 CA . HIV WT is represented by black lines/circles, the Q63/67A CA mutant by red line/diamonds, and the T54A/N57A CA mutant by blue line/triangles. The results shown are from three independently performed experiments and the standard deviation at each time point is shown.

    Article Snippet: For infections, HeLa target cells were seeded on fibronectin-treated coverslips and spinoculated for 2 h at 17 °C in the presence or absence of Bafilomycin A (Sigma).

    Techniques: In Situ, Labeling, Infection, Staining, Mutagenesis, Standard Deviation

    EV71 induces tSG formation via the PKR-eIF2α pathway. (A) KD of PKR effects on EV71-2A C110S -induced phosphorylation of PKR and eIF2α. shNC- and shPKR-HeLa cells were infected with EV71 (MOI = 1, as control) or EV71-2A C110S (MOI = 3) for 12 h and subjected to WB. Tubulin served as a loading control. (B) KD of PKR effects on EV71-2A C110S -induced tSG formation. Cells were infected as in A and subjected to IF assay. “+” indicates the infected cells, and yellow arrows indicate the uninfected cells.(C) Quantitative analysis of cells with TIA-1 foci in B. n = 3, 300 cells/condition were counted, mean±SD; n.s., no statistical significance, ***p

    Journal: PLoS Pathogens

    Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

    doi: 10.1371/journal.ppat.1006901

    Figure Lengend Snippet: EV71 induces tSG formation via the PKR-eIF2α pathway. (A) KD of PKR effects on EV71-2A C110S -induced phosphorylation of PKR and eIF2α. shNC- and shPKR-HeLa cells were infected with EV71 (MOI = 1, as control) or EV71-2A C110S (MOI = 3) for 12 h and subjected to WB. Tubulin served as a loading control. (B) KD of PKR effects on EV71-2A C110S -induced tSG formation. Cells were infected as in A and subjected to IF assay. “+” indicates the infected cells, and yellow arrows indicate the uninfected cells.(C) Quantitative analysis of cells with TIA-1 foci in B. n = 3, 300 cells/condition were counted, mean±SD; n.s., no statistical significance, ***p

    Article Snippet: Other stably expressing cells (eIF4GI-HA-HeLa, eIF4GIG689E -HA-HeLa), cells with KD of PKR (shPKR-HeLa) or negative control (shNC-HeLa) cells derived from HeLa cells were maintained in DMEM with 10% FBS, 100 U/ml penicillin/streptomycin, and 1 μg/ml puromycin (Sigma-Aldrich) at 37°C and 5% CO2 .

    Techniques: Infection, Western Blot

    2A induces aSGs to stall host mRNAs and release viral mRNAs. (A-C) Effects of EV71-induced aSGs and EV71 C110S -induced tSGs on viral translation. Graphic description of EV71-UTR driven Renilla luciferase reporter expression (A) and reporter assays in HeLa cells (B and C). Cells were infected with EV71 for 3h to induce aSG formation or infected with EV71-2A C110S for 6h to induce tSG formation before transfection with UTR EV71 -Rluc mRNAs, and the luciferase activity was measured at indicated time post-transfection. The EV71-2A C110S -infected eIF4GI/eIF4GI G689E -HA-HeLa cells and EV71-infected shNC/shPKR-HeLa cells were negative controls. n = 3, mean±SD, n.s., no statistical significance; *p

    Journal: PLoS Pathogens

    Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

    doi: 10.1371/journal.ppat.1006901

    Figure Lengend Snippet: 2A induces aSGs to stall host mRNAs and release viral mRNAs. (A-C) Effects of EV71-induced aSGs and EV71 C110S -induced tSGs on viral translation. Graphic description of EV71-UTR driven Renilla luciferase reporter expression (A) and reporter assays in HeLa cells (B and C). Cells were infected with EV71 for 3h to induce aSG formation or infected with EV71-2A C110S for 6h to induce tSG formation before transfection with UTR EV71 -Rluc mRNAs, and the luciferase activity was measured at indicated time post-transfection. The EV71-2A C110S -infected eIF4GI/eIF4GI G689E -HA-HeLa cells and EV71-infected shNC/shPKR-HeLa cells were negative controls. n = 3, mean±SD, n.s., no statistical significance; *p

    Article Snippet: Other stably expressing cells (eIF4GI-HA-HeLa, eIF4GIG689E -HA-HeLa), cells with KD of PKR (shPKR-HeLa) or negative control (shNC-HeLa) cells derived from HeLa cells were maintained in DMEM with 10% FBS, 100 U/ml penicillin/streptomycin, and 1 μg/ml puromycin (Sigma-Aldrich) at 37°C and 5% CO2 .

    Techniques: Luciferase, Expressing, Infection, Transfection, Activity Assay

    Functional characterization of shRNA-expressing plasmids . (A) HeLa cells were co-transfected with 50 ng of plasmids expressing shRNA targeting firefly luciferase, 200 ng of target pGL2 plasmid and 1 ng of phRL-SV40. Firefly luciferase (FF) activity normalized according to non-targeted Renilla luciferase activity is shown. Firefly luciferase activity in control sample (without a shRNA-expressing plasmid) was set to 1. Values are expressed as mean +/- SEM from samples transfected at least in triplicates. (B) pTMP and pLMP plasmids carrying loxP sites were transformed either to regular or Cre recombinase-expressing E. coli strains. Electrophoresis of isolated plasmid DNA is shown. The recombined plasmid after Cre-mediated recombination is marked by an arrow. (C) HeLa and HEK293 cells were co-transfected with 10-200 ng of plasmids expressing shRNA targeting firefly luciferase, 200 ng of target pGL2 plasmid, and 1 ng of phRL-SV40. Relative firefly luciferase activity compared to control cells is shown. Firefly luciferase activity in the control sample (omitting shRNA-expressing plasmid) was set to 1. Values are expressed as mean +/- SEM from samples transfected at least in triplicates.

    Journal: Journal of Negative Results in Biomedicine

    Article Title: Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

    doi: 10.1186/1477-5751-9-8

    Figure Lengend Snippet: Functional characterization of shRNA-expressing plasmids . (A) HeLa cells were co-transfected with 50 ng of plasmids expressing shRNA targeting firefly luciferase, 200 ng of target pGL2 plasmid and 1 ng of phRL-SV40. Firefly luciferase (FF) activity normalized according to non-targeted Renilla luciferase activity is shown. Firefly luciferase activity in control sample (without a shRNA-expressing plasmid) was set to 1. Values are expressed as mean +/- SEM from samples transfected at least in triplicates. (B) pTMP and pLMP plasmids carrying loxP sites were transformed either to regular or Cre recombinase-expressing E. coli strains. Electrophoresis of isolated plasmid DNA is shown. The recombined plasmid after Cre-mediated recombination is marked by an arrow. (C) HeLa and HEK293 cells were co-transfected with 10-200 ng of plasmids expressing shRNA targeting firefly luciferase, 200 ng of target pGL2 plasmid, and 1 ng of phRL-SV40. Relative firefly luciferase activity compared to control cells is shown. Firefly luciferase activity in the control sample (omitting shRNA-expressing plasmid) was set to 1. Values are expressed as mean +/- SEM from samples transfected at least in triplicates.

    Article Snippet: Transformed cell lines HeLa, HEK293, and NIH3T3 cells were cultured in Dulbecco's Modified Eagle medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Gibco), Penicillin 100 U/ml and Streptomycin 100 μg/ml (Gibco).

    Techniques: Functional Assay, shRNA, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Transformation Assay, Electrophoresis, Isolation

    (a) Growth profile for P. stuartii NK (37°C, LB). (b) Adherence of bacteria P. stuartii NK to HeLa-M cells, depending on the age of the bacterial culture. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 50 bacterial cells to 1 eukaryotic cell for 2 hours at 37°C.

    Journal: BioMed Research International

    Article Title: The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion

    doi: 10.1155/2018/3589135

    Figure Lengend Snippet: (a) Growth profile for P. stuartii NK (37°C, LB). (b) Adherence of bacteria P. stuartii NK to HeLa-M cells, depending on the age of the bacterial culture. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 50 bacterial cells to 1 eukaryotic cell for 2 hours at 37°C.

    Article Snippet: Scanning Electron Microscopy For SEM, HeLa-M cells were grown on glass coverslips and infected with bacteria for 30–120 min at 37°C and subsequently were washed and fixed in 2% glutaraldehyde in PBS buffer (Sigma, USA).

    Techniques: Infection

    Confocal microscopy of P. stuartii NK invasion into HeLa-M cells by confocal microscopy. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 50 bacterial cells to 1 eukaryotic cell for 2 hours at 37°C. (a) Control (HeLa-M cells were not infected with bacteria); (b) Experiment (HeLa-M cells were infected with bacteria for 120 minutes). Actin cytoskeleton was stained with rhodamine-phalloidin (red); DNA of cell nuclei and bacteria were stained with DAPI (blue). Inset shows intracellular bacteria (white arrows).

    Journal: BioMed Research International

    Article Title: The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion

    doi: 10.1155/2018/3589135

    Figure Lengend Snippet: Confocal microscopy of P. stuartii NK invasion into HeLa-M cells by confocal microscopy. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 50 bacterial cells to 1 eukaryotic cell for 2 hours at 37°C. (a) Control (HeLa-M cells were not infected with bacteria); (b) Experiment (HeLa-M cells were infected with bacteria for 120 minutes). Actin cytoskeleton was stained with rhodamine-phalloidin (red); DNA of cell nuclei and bacteria were stained with DAPI (blue). Inset shows intracellular bacteria (white arrows).

    Article Snippet: Scanning Electron Microscopy For SEM, HeLa-M cells were grown on glass coverslips and infected with bacteria for 30–120 min at 37°C and subsequently were washed and fixed in 2% glutaraldehyde in PBS buffer (Sigma, USA).

    Techniques: Confocal Microscopy, Infection, Staining

    Efficiency of P. stuartii NK invasion into HeLa-M cells depending on the multiplicity of infection, 12-hour culture. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at the different ratios of bacterial to eukaryotic cells for 2 hours at 37°C.

    Journal: BioMed Research International

    Article Title: The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion

    doi: 10.1155/2018/3589135

    Figure Lengend Snippet: Efficiency of P. stuartii NK invasion into HeLa-M cells depending on the multiplicity of infection, 12-hour culture. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at the different ratios of bacterial to eukaryotic cells for 2 hours at 37°C.

    Article Snippet: Scanning Electron Microscopy For SEM, HeLa-M cells were grown on glass coverslips and infected with bacteria for 30–120 min at 37°C and subsequently were washed and fixed in 2% glutaraldehyde in PBS buffer (Sigma, USA).

    Techniques: Infection

    SEM-images of P. stuartii NK adhesion onto HeLa-M cells. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 50 bacterial cells to 1 eukaryotic cell at 37°C. (a) Control (HeLa-M cells were not infected with bacteria); (b) 30 minutes of postinfection with P. stuartii NK; (c) 60 minutes of postinfection with P. stuartii NK. Magnification 15. 00 K X.

    Journal: BioMed Research International

    Article Title: The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion

    doi: 10.1155/2018/3589135

    Figure Lengend Snippet: SEM-images of P. stuartii NK adhesion onto HeLa-M cells. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 50 bacterial cells to 1 eukaryotic cell at 37°C. (a) Control (HeLa-M cells were not infected with bacteria); (b) 30 minutes of postinfection with P. stuartii NK; (c) 60 minutes of postinfection with P. stuartii NK. Magnification 15. 00 K X.

    Article Snippet: Scanning Electron Microscopy For SEM, HeLa-M cells were grown on glass coverslips and infected with bacteria for 30–120 min at 37°C and subsequently were washed and fixed in 2% glutaraldehyde in PBS buffer (Sigma, USA).

    Techniques: Infection

    Efficiency of P. stuartii NK invasion into HeLa-M cells depending on the stage of bacterial culture growth. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 100 bacterial cells to 1 eukaryotic cell for 2 hours at 37°C.

    Journal: BioMed Research International

    Article Title: The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion

    doi: 10.1155/2018/3589135

    Figure Lengend Snippet: Efficiency of P. stuartii NK invasion into HeLa-M cells depending on the stage of bacterial culture growth. Subconfluent HeLa-M monolayers were infected with P. stuartii NK at a ratio of 100 bacterial cells to 1 eukaryotic cell for 2 hours at 37°C.

    Article Snippet: Scanning Electron Microscopy For SEM, HeLa-M cells were grown on glass coverslips and infected with bacteria for 30–120 min at 37°C and subsequently were washed and fixed in 2% glutaraldehyde in PBS buffer (Sigma, USA).

    Techniques: Infection

    Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. SAv-HRP, streptavidin–horseradish peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3

    Journal: Nature Communications

    Article Title: Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide

    doi: 10.1038/s41467-018-04343-0

    Figure Lengend Snippet: Target-selective protein labelling with LDNASA chemistry. a Western blotting analysis of FKBP12 labelling in cell lysates. HeLa cell lysate was mixed with recombinant FKBP12 (1 µM) and incubated with reagent in 50 mM HEPES buffer, pH 7.2, 37 °C, 1 h. SAv-HRP, streptavidin–horseradish peroxidase conjugate; CBB, Coomassie brilliant blue staining. The high background (smear) signals of lane 6 and 7 may suggest unspecific labelling due to a high concentration of reagent (20 µM), whereas such background signals were less in lanes 1–5 (1–5 µM of reagent). b Western blotting analysis of endogenous FKBP12 labelling in live C2C12 cells. The cells were treated with 1 or 5 (1 µM) for 0–120 min at 37 °C in medium. c Time course plot of the endogenous FKBP12 labelling with 1 . d Molecular structures of LDNASA 9 and LDAI 10 for FR labelling. e In-gel fluorescence and western blotting analysis of endogenous FR labelling in KB cells. The cells were treated with 9 or 10 (1 µM) for 0–60 min at 37 °C in medium. FA, Folic acid (competitive inhibitor). f Time profile of the endogenous FR labelling with 9 . I and I 30 min are band intensities of Oregon Green-labelled FR at the indicated time point and at 30 min, respectively. Error bars represent s.d., n = 3

    Article Snippet: FKBP12 labelling in HeLa cell lysate HeLa cells (kindly gifted from Prof. Yoshiki Katayama) (1×107 cells) were suspended in HEPES buffer (50 mM, pH 7.2) containing 1% protease inhibitor cocktail set III (Calbiochem) and lysed by Potter-Elvehjem homogeniser at 4 °C.

    Techniques: Western Blot, Recombinant, Incubation, Staining, Concentration Assay, Fluorescence

    DARPins selected to bind to lamin A can alter lamin assembly in vitro and in vivo. Left panels, lamin A in vitro assembly was performed in the absence of DARPins (no DARPin, buffer), in the presence of a control DARPin (E3_5) or in the presence of the indicated DARPins that specifically bound to human lamin A. Samples were centrifuged for 35 min at 50,000 g , and the supernatant (SN) and pellet (P) fractions were analyzed by SDS PAGE and staining with Coomassie Blue. Right panels, confocal images of HeLa-K cells that stably expressed DARPins and were immunostained for lamin A/C (red). DARPin-expressing cells were identified by the bicistronic expression of EGFP (not shown). Scale bar: 10 µm.

    Journal: Journal of Cell Science

    Article Title: Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins

    doi: 10.1242/jcs.171843

    Figure Lengend Snippet: DARPins selected to bind to lamin A can alter lamin assembly in vitro and in vivo. Left panels, lamin A in vitro assembly was performed in the absence of DARPins (no DARPin, buffer), in the presence of a control DARPin (E3_5) or in the presence of the indicated DARPins that specifically bound to human lamin A. Samples were centrifuged for 35 min at 50,000 g , and the supernatant (SN) and pellet (P) fractions were analyzed by SDS PAGE and staining with Coomassie Blue. Right panels, confocal images of HeLa-K cells that stably expressed DARPins and were immunostained for lamin A/C (red). DARPin-expressing cells were identified by the bicistronic expression of EGFP (not shown). Scale bar: 10 µm.

    Article Snippet: U2OS cells, HDFs and HeLa-K cells were modified through lentiviral infection [in the case of HeLa-K, cells were modified for stable knockdown of lamin A/C (Sigma mission, clone number .1-752s1c1) and for the respective scrambled control] using HEK-293T cells as packaging cells and the packaging vectors dMD2.G and psPAX2 (Addgene), and following the Purefection protocol for Lentiviral packaging (System Biosciences).

    Techniques: In Vitro, In Vivo, SDS Page, Staining, Stable Transfection, Expressing

    A-type lamins delocalize to the nucleoplasm post-mitotically and retain a high level of phosphorylation. (A) HeLa-K cells that stably expressed GFP–lamin-A were incubated with mCherry-tagged DARPins E3_5 or LaA_1, and Hoechst 33342 stain for up to 60 min, after brief incubation with Triton X–100 to allow penetration of the DARPins into the cells. Scale bar: 20 µm. (B) Live-cell imaging of HeLa-K cells that stably expressed GFP–laminA and had been transfected with mCherry–DARPin LaA_1. GFP–lamin-A reassociated with the lamina in non-transfected daughter cells (marked with asterisks), whereas expression of mCherry- LaA_1 blocked GFP–lamin-A reassembly after mitosis (marked with arrowheads). Scale bar: 20 µm. (C) Western blot analysis of wild-type and modified U2OS cells to detect A-type lamins, as well as A-type lamins phosphorylated at amino acid Ser22. Nocodazole-treated cells were used as a control for phosphorylation, β-actin was used as loading control. (D) Western blot analysis of wild-type and modified U2OS cells treated with cycloheximide for the indicated time points or DMSO to detect degradation of A-type lamins. β-actin was used as control for a stable protein, and the Coomassie-stained membrane shows the efficiency of cycloheximide treatment. In D, the upper band represents lamin A and the lower band represents lamin C.

    Journal: Journal of Cell Science

    Article Title: Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins

    doi: 10.1242/jcs.171843

    Figure Lengend Snippet: A-type lamins delocalize to the nucleoplasm post-mitotically and retain a high level of phosphorylation. (A) HeLa-K cells that stably expressed GFP–lamin-A were incubated with mCherry-tagged DARPins E3_5 or LaA_1, and Hoechst 33342 stain for up to 60 min, after brief incubation with Triton X–100 to allow penetration of the DARPins into the cells. Scale bar: 20 µm. (B) Live-cell imaging of HeLa-K cells that stably expressed GFP–laminA and had been transfected with mCherry–DARPin LaA_1. GFP–lamin-A reassociated with the lamina in non-transfected daughter cells (marked with asterisks), whereas expression of mCherry- LaA_1 blocked GFP–lamin-A reassembly after mitosis (marked with arrowheads). Scale bar: 20 µm. (C) Western blot analysis of wild-type and modified U2OS cells to detect A-type lamins, as well as A-type lamins phosphorylated at amino acid Ser22. Nocodazole-treated cells were used as a control for phosphorylation, β-actin was used as loading control. (D) Western blot analysis of wild-type and modified U2OS cells treated with cycloheximide for the indicated time points or DMSO to detect degradation of A-type lamins. β-actin was used as control for a stable protein, and the Coomassie-stained membrane shows the efficiency of cycloheximide treatment. In D, the upper band represents lamin A and the lower band represents lamin C.

    Article Snippet: U2OS cells, HDFs and HeLa-K cells were modified through lentiviral infection [in the case of HeLa-K, cells were modified for stable knockdown of lamin A/C (Sigma mission, clone number .1-752s1c1) and for the respective scrambled control] using HEK-293T cells as packaging cells and the packaging vectors dMD2.G and psPAX2 (Addgene), and following the Purefection protocol for Lentiviral packaging (System Biosciences).

    Techniques: Stable Transfection, Incubation, Staining, Live Cell Imaging, Transfection, Expressing, Western Blot, Modification

    Nuclear mechanical properties are impaired if A-type lamins are not incorporated into the lamina. (A) Quantitative analysis of nuclear roundness by assessing the nuclear contour ratio of wild-type HeLa-K cells and cells that stably expressed GFP–lamin-A at low and high expression levels (LaA OE 1 and LaA OE 2, respectively), a scrambled siRNA, an siRNA to induce LMNA silencing (LMNA kd), the control DARPin E3_5, or the indicated different lamin-A-specific DARPins. Values represent the average nuclear contour ratio of > 300 nuclei±s.e.m. (B) Changes in the nuclear shape of unstretched (pre-strain, upper panel) and fully stretched (full strain, middle panel) wild-type and the modified HeLa-K cells. Images in the lower panels represent nuclei under strain, overlaid with the nuclear contour from the pre-stretch state in red. Scale bar: 5 µm. (C) Statistical analysis of nuclear deformation of wild-type and modified HeLa-K cells. Values represent the average normalized nuclear strain (inferred from the ratio of induced nuclear strain to the applied membrane strain) of ≥100 nuclei±s.e.m. Statistical significance in A and C was calculated using a one-way ANOVA test followed by Bonferroni's multiple comparison test. A P -value of > 0.05 was considered as not significant (n.s.); *** P ≤0.001.

    Journal: Journal of Cell Science

    Article Title: Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins

    doi: 10.1242/jcs.171843

    Figure Lengend Snippet: Nuclear mechanical properties are impaired if A-type lamins are not incorporated into the lamina. (A) Quantitative analysis of nuclear roundness by assessing the nuclear contour ratio of wild-type HeLa-K cells and cells that stably expressed GFP–lamin-A at low and high expression levels (LaA OE 1 and LaA OE 2, respectively), a scrambled siRNA, an siRNA to induce LMNA silencing (LMNA kd), the control DARPin E3_5, or the indicated different lamin-A-specific DARPins. Values represent the average nuclear contour ratio of > 300 nuclei±s.e.m. (B) Changes in the nuclear shape of unstretched (pre-strain, upper panel) and fully stretched (full strain, middle panel) wild-type and the modified HeLa-K cells. Images in the lower panels represent nuclei under strain, overlaid with the nuclear contour from the pre-stretch state in red. Scale bar: 5 µm. (C) Statistical analysis of nuclear deformation of wild-type and modified HeLa-K cells. Values represent the average normalized nuclear strain (inferred from the ratio of induced nuclear strain to the applied membrane strain) of ≥100 nuclei±s.e.m. Statistical significance in A and C was calculated using a one-way ANOVA test followed by Bonferroni's multiple comparison test. A P -value of > 0.05 was considered as not significant (n.s.); *** P ≤0.001.

    Article Snippet: U2OS cells, HDFs and HeLa-K cells were modified through lentiviral infection [in the case of HeLa-K, cells were modified for stable knockdown of lamin A/C (Sigma mission, clone number .1-752s1c1) and for the respective scrambled control] using HEK-293T cells as packaging cells and the packaging vectors dMD2.G and psPAX2 (Addgene), and following the Purefection protocol for Lentiviral packaging (System Biosciences).

    Techniques: Stable Transfection, Expressing, Modification

    Over-expressed RelA peptide co-localizes with SETD6 and inhibits its catalytic activity in cells ( A ) The 15 amino acids (302-316) sequence of the RelA peptide. Methylated lysine residue (K310) is labeled in red. ( B ) MCF7 cells were transfected with increasing amounts of the Flag-RelA302-316 plasmid. 24 hours post-transfection, cells were harvested, RNA was extracted and used for a qPCR analysis of the indicated genes. mRNA levels were normalized to GAPDH and to control cells. Error bars represent triplicates. ( C ) HeLa or MDA-MB-231 cells were plated at 15,000 cells/well and transfected either with Flag-RelA302-316 or an empty plasmid. 48 hours after transfection viability was measured using the PrestoBlue reagent. Standard deviation represents quadruplicates of two independent experiments. HeLa cells were transfected with the indicated combinations of the GFP-RelA302-316, mCherry-H2B ( D ) and mCherry-SETD6 ( E ). 24 hours after transfection cells were visualized by fluorescent microscopy.

    Journal: Oncotarget

    Article Title: Peptide inhibition of the SETD6 methyltransferase catalytic activity

    doi: 10.18632/oncotarget.23591

    Figure Lengend Snippet: Over-expressed RelA peptide co-localizes with SETD6 and inhibits its catalytic activity in cells ( A ) The 15 amino acids (302-316) sequence of the RelA peptide. Methylated lysine residue (K310) is labeled in red. ( B ) MCF7 cells were transfected with increasing amounts of the Flag-RelA302-316 plasmid. 24 hours post-transfection, cells were harvested, RNA was extracted and used for a qPCR analysis of the indicated genes. mRNA levels were normalized to GAPDH and to control cells. Error bars represent triplicates. ( C ) HeLa or MDA-MB-231 cells were plated at 15,000 cells/well and transfected either with Flag-RelA302-316 or an empty plasmid. 48 hours after transfection viability was measured using the PrestoBlue reagent. Standard deviation represents quadruplicates of two independent experiments. HeLa cells were transfected with the indicated combinations of the GFP-RelA302-316, mCherry-H2B ( D ) and mCherry-SETD6 ( E ). 24 hours after transfection cells were visualized by fluorescent microscopy.

    Article Snippet: Cell lines, transfection and treatments HeLa cells, human embryonic kidney cells (HEK293T), A549 cells and MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) with 10% fetal bovine serum (Gibco), 2mg/ml L-glutamine (Sigma), penicillin-streptomycin (Sigma), and non-essential amino acids (Sigma).

    Techniques: Activity Assay, Sequencing, Methylation, Labeling, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Standard Deviation, Microscopy

    The vp22-RelA peptide penetrates cells and interacts with SETD6 FITC labeled vp22-RelA peptide was introduced into HEK293T ( A ) or A549 ( B ) cells transfected with mCherry-SETD6. Cells were fixed, mounted with DAPI containing mounting solution and visualized using confocal microscopy. Lysates of SETD6 KO HeLa cells stably expressing an empty plasmid or the Flag-SETD6 plasmid were immunoprecipitated with biotin-labeled vp22-RelA302-316 ( C ), or the stably expressing Flag-SETD6 HeLa KO SETD6 cells were immunoprecipitated with biotin-labeled vp22 or vp22-RelA302-316 ( D ) and blotted with Flag antibody to test Flag-SETD6 pull-down. Levels of Flag-SETD6 and GAPDH (loading control) are shown in the lower panels of (C and D).

    Journal: Oncotarget

    Article Title: Peptide inhibition of the SETD6 methyltransferase catalytic activity

    doi: 10.18632/oncotarget.23591

    Figure Lengend Snippet: The vp22-RelA peptide penetrates cells and interacts with SETD6 FITC labeled vp22-RelA peptide was introduced into HEK293T ( A ) or A549 ( B ) cells transfected with mCherry-SETD6. Cells were fixed, mounted with DAPI containing mounting solution and visualized using confocal microscopy. Lysates of SETD6 KO HeLa cells stably expressing an empty plasmid or the Flag-SETD6 plasmid were immunoprecipitated with biotin-labeled vp22-RelA302-316 ( C ), or the stably expressing Flag-SETD6 HeLa KO SETD6 cells were immunoprecipitated with biotin-labeled vp22 or vp22-RelA302-316 ( D ) and blotted with Flag antibody to test Flag-SETD6 pull-down. Levels of Flag-SETD6 and GAPDH (loading control) are shown in the lower panels of (C and D).

    Article Snippet: Cell lines, transfection and treatments HeLa cells, human embryonic kidney cells (HEK293T), A549 cells and MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) with 10% fetal bovine serum (Gibco), 2mg/ml L-glutamine (Sigma), penicillin-streptomycin (Sigma), and non-essential amino acids (Sigma).

    Techniques: Labeling, Transfection, Confocal Microscopy, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

    The vp22-RelA peptide blocks SETD6 catalytic activity in vitro and in cells (A+B) In vitro methylation assay in the presence of 3 H-labeled S-adenosyl methionine testing GST-SETD6 auto-methylation activity ( A ) or methylation of recombinant His-Plk1 by GST-SETD6 ( B ) while introducing increasing amounts of the vp22-RelA peptide. The vp22 peptide was used as negative control. Coomassie stain of the recombinant proteins used in the reactions is shown on the bottom of each experiment. ( C ) Methylated proteins were pulled-down from cellular extracts of HEK293T cells with His-MBT to detect differences in auto-methylation signal of endogenous SETD6 upon treatment with the vp22-RelA peptide. As negative control, pull-down was also performed with HEK293T cells treated with the vp22 peptide. ( D ) HeLa SETD6 KO cells stably expressing an empty plasmid or the Flag-SETD6 plasmid were treated or not with 40 uM of the vp22 or vp22-RelA302-316 peptides in serum free medium for 4 h, after which cells were washed and fresh complete medium was added overnight. Extracts were immunoprecipitated using pan-methyl antibody and blotted with Plk1 antibody. Endogenous levels of Plk1, SETD6 and GAPDH (loading control) are shown in the lower panel.

    Journal: Oncotarget

    Article Title: Peptide inhibition of the SETD6 methyltransferase catalytic activity

    doi: 10.18632/oncotarget.23591

    Figure Lengend Snippet: The vp22-RelA peptide blocks SETD6 catalytic activity in vitro and in cells (A+B) In vitro methylation assay in the presence of 3 H-labeled S-adenosyl methionine testing GST-SETD6 auto-methylation activity ( A ) or methylation of recombinant His-Plk1 by GST-SETD6 ( B ) while introducing increasing amounts of the vp22-RelA peptide. The vp22 peptide was used as negative control. Coomassie stain of the recombinant proteins used in the reactions is shown on the bottom of each experiment. ( C ) Methylated proteins were pulled-down from cellular extracts of HEK293T cells with His-MBT to detect differences in auto-methylation signal of endogenous SETD6 upon treatment with the vp22-RelA peptide. As negative control, pull-down was also performed with HEK293T cells treated with the vp22 peptide. ( D ) HeLa SETD6 KO cells stably expressing an empty plasmid or the Flag-SETD6 plasmid were treated or not with 40 uM of the vp22 or vp22-RelA302-316 peptides in serum free medium for 4 h, after which cells were washed and fresh complete medium was added overnight. Extracts were immunoprecipitated using pan-methyl antibody and blotted with Plk1 antibody. Endogenous levels of Plk1, SETD6 and GAPDH (loading control) are shown in the lower panel.

    Article Snippet: Cell lines, transfection and treatments HeLa cells, human embryonic kidney cells (HEK293T), A549 cells and MDA-MB-231 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) with 10% fetal bovine serum (Gibco), 2mg/ml L-glutamine (Sigma), penicillin-streptomycin (Sigma), and non-essential amino acids (Sigma).

    Techniques: Activity Assay, In Vitro, Methylation, Labeling, Recombinant, Negative Control, Staining, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

    ( A – C ) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide) and ( D – F ) Sulforhodamine B (SRB) and Trypan Blue (TB) assays were performed on control (SCR) and on RAB7Ai 2008, HeLa and A431 cells after treatment with cisplatin (CDDP) at the indicated concentrations. ( G – I ) C13 (mock) or C13 expressing HA-RAB7A cells, HeLa and A431 Pt control (mock) and expressing HA-RAB7A were either untreated or treated with CDDP at the indicated concentrations and cytotoxicity was analyzed with MTT assay. ( J ) C13 (mock) or expressing the HA-RAB7AQ67L mutant protein were either untreated or treated with CDDP for 24 h and subjected to MTT assay. ( K – M ) MTT assay was performed on control (SCR) and on RAB7Ai 2008, HeLa and A431 cells after treatment with CDDP for 48 h at the indicated concentrations. Each point represents mean ± SE of at least three independent experiments run in eight replicates. Statistical significance was measured by comparing values with values of control untreated cells. (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001).

    Journal: Cancers

    Article Title: Modulation of RAB7A Protein Expression Determines Resistance to Cisplatin through Late Endocytic Pathway Impairment and Extracellular Vesicular Secretion

    doi: 10.3390/cancers11010052

    Figure Lengend Snippet: ( A – C ) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide) and ( D – F ) Sulforhodamine B (SRB) and Trypan Blue (TB) assays were performed on control (SCR) and on RAB7Ai 2008, HeLa and A431 cells after treatment with cisplatin (CDDP) at the indicated concentrations. ( G – I ) C13 (mock) or C13 expressing HA-RAB7A cells, HeLa and A431 Pt control (mock) and expressing HA-RAB7A were either untreated or treated with CDDP at the indicated concentrations and cytotoxicity was analyzed with MTT assay. ( J ) C13 (mock) or expressing the HA-RAB7AQ67L mutant protein were either untreated or treated with CDDP for 24 h and subjected to MTT assay. ( K – M ) MTT assay was performed on control (SCR) and on RAB7Ai 2008, HeLa and A431 cells after treatment with CDDP for 48 h at the indicated concentrations. Each point represents mean ± SE of at least three independent experiments run in eight replicates. Statistical significance was measured by comparing values with values of control untreated cells. (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001).

    Article Snippet: Cells Lines HeLa cells (human cervix carcinoma) and their CDDP-resistant counterpart (HeLa Pt) were grown in DMEM (Sigma-Aldrich, St. Louis, MO, USA) while 2008 (human cervical cancer cells), A431(human cervix squamous carcinoma) and their CDDP-resistant sublines C13 and A431 Pt were maintained in RPMI-1640 medium (Sigma-Aldrich).

    Techniques: MTT Assay, Sulforhodamine B Assay, Expressing, Mutagenesis

    Effect of prolonged Cdk1 inhibition on Gwl dephosphorylation in Fcp1-depleted cells. Control (Cont.) or Fcp1-depleted (Fcp1) HeLa cells were arrested at prometaphase, split into two samples, one sample receiving vehicle (-), the other RO3306 (+), and further incubated for 30 min. Cell lysates were processed for mock (Mk) or Gwl IP that were then probed for indicated antigens. Data shown are representative of at least four independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.010

    Journal: eLife

    Article Title: Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    doi: 10.7554/eLife.10399

    Figure Lengend Snippet: Effect of prolonged Cdk1 inhibition on Gwl dephosphorylation in Fcp1-depleted cells. Control (Cont.) or Fcp1-depleted (Fcp1) HeLa cells were arrested at prometaphase, split into two samples, one sample receiving vehicle (-), the other RO3306 (+), and further incubated for 30 min. Cell lysates were processed for mock (Mk) or Gwl IP that were then probed for indicated antigens. Data shown are representative of at least four independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.010

    Article Snippet: For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO).

    Techniques: Inhibition, De-Phosphorylation Assay, Incubation

    PRC1 localization in Fcp1-depleted cells. ( A ) Asynchronously growing control (Cont. siRNAs) or Fcp1-siRNAs-treated (Fcp1 siRNAs) HeLa cells were fixed and stained for PRC1 (red), α-tubulin (green) and DNA (blue). ( B ) Control (Cont. siRNAs) or Fcp1-siRNAs-treated (Fcp1 siRNAs) HeLa cells, previously arrested at prometaphase, were released into fresh medium in the presence of the proteasome inhibitor MG132. After 30 min incubation, the Cdk1 inhibitor RO3306 was added, cells were fixed and stained for PRC1 (red), α-tubulin (green) and DNA (blue) at indicated time points of further incubation. Scale bars, 10 μm. Data shown are representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.10399.006

    Journal: eLife

    Article Title: Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    doi: 10.7554/eLife.10399

    Figure Lengend Snippet: PRC1 localization in Fcp1-depleted cells. ( A ) Asynchronously growing control (Cont. siRNAs) or Fcp1-siRNAs-treated (Fcp1 siRNAs) HeLa cells were fixed and stained for PRC1 (red), α-tubulin (green) and DNA (blue). ( B ) Control (Cont. siRNAs) or Fcp1-siRNAs-treated (Fcp1 siRNAs) HeLa cells, previously arrested at prometaphase, were released into fresh medium in the presence of the proteasome inhibitor MG132. After 30 min incubation, the Cdk1 inhibitor RO3306 was added, cells were fixed and stained for PRC1 (red), α-tubulin (green) and DNA (blue) at indicated time points of further incubation. Scale bars, 10 μm. Data shown are representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.10399.006

    Article Snippet: For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO).

    Techniques: Staining, Incubation

    Cdk1-dependent phosphorylations of Gwl. ( A ) Mock- (Mk), V5-GwlWT-, V5-GwlS90A- and V5-GwlS453A-transfected HeLa cells were arrested at prometaphase, lysed and processed for V5 IP. IPs were separated in parallel SDS/PAGE and probed for indicated antigens. ( B ). Mock- (Mk) and V5-GwlWT-transfected HeLa cells were arrested at prometaphase and released. Cells were taken after 120 min of incubation, to allow complete transition into the G1 cell cycle phase, and lysates processed for V5 IP. IPs were split into two portions and incubated in kinase reactions at 37°C for 20 min – or + active Cdk1 (Cyclin A-Cdk1, 126 ng per reaction), before being separated in parallel SDS/PAGE and probed for indicated antigens. Data shown are representative of at least four independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.009

    Journal: eLife

    Article Title: Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    doi: 10.7554/eLife.10399

    Figure Lengend Snippet: Cdk1-dependent phosphorylations of Gwl. ( A ) Mock- (Mk), V5-GwlWT-, V5-GwlS90A- and V5-GwlS453A-transfected HeLa cells were arrested at prometaphase, lysed and processed for V5 IP. IPs were separated in parallel SDS/PAGE and probed for indicated antigens. ( B ). Mock- (Mk) and V5-GwlWT-transfected HeLa cells were arrested at prometaphase and released. Cells were taken after 120 min of incubation, to allow complete transition into the G1 cell cycle phase, and lysates processed for V5 IP. IPs were split into two portions and incubated in kinase reactions at 37°C for 20 min – or + active Cdk1 (Cyclin A-Cdk1, 126 ng per reaction), before being separated in parallel SDS/PAGE and probed for indicated antigens. Data shown are representative of at least four independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.009

    Article Snippet: For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO).

    Techniques: Transfection, SDS Page, Incubation

    Fcp1 affects Ensa/ARPP19 kinase ability of Gwl. ( A ) V5 IP from V5-GwlWT-transfected, prometaphase-arrested, HeLa cells was divided into three sets and incubated for phosphatase reactions with buffer (Cont.), Fcp1WT or Fcp1CD proteins (Mk IP; 1/3 mock IP incubated with buffer). IPs were washed, each set split into three portions and incubated in kinase reactions at 37°C with recombinant Ensa protein. ( B ) Gwl IP from prometaphase-arrested HeLa cell lysates was divided into three sets. Each set was incubated in phosphatase reactions containinig either just buffer (Cont.), Fcp1WT or Fcp1CD proteins (Mk IP; 1/3 mock IP incubated with buffer). After phosphatase reaction, IPs were washed, each set split into three portions and incubated in kinase reactions at 37°C with recombinant ARPP19 protein. ( C ) V5 IP from lysates of prometaphase-arrested HeLa cells, previously transfected with V5-GwlWT, V5-GwlS453A or V5-GwlS452A, were split into three portions and incubated in kinase reactions at 37°C with recombinant Ensa protein. Kinase reactions were stopped at indicated time points and probed for indicated antigens (Mk IPs were incubated for 15 min). Graphs show quantitation (arbitrary units) of pS67-Ensa and of pS62-ARPP19 optical density. Data shown are representative of three independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.014

    Journal: eLife

    Article Title: Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    doi: 10.7554/eLife.10399

    Figure Lengend Snippet: Fcp1 affects Ensa/ARPP19 kinase ability of Gwl. ( A ) V5 IP from V5-GwlWT-transfected, prometaphase-arrested, HeLa cells was divided into three sets and incubated for phosphatase reactions with buffer (Cont.), Fcp1WT or Fcp1CD proteins (Mk IP; 1/3 mock IP incubated with buffer). IPs were washed, each set split into three portions and incubated in kinase reactions at 37°C with recombinant Ensa protein. ( B ) Gwl IP from prometaphase-arrested HeLa cell lysates was divided into three sets. Each set was incubated in phosphatase reactions containinig either just buffer (Cont.), Fcp1WT or Fcp1CD proteins (Mk IP; 1/3 mock IP incubated with buffer). After phosphatase reaction, IPs were washed, each set split into three portions and incubated in kinase reactions at 37°C with recombinant ARPP19 protein. ( C ) V5 IP from lysates of prometaphase-arrested HeLa cells, previously transfected with V5-GwlWT, V5-GwlS453A or V5-GwlS452A, were split into three portions and incubated in kinase reactions at 37°C with recombinant Ensa protein. Kinase reactions were stopped at indicated time points and probed for indicated antigens (Mk IPs were incubated for 15 min). Graphs show quantitation (arbitrary units) of pS67-Ensa and of pS62-ARPP19 optical density. Data shown are representative of three independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.014

    Article Snippet: For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO).

    Techniques: Transfection, Incubation, Recombinant, Quantitation Assay

    Fcp1-dependency of mitotic exit dephosphorylations. Control (Cont.) or Fcp1-depleted (Fcp1), as well as Fcp1-depleted complemented with wild type Fcp1 (Fcp1WT), HeLa cells were arrested at prometaphase. Lysates from cells released from prometaphase arrest and sampled at indicated time points of incubation were probed for indicated antigens. Fcp1-depletion efficiency is depicted in Figure 1B . The data shown are representative of three independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.004

    Journal: eLife

    Article Title: Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    doi: 10.7554/eLife.10399

    Figure Lengend Snippet: Fcp1-dependency of mitotic exit dephosphorylations. Control (Cont.) or Fcp1-depleted (Fcp1), as well as Fcp1-depleted complemented with wild type Fcp1 (Fcp1WT), HeLa cells were arrested at prometaphase. Lysates from cells released from prometaphase arrest and sampled at indicated time points of incubation were probed for indicated antigens. Fcp1-depletion efficiency is depicted in Figure 1B . The data shown are representative of three independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.004

    Article Snippet: For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO).

    Techniques: Incubation

    Mitotic phenotypes in Fcp1-depleted cells. Asynchronously growing control-siRNAs- (Cont. siRNAs) or Fcp1-siRNAs-treated (Fcp1 siRNAs) HeLa cells were taken at the indicated time points (hours, hrs) from the siRNAs treatment. Upper left panels, cell lysates were separated on SDS/PAGE and probed for indicated antigens including clived caspase 3 (Clived Casp 3). Lower left panels, cells were fixed and stained for DNA (blue) and α-tubulin (green). Rigth graphs, percentage of mitotic, binucleated and multinucleated cells, scored visually from three fields per sample after staining as described for the lower left panels (about 100 cells per field; cells were scored as mitotic from late prophase to anaphase-telophase) and of death cells, scored visually by trypan blue inclusion from triplicates per sample. Scale bars, 10 μm. Data shown are representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.10399.007

    Journal: eLife

    Article Title: Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    doi: 10.7554/eLife.10399

    Figure Lengend Snippet: Mitotic phenotypes in Fcp1-depleted cells. Asynchronously growing control-siRNAs- (Cont. siRNAs) or Fcp1-siRNAs-treated (Fcp1 siRNAs) HeLa cells were taken at the indicated time points (hours, hrs) from the siRNAs treatment. Upper left panels, cell lysates were separated on SDS/PAGE and probed for indicated antigens including clived caspase 3 (Clived Casp 3). Lower left panels, cells were fixed and stained for DNA (blue) and α-tubulin (green). Rigth graphs, percentage of mitotic, binucleated and multinucleated cells, scored visually from three fields per sample after staining as described for the lower left panels (about 100 cells per field; cells were scored as mitotic from late prophase to anaphase-telophase) and of death cells, scored visually by trypan blue inclusion from triplicates per sample. Scale bars, 10 μm. Data shown are representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.10399.007

    Article Snippet: For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO).

    Techniques: SDS Page, Staining

    Effects of prolonged Cdk1 inhibition on Gwl activity in Fcp1-depleted cells. Gwl IPs from cell lysates of prometaphase-arrested Fcp1 siRNAs-treated (Fcp1) HeLa cells treated – or + RO3306 for 30 min, the same cell lysates of the experiment described in Figure 2—figure supplement 2 , were each split into three portions and incubated in kinase reactions at 37°C with recombinant Ensa protein. Kinase reactions were stopped at indicated time points of incubation and probed for indicated antigens (Mk IPs were incubated for 15 min). Graphs show quantitation (arbitrary units) of pS67-Ensa optical density. Data shown are representative of three independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.015

    Journal: eLife

    Article Title: Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    doi: 10.7554/eLife.10399

    Figure Lengend Snippet: Effects of prolonged Cdk1 inhibition on Gwl activity in Fcp1-depleted cells. Gwl IPs from cell lysates of prometaphase-arrested Fcp1 siRNAs-treated (Fcp1) HeLa cells treated – or + RO3306 for 30 min, the same cell lysates of the experiment described in Figure 2—figure supplement 2 , were each split into three portions and incubated in kinase reactions at 37°C with recombinant Ensa protein. Kinase reactions were stopped at indicated time points of incubation and probed for indicated antigens (Mk IPs were incubated for 15 min). Graphs show quantitation (arbitrary units) of pS67-Ensa optical density. Data shown are representative of three independent experiments per type. DOI: http://dx.doi.org/10.7554/eLife.10399.015

    Article Snippet: For asynchronous siRNAs treatment, Hela cells were transfected with non-targeting or human Fcp1 3’ UTR-targeting (5’-guaagugacagguguuaaa-3’) siRNAs (Dharmacon Inc., Lafayette, CO).

    Techniques: Inhibition, Activity Assay, Incubation, Recombinant, Quantitation Assay

    Two dimensional gel electrophoresis of hTERT overexpressing (A) U2OS and (B) HeLa cell line. Total proteins were extracted from hTERT overexpressing U2OS and HeLa cells and separated non-linearly on IPG strip of PH 3–10, followed by electrophoresis through 12% polyacrylamide gels. The gels were further stained and analyzed by image master 2D platinum software.

    Journal: PLoS ONE

    Article Title: Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells

    doi: 10.1371/journal.pone.0181027

    Figure Lengend Snippet: Two dimensional gel electrophoresis of hTERT overexpressing (A) U2OS and (B) HeLa cell line. Total proteins were extracted from hTERT overexpressing U2OS and HeLa cells and separated non-linearly on IPG strip of PH 3–10, followed by electrophoresis through 12% polyacrylamide gels. The gels were further stained and analyzed by image master 2D platinum software.

    Article Snippet: Cell culture Two cell lines Viz., 1) U2OS (an hTERT negative human osteosarcoma cell line) and 2) HeLa cells (an hTERT expressing cervical cancer cell line) were obtained from National Centre for Cell Science, Pune and grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, South Logan Utah,) with 10% fetal bovine serum (FBS) (Himedia).

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Stripping Membranes, Staining, Software

    Validation of upregulation of heat shock proteins in U2OS and HeLa cell lines. The hTERT induced upregulation of Hsp60 and hsp70 were confirmed by qRT-PCR in (A) U2OS and (B) HeLa cell lines respectively. C) Western blotting showing upregulation of Hsp60 and Hsp70 at protein level. D) Histogram shows the results by applying ImageJ software. D) To confirm the upregulation of Hsp90 in U2OS cell line E) QRT-PCR and F) western blotting is performed.

    Journal: PLoS ONE

    Article Title: Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells

    doi: 10.1371/journal.pone.0181027

    Figure Lengend Snippet: Validation of upregulation of heat shock proteins in U2OS and HeLa cell lines. The hTERT induced upregulation of Hsp60 and hsp70 were confirmed by qRT-PCR in (A) U2OS and (B) HeLa cell lines respectively. C) Western blotting showing upregulation of Hsp60 and Hsp70 at protein level. D) Histogram shows the results by applying ImageJ software. D) To confirm the upregulation of Hsp90 in U2OS cell line E) QRT-PCR and F) western blotting is performed.

    Article Snippet: Cell culture Two cell lines Viz., 1) U2OS (an hTERT negative human osteosarcoma cell line) and 2) HeLa cells (an hTERT expressing cervical cancer cell line) were obtained from National Centre for Cell Science, Pune and grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, South Logan Utah,) with 10% fetal bovine serum (FBS) (Himedia).

    Techniques: Quantitative RT-PCR, Western Blot, Software

    hTERT overexpression in HeLa and U2OS cell lines. hTERT is overexpressed in U2OS and HeLa cell lines. (A D) mRNA level of hTERT in U2OS HeLa cell lines was determined by quantitative real-time PCR. (B E) Western blotting confirms overexpression of hTERT in U2OS and HeLa cell line. (C F) Histograms depict densitometric quantification of the hTERT overexpression of three corresponding independent Western blot experiments in U2OS and HeLa cell lines.

    Journal: PLoS ONE

    Article Title: Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells

    doi: 10.1371/journal.pone.0181027

    Figure Lengend Snippet: hTERT overexpression in HeLa and U2OS cell lines. hTERT is overexpressed in U2OS and HeLa cell lines. (A D) mRNA level of hTERT in U2OS HeLa cell lines was determined by quantitative real-time PCR. (B E) Western blotting confirms overexpression of hTERT in U2OS and HeLa cell line. (C F) Histograms depict densitometric quantification of the hTERT overexpression of three corresponding independent Western blot experiments in U2OS and HeLa cell lines.

    Article Snippet: Cell culture Two cell lines Viz., 1) U2OS (an hTERT negative human osteosarcoma cell line) and 2) HeLa cells (an hTERT expressing cervical cancer cell line) were obtained from National Centre for Cell Science, Pune and grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, South Logan Utah,) with 10% fetal bovine serum (FBS) (Himedia).

    Techniques: Over Expression, Real-time Polymerase Chain Reaction, Western Blot

    Validation of GAPDH in HeLa and U2OS cell lines. QRT-PCR was performed to check the hTERT induced upregulation of GAPDH in (A) U2OS and (B) HeLa cell lines. C) Represents results of Western blotting to check the up-regulation of GAPDH at protein level while (D) represents histograms of respective blots showing no prominent difference in expression of GAPDH at protein level.

    Journal: PLoS ONE

    Article Title: Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells

    doi: 10.1371/journal.pone.0181027

    Figure Lengend Snippet: Validation of GAPDH in HeLa and U2OS cell lines. QRT-PCR was performed to check the hTERT induced upregulation of GAPDH in (A) U2OS and (B) HeLa cell lines. C) Represents results of Western blotting to check the up-regulation of GAPDH at protein level while (D) represents histograms of respective blots showing no prominent difference in expression of GAPDH at protein level.

    Article Snippet: Cell culture Two cell lines Viz., 1) U2OS (an hTERT negative human osteosarcoma cell line) and 2) HeLa cells (an hTERT expressing cervical cancer cell line) were obtained from National Centre for Cell Science, Pune and grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, South Logan Utah,) with 10% fetal bovine serum (FBS) (Himedia).

    Techniques: Quantitative RT-PCR, Western Blot, Expressing

    hTERT overexpression enhances the migration of cancer cells. A) Microscopic images of in vitro wound healing at 0, 12, 24, 36 and 48 h after the creation of wounds in U2OS cells. B) Histogram represents quantification of the effect of hTERT overexpression on cell mobility (% migration) in U2OS cell line. C) Microscopic images of in vitro wound healing at 0, 12, 24, 36 and 48 h after the creation of wounds in HeLa cells. D) Histogram representing quantification of the effect of hTERT overexpression on cell mobility (% migration) in HeLa cell line.

    Journal: PLoS ONE

    Article Title: Proteomic identification of proteins differentially expressed following overexpression of hTERT (human telomerase reverse transcriptase) in cancer cells

    doi: 10.1371/journal.pone.0181027

    Figure Lengend Snippet: hTERT overexpression enhances the migration of cancer cells. A) Microscopic images of in vitro wound healing at 0, 12, 24, 36 and 48 h after the creation of wounds in U2OS cells. B) Histogram represents quantification of the effect of hTERT overexpression on cell mobility (% migration) in U2OS cell line. C) Microscopic images of in vitro wound healing at 0, 12, 24, 36 and 48 h after the creation of wounds in HeLa cells. D) Histogram representing quantification of the effect of hTERT overexpression on cell mobility (% migration) in HeLa cell line.

    Article Snippet: Cell culture Two cell lines Viz., 1) U2OS (an hTERT negative human osteosarcoma cell line) and 2) HeLa cells (an hTERT expressing cervical cancer cell line) were obtained from National Centre for Cell Science, Pune and grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, South Logan Utah,) with 10% fetal bovine serum (FBS) (Himedia).

    Techniques: Over Expression, Migration, In Vitro

    Proximity Ligation Assay on Scramble/Cas9 HeLa cells showing interactions between TMEM97 and PGRMC1, LDLR and PGRMC1, and LDLR and TMEM97. ( A ) standard, non-lipoprotein depleted conditions, ( B ) sterol-starved conditions, and ( C ) sterol-starved conditions followed with treatment with 50 μg/mL LDL for 3 hours. Inserts represent an enlarged region focusing on a single cell.

    Journal: Scientific Reports

    Article Title: Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex

    doi: 10.1038/s41598-018-35430-3

    Figure Lengend Snippet: Proximity Ligation Assay on Scramble/Cas9 HeLa cells showing interactions between TMEM97 and PGRMC1, LDLR and PGRMC1, and LDLR and TMEM97. ( A ) standard, non-lipoprotein depleted conditions, ( B ) sterol-starved conditions, and ( C ) sterol-starved conditions followed with treatment with 50 μg/mL LDL for 3 hours. Inserts represent an enlarged region focusing on a single cell.

    Article Snippet: The control cell line was generated by two sequential procedures: (1) transfecting HeLa cells with the non-specific 20-nucleotide RNA CRISPR/Cas9 Plasmid (Santa Cruz biotechnology), and (2) transducing the resulting cells with lentivirus containing lentiCas9-Blast plasmid and then selecting cells for stable expression of Cas9.

    Techniques: Proximity Ligation Assay

    SIRPα proteolysis modulates inflammatory signaling. a , HeLa cells were incubated with TNFα (10 ng/ml) for the indicated times. Cells were harvested and probed for pIKKα/β and total IKKα. b , HeLa cells were transfected

    Journal: The Journal of Biological Chemistry

    Article Title: Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling *

    doi: 10.1074/jbc.M115.682914

    Figure Lengend Snippet: SIRPα proteolysis modulates inflammatory signaling. a , HeLa cells were incubated with TNFα (10 ng/ml) for the indicated times. Cells were harvested and probed for pIKKα/β and total IKKα. b , HeLa cells were transfected

    Article Snippet: THP-1 and HeLa cells were transfected with 10–30 n m DsiRNA using GeneMute siRNA transfection reagent (SignaGen) according to the manufacturer's instructions.

    Techniques: Incubation, Transfection

    Expression and localization of NKAP. ( A ) Presence of NKAP in mouse tissues. Homogenates of adult mouse tissues as indicated were separated by SDS-PAGE (12% acrylamide) and the corresponding blot probed with NKAP-specific mAb K85-80-5. ( B ) Localization of NKAP during mitosis. HeLa cells were synchronized using nocodazole to block progression of the cell cycle and then released and fixed using 4% paraformaldehyde (PFA). Cells were stained with mAb K85-80-5, and nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). The phases of the cell cycle are indicated. Bar, 10 µm.

    Journal: Nucleic Acids Research

    Article Title: NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

    doi: 10.1093/nar/gkt1311

    Figure Lengend Snippet: Expression and localization of NKAP. ( A ) Presence of NKAP in mouse tissues. Homogenates of adult mouse tissues as indicated were separated by SDS-PAGE (12% acrylamide) and the corresponding blot probed with NKAP-specific mAb K85-80-5. ( B ) Localization of NKAP during mitosis. HeLa cells were synchronized using nocodazole to block progression of the cell cycle and then released and fixed using 4% paraformaldehyde (PFA). Cells were stained with mAb K85-80-5, and nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). The phases of the cell cycle are indicated. Bar, 10 µm.

    Article Snippet: Chromatin immunoprecipitation and DNA sequencing Chromatin immunoprecipitation (ChIP) was performed in HeLa cells and HeLa cells expressing GFP-NKAP using a kit (ChIP-IT Express, 53008, Active Motif) according to the manufacturer’s protocol.

    Techniques: Expressing, SDS Page, Blocking Assay, Staining

    Overexpression of NKAP and RS+Basic alters SRSF2 localization. ( A ) HeLa cells expressing GFP-NKAP were stained for SRSF2. Cells expressing moderate amounts (upper panel) and strongly overexpressing GFP-NKAP are shown (middle and lower panel). ( B ) Expression of GFP-RS+Basic in HeLa cells. Upper panel, cells expressing moderate amounts, middle and lower panel, overexpression of GFP-RS+Basic. Bar, 10 µm. ( C ) The distribution of SRSF1 and speckle number was altered when GFP-NKAP was strongly overexpressed. ( D ) Overexpression of RS+Basic led to altered localization of SRSF1.

    Journal: Nucleic Acids Research

    Article Title: NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

    doi: 10.1093/nar/gkt1311

    Figure Lengend Snippet: Overexpression of NKAP and RS+Basic alters SRSF2 localization. ( A ) HeLa cells expressing GFP-NKAP were stained for SRSF2. Cells expressing moderate amounts (upper panel) and strongly overexpressing GFP-NKAP are shown (middle and lower panel). ( B ) Expression of GFP-RS+Basic in HeLa cells. Upper panel, cells expressing moderate amounts, middle and lower panel, overexpression of GFP-RS+Basic. Bar, 10 µm. ( C ) The distribution of SRSF1 and speckle number was altered when GFP-NKAP was strongly overexpressed. ( D ) Overexpression of RS+Basic led to altered localization of SRSF1.

    Article Snippet: Chromatin immunoprecipitation and DNA sequencing Chromatin immunoprecipitation (ChIP) was performed in HeLa cells and HeLa cells expressing GFP-NKAP using a kit (ChIP-IT Express, 53008, Active Motif) according to the manufacturer’s protocol.

    Techniques: Over Expression, Expressing, Staining

    Subcellular localization of NKAP. ( A ) Schematic representation of NKAP polypeptides used in this study. ( B ) Full length NKAP, Rs + Basic domain, RS domain, Basic domain and DUF 926 domain were tagged with N-terminal GFP and expressed in HeLa cells that were fixed and stained with DAPI. Bar, 10 µm.

    Journal: Nucleic Acids Research

    Article Title: NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

    doi: 10.1093/nar/gkt1311

    Figure Lengend Snippet: Subcellular localization of NKAP. ( A ) Schematic representation of NKAP polypeptides used in this study. ( B ) Full length NKAP, Rs + Basic domain, RS domain, Basic domain and DUF 926 domain were tagged with N-terminal GFP and expressed in HeLa cells that were fixed and stained with DAPI. Bar, 10 µm.

    Article Snippet: Chromatin immunoprecipitation and DNA sequencing Chromatin immunoprecipitation (ChIP) was performed in HeLa cells and HeLa cells expressing GFP-NKAP using a kit (ChIP-IT Express, 53008, Active Motif) according to the manufacturer’s protocol.

    Techniques: Staining

    NKAP localizes to the nuclear speckles. ( A ) HeLa cells overexpressing GFP-NKAP, GFP-RS+Basic, GFP-RS and GFP-Basic were fixed with 4% PFA and stained for PML bodies and nuclear speckles by pAB PML and mAb SRSF2, respectively. DNA was stained with DAPI. ( B ) Arrest of transcription affects NKAP and RS + Basic distribution. HeLa cells expressing GFP-NKAP and GFP-RS+Basic were treated with actinomycin D, fixed with 4% PFA and stained for SRSF2. Bar, 10 µm.

    Journal: Nucleic Acids Research

    Article Title: NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

    doi: 10.1093/nar/gkt1311

    Figure Lengend Snippet: NKAP localizes to the nuclear speckles. ( A ) HeLa cells overexpressing GFP-NKAP, GFP-RS+Basic, GFP-RS and GFP-Basic were fixed with 4% PFA and stained for PML bodies and nuclear speckles by pAB PML and mAb SRSF2, respectively. DNA was stained with DAPI. ( B ) Arrest of transcription affects NKAP and RS + Basic distribution. HeLa cells expressing GFP-NKAP and GFP-RS+Basic were treated with actinomycin D, fixed with 4% PFA and stained for SRSF2. Bar, 10 µm.

    Article Snippet: Chromatin immunoprecipitation and DNA sequencing Chromatin immunoprecipitation (ChIP) was performed in HeLa cells and HeLa cells expressing GFP-NKAP using a kit (ChIP-IT Express, 53008, Active Motif) according to the manufacturer’s protocol.

    Techniques: Staining, Expressing

    RS+Basic interacts with FUS. ( A ) Immunoprecipitation of FUS using anti-NKAP monoclonal antibody K85-80-5. ( B ) The GST-RS+Basic of NKAP precipitates FLAG-FUS recognized by polyclonal FLAG antibodies. GST, GST-RS+Basic and GST-DUF were visualized by Ponceau S staining. Proteins were separated by 12% SDS-PAGE. ( C ) HeLa cells were transfected with GFP-NKAP and FLAG-FUS, fixed and stained with FLAG antibodies. Secondary antibody conjugated with Alexa 568 was used. Nuclei were visualized with DAPI. Bar, 10 µm.

    Journal: Nucleic Acids Research

    Article Title: NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

    doi: 10.1093/nar/gkt1311

    Figure Lengend Snippet: RS+Basic interacts with FUS. ( A ) Immunoprecipitation of FUS using anti-NKAP monoclonal antibody K85-80-5. ( B ) The GST-RS+Basic of NKAP precipitates FLAG-FUS recognized by polyclonal FLAG antibodies. GST, GST-RS+Basic and GST-DUF were visualized by Ponceau S staining. Proteins were separated by 12% SDS-PAGE. ( C ) HeLa cells were transfected with GFP-NKAP and FLAG-FUS, fixed and stained with FLAG antibodies. Secondary antibody conjugated with Alexa 568 was used. Nuclei were visualized with DAPI. Bar, 10 µm.

    Article Snippet: Chromatin immunoprecipitation and DNA sequencing Chromatin immunoprecipitation (ChIP) was performed in HeLa cells and HeLa cells expressing GFP-NKAP using a kit (ChIP-IT Express, 53008, Active Motif) according to the manufacturer’s protocol.

    Techniques: Immunoprecipitation, Staining, SDS Page, Transfection

    Binding of proteins to AREs within the 3′-UTR of PTHrP 1-141 mRNA. A . EMSA using ARE-containing region of the 3′-UTR and S100 extract from HeLa cells. 20 μg of HeLa S100 cytoplasmic extract were incubated with a 32 P-labeled RNA

    Journal: Molecular and cellular endocrinology

    Article Title: AU-RICH ELEMENTS IN THE 3?-UTR REGULATE THE STABILITY OF THE 141 AMINO ACID ISOFORM OF PARATHYROID HORMONE-RELATED PROTEIN mRNA

    doi: 10.1016/j.mce.2012.08.015

    Figure Lengend Snippet: Binding of proteins to AREs within the 3′-UTR of PTHrP 1-141 mRNA. A . EMSA using ARE-containing region of the 3′-UTR and S100 extract from HeLa cells. 20 μg of HeLa S100 cytoplasmic extract were incubated with a 32 P-labeled RNA

    Article Snippet: S100 extracts from HeLa cells were purchased from Biovest (Worcester, MA).

    Techniques: Binding Assay, Incubation, Labeling

    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

    Ubc9 co-localizes with SUMO1-modified RanGAP1 and RanBP2 at both NPCs and ALPCs. (A) HeLa cells were analyzed by immunofluorescence microscopy using anti-Ubc9 antibody and mAb414. (B and C) HeLa cells were transfected with the construct encoding Myc-tagged Ubc9, double-stained with mouse anti-Myc mAb (9E10) and rabbit anti-RanGAP1 antibodies or with rabbit anti-Myc antibody and mouse anti-RanBP2 mAb, and then analyzed by immunofluorescence microscopy. Bar, 10 μm. The enlarged versions of inlets are shown at the top-right corner of each image.

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: Ubc9 co-localizes with SUMO1-modified RanGAP1 and RanBP2 at both NPCs and ALPCs. (A) HeLa cells were analyzed by immunofluorescence microscopy using anti-Ubc9 antibody and mAb414. (B and C) HeLa cells were transfected with the construct encoding Myc-tagged Ubc9, double-stained with mouse anti-Myc mAb (9E10) and rabbit anti-RanGAP1 antibodies or with rabbit anti-Myc antibody and mouse anti-RanBP2 mAb, and then analyzed by immunofluorescence microscopy. Bar, 10 μm. The enlarged versions of inlets are shown at the top-right corner of each image.

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Modification, Immunofluorescence, Microscopy, Transfection, Construct, Staining

    CRM1-mediated export complexes accumulate at ALPCs when the disassembly of these export complexes is inhibited by transient expression of RanQ69L mutant. (A and B) HeLa cells were transfected with the construct encoding FLAG-tagged Ran wild-type (WT) or RanQ69L GTPase mutant (Mut), double-labeled with anti-FLAG and RanGAP1 antibodies (A) or mAb414 (B) and analyzed by immunofluorescence microscopy. (C-E) HeLa cells were co-transfected with the constructs encoding Rev-GR-GFP and FLAG-tagged Ran WT or RanQ69L. The transfected cells were incubated with 1 μM dexamethasone to induce the nuclear accumulation of Rev-GR-GFP, washed with fresh medium to remove dexamethasone to initiate nuclear export, stained with anti-RanGAP1 antibodies (C), anti-FLAG antibodies (D), or both anti-RanGAP1 and anti-FLAG antibodies (E), and analyzed by immunofluorescence microscopy. Bar, 10 μm. The boxes at the top-left corner of each image reveal the enlarged version of inlets.

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: CRM1-mediated export complexes accumulate at ALPCs when the disassembly of these export complexes is inhibited by transient expression of RanQ69L mutant. (A and B) HeLa cells were transfected with the construct encoding FLAG-tagged Ran wild-type (WT) or RanQ69L GTPase mutant (Mut), double-labeled with anti-FLAG and RanGAP1 antibodies (A) or mAb414 (B) and analyzed by immunofluorescence microscopy. (C-E) HeLa cells were co-transfected with the constructs encoding Rev-GR-GFP and FLAG-tagged Ran WT or RanQ69L. The transfected cells were incubated with 1 μM dexamethasone to induce the nuclear accumulation of Rev-GR-GFP, washed with fresh medium to remove dexamethasone to initiate nuclear export, stained with anti-RanGAP1 antibodies (C), anti-FLAG antibodies (D), or both anti-RanGAP1 and anti-FLAG antibodies (E), and analyzed by immunofluorescence microscopy. Bar, 10 μm. The boxes at the top-left corner of each image reveal the enlarged version of inlets.

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Expressing, Mutagenesis, Transfection, Construct, Labeling, Immunofluorescence, Microscopy, Incubation, Staining

    ALPCs function as intermediate docking sites for importin α/β-mediated import complexes during nuclear import. HeLa cells were transfected with siRNAs specific to ELYS to upregulate annulate lamellae and then with the construct encoding Rev-GR-GFP fusion proteins. The transfected cells were incubated with LMB for 2 h to inhibit CRM1-mediated export, treated with both dexamethasone (1 μM) and LMB to induce importin α/β-mediated import for the indicated times, and analyzed by immunofluorescence microscopy. At least 50 Rev-GR-GFP cells were analyzed for each time point of dexamethasone treatment to select o representative cell as shown in this figure. The arrows indicated the sites of ALPCs. Bar, 10 μm.

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: ALPCs function as intermediate docking sites for importin α/β-mediated import complexes during nuclear import. HeLa cells were transfected with siRNAs specific to ELYS to upregulate annulate lamellae and then with the construct encoding Rev-GR-GFP fusion proteins. The transfected cells were incubated with LMB for 2 h to inhibit CRM1-mediated export, treated with both dexamethasone (1 μM) and LMB to induce importin α/β-mediated import for the indicated times, and analyzed by immunofluorescence microscopy. At least 50 Rev-GR-GFP cells were analyzed for each time point of dexamethasone treatment to select o representative cell as shown in this figure. The arrows indicated the sites of ALPCs. Bar, 10 μm.

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Transfection, Construct, Incubation, Immunofluorescence, Microscopy

    Upregulation of annulate lamellae causes a redistribution of both pore complexes and nuclear transport receptors from the nuclear envelope to annulate lamellae. (A) HeLa cells were treated with vinblastine or DMSO as a control and analyzed by immunofluorescence microscopy with mAb414 and anti-RanGAP1 antibodies. (B-F) HeLa cells were transfected with control or ELYS-specific siRNAs, double-labeled with mAb414 and anti-ELYS antibodies (B), mAb414 and anti-RanGAP1 antibodies (C), anti-importin α and anti-RanGAP1 antibodies (D), anti-importin β and anti-RanGAP1 antibodies (E), and anti-CRM1 and anti-RanGAP1 antibodies (F) followed by immunofluorescence microscopy. The boxes at the corner of each image represent the enlarged version of inlets. Bar, 10 μm.

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: Upregulation of annulate lamellae causes a redistribution of both pore complexes and nuclear transport receptors from the nuclear envelope to annulate lamellae. (A) HeLa cells were treated with vinblastine or DMSO as a control and analyzed by immunofluorescence microscopy with mAb414 and anti-RanGAP1 antibodies. (B-F) HeLa cells were transfected with control or ELYS-specific siRNAs, double-labeled with mAb414 and anti-ELYS antibodies (B), mAb414 and anti-RanGAP1 antibodies (C), anti-importin α and anti-RanGAP1 antibodies (D), anti-importin β and anti-RanGAP1 antibodies (E), and anti-CRM1 and anti-RanGAP1 antibodies (F) followed by immunofluorescence microscopy. The boxes at the corner of each image represent the enlarged version of inlets. Bar, 10 μm.

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Immunofluorescence, Microscopy, Transfection, Labeling

    SUMO1-modified RanGAP1 is equally distributed between the nuclear and cytosolic fractions in a variety of mammalian cells. (A) HeLa cells were fractionated by two different methods using either digitonin or NP-40 as non-ionic detergent. The nuclear and cytosolic fractions were analyzed by immunoblotting with antibodies specific to SUMO-1, RanGAP1, α-tubulin as a marker for cytosolic proteins, lamin B as a marker for nuclear proteins, or POM121 as a marker for the NPC proteins. (B and C) Different types of tumor/cancer cells including HeLa, BRL, 293T and U2OS (B) and normal/non-tumorigenic fibroblasts including human GM03652 and mouse NIH3T3 (C) were fractionated by NP-40 method and then analyzed by immunoblotting with the indicated antibodies. (D) Total cell lysates of HeLa, PN and SMC cells were used for immunoblot analysis with the indicated antibodies.

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: SUMO1-modified RanGAP1 is equally distributed between the nuclear and cytosolic fractions in a variety of mammalian cells. (A) HeLa cells were fractionated by two different methods using either digitonin or NP-40 as non-ionic detergent. The nuclear and cytosolic fractions were analyzed by immunoblotting with antibodies specific to SUMO-1, RanGAP1, α-tubulin as a marker for cytosolic proteins, lamin B as a marker for nuclear proteins, or POM121 as a marker for the NPC proteins. (B and C) Different types of tumor/cancer cells including HeLa, BRL, 293T and U2OS (B) and normal/non-tumorigenic fibroblasts including human GM03652 and mouse NIH3T3 (C) were fractionated by NP-40 method and then analyzed by immunoblotting with the indicated antibodies. (D) Total cell lysates of HeLa, PN and SMC cells were used for immunoblot analysis with the indicated antibodies.

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Modification, Marker

    Upregulation of annulate lamellae by RNAi-knockdown of ELYS decreases the rates of both nuclear import and export. HeLa cells were transfected with control or ELYS-specific siRNAs and then with the construct encoding Rev-GR-GFP. (A and B) To evaluate the effect of ELYS RNAi on the rate of nuclear import, the transfected cells were treated with 0.25 μM of dexamethasone for the indicated times and analyzed by fluorescence microscopy (A). The histogram shows the nuclear to total signal ratios of Rev-GR-GFP (B). (C and D) To test if ELYS RNAi affects the rate of nuclear export, the transfected cells were treated with 0.25 μM of dexamethasone for 3 h to induce the nuclear accumulation of Rev-GR-GFP, washed with PBS, incubated with fresh medium for the indicated times, and analyzed by fluorescence microscopy (C). The histogram shows the cytoplasmic to total signal ratio of Rev-GR-GFP (D). Each bar indicates the mean value ± SEM ( N = 60, Student’s t test) (B and D). Bar, 10 μm (A and C).

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: Upregulation of annulate lamellae by RNAi-knockdown of ELYS decreases the rates of both nuclear import and export. HeLa cells were transfected with control or ELYS-specific siRNAs and then with the construct encoding Rev-GR-GFP. (A and B) To evaluate the effect of ELYS RNAi on the rate of nuclear import, the transfected cells were treated with 0.25 μM of dexamethasone for the indicated times and analyzed by fluorescence microscopy (A). The histogram shows the nuclear to total signal ratios of Rev-GR-GFP (B). (C and D) To test if ELYS RNAi affects the rate of nuclear export, the transfected cells were treated with 0.25 μM of dexamethasone for 3 h to induce the nuclear accumulation of Rev-GR-GFP, washed with PBS, incubated with fresh medium for the indicated times, and analyzed by fluorescence microscopy (C). The histogram shows the cytoplasmic to total signal ratio of Rev-GR-GFP (D). Each bar indicates the mean value ± SEM ( N = 60, Student’s t test) (B and D). Bar, 10 μm (A and C).

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Transfection, Construct, Fluorescence, Microscopy, Incubation

    RanBP2 is required for the localization of RanGTP and CRM1 at NPCs and ALPCs. (A) HeLa cells were first transfected with control or RanBP2-specific siRNAs for 48 h and then the constructs encoding FLAG-tagged RanQ69L for 24 h followed by immunofluorescence microscopy with antibodies specific to RanBP2 and FLAG. (B) HeLa cells were transfected with control or RanBP2-specific siRNAs for 72 h and then analyzed by immunofluorescence microscopy with antibodies specific to RanBP2 and CRM1. In RanBP2 RNAi cells (lower panels), “-” indicates the cell with depletion of RanBP2, and “+” indicates the cell with levels of RanBP2 that are similar to those in control cells (upper panels). The boxes at the bottom corner of each image show the enlarged version of inlets. Bar, 10 μm.

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: RanBP2 is required for the localization of RanGTP and CRM1 at NPCs and ALPCs. (A) HeLa cells were first transfected with control or RanBP2-specific siRNAs for 48 h and then the constructs encoding FLAG-tagged RanQ69L for 24 h followed by immunofluorescence microscopy with antibodies specific to RanBP2 and FLAG. (B) HeLa cells were transfected with control or RanBP2-specific siRNAs for 72 h and then analyzed by immunofluorescence microscopy with antibodies specific to RanBP2 and CRM1. In RanBP2 RNAi cells (lower panels), “-” indicates the cell with depletion of RanBP2, and “+” indicates the cell with levels of RanBP2 that are similar to those in control cells (upper panels). The boxes at the bottom corner of each image show the enlarged version of inlets. Bar, 10 μm.

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Transfection, Construct, Immunofluorescence, Microscopy

    Both covalent SUMOylation and non-covalent interaction with RanBP2 are required for RanGAP1 localization to ALPCs. (A) HeLa cells were transfected with the constructs encoding Myc-tagged RanGAP1 wild-type (WT) or SUMOylation-deficient K526R mutant (Mut) and analyzed by immunofluorescence microscopy with antibodies specific to Myc and RanBP2. The boxes at the bottom corner of each image show the enlarged version of inlets. Bar, 10 μm. (B) The transfected cells were analyzed by immunoblotting with antibodies specific to RanGAP1, Myc and α-tubulin. (C) HeLa cells were transfected with control or RanBP2-specific siRNAs, double-stained with antibodies specific to RanBP2 and RanGAP1, and analyzed by immunofluorescence microscopy. In the lower panel, white dashed lines indicate the borders of RanBP2 RNAi cells, in which “-” indicates a significant knockdown of RanBP2 and “+” indicates that the signals of RanBP2 are comparable to those in control RNAi cells (upper panel). Bar, 10 μm. (D) The cells transfected with control or RanBP2-specific siRNAs were analyzed by immunoblotting with the indicated antibodies.

    Journal: PLoS ONE

    Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    doi: 10.1371/journal.pone.0144508

    Figure Lengend Snippet: Both covalent SUMOylation and non-covalent interaction with RanBP2 are required for RanGAP1 localization to ALPCs. (A) HeLa cells were transfected with the constructs encoding Myc-tagged RanGAP1 wild-type (WT) or SUMOylation-deficient K526R mutant (Mut) and analyzed by immunofluorescence microscopy with antibodies specific to Myc and RanBP2. The boxes at the bottom corner of each image show the enlarged version of inlets. Bar, 10 μm. (B) The transfected cells were analyzed by immunoblotting with antibodies specific to RanGAP1, Myc and α-tubulin. (C) HeLa cells were transfected with control or RanBP2-specific siRNAs, double-stained with antibodies specific to RanBP2 and RanGAP1, and analyzed by immunofluorescence microscopy. In the lower panel, white dashed lines indicate the borders of RanBP2 RNAi cells, in which “-” indicates a significant knockdown of RanBP2 and “+” indicates that the signals of RanBP2 are comparable to those in control RNAi cells (upper panel). Bar, 10 μm. (D) The cells transfected with control or RanBP2-specific siRNAs were analyzed by immunoblotting with the indicated antibodies.

    Article Snippet: Analyses of nuclear import and export complexes at ALPCs To test if the importin α/β-mediated import complexes are assembled at ALPCs, HeLa cells were first transfected with siRNAs specific to ELYS for 48 h to upregulate annulate lamellae and then the pXRGG plasmids encoding Rev-GR-GFP for 22 h. The transfected cells were treated with 200 nM leptomycin B (LMB) (Enzo Life Sciences) for 2 h to inhibit CRM1-mediated export, incubated with 1 μM dexamethasone and 200 nM of LMB for 0, 2.5, 5, 7.5, 15, 30 min to induce nuclear import of Rev-GR-GFP, and analyzed by immunofluorescence microcopy using mAb414.

    Techniques: Transfection, Construct, Mutagenesis, Immunofluorescence, Microscopy, Staining

    SIRT1 dissociates with CHK2 for its activation upon oxidative stress. a SIRT1 bound CHK2 under control conditions but dissociated with CHK2 after treatment with 1 mM of H 2 O 2 in HeLa cells. b The acetylated CHK2 level was elevated in response to oxidative stress in HeLa cells. c DDR pathways were activated upon oxidative stress. The phosphorylation levels of CHK2 (p-CHK2) and p53 (p-p53) were highly increased in response to oxidative stress in HCT116 cells. IP immunoprecipitation, WB Western blot. Western blots in figures are representative of more than three independent experiments

    Journal: Experimental & Molecular Medicine

    Article Title: Deacetylation of CHK2 by SIRT1 protects cells from oxidative stress-dependent DNA damage response

    doi: 10.1038/s12276-019-0232-4

    Figure Lengend Snippet: SIRT1 dissociates with CHK2 for its activation upon oxidative stress. a SIRT1 bound CHK2 under control conditions but dissociated with CHK2 after treatment with 1 mM of H 2 O 2 in HeLa cells. b The acetylated CHK2 level was elevated in response to oxidative stress in HeLa cells. c DDR pathways were activated upon oxidative stress. The phosphorylation levels of CHK2 (p-CHK2) and p53 (p-p53) were highly increased in response to oxidative stress in HCT116 cells. IP immunoprecipitation, WB Western blot. Western blots in figures are representative of more than three independent experiments

    Article Snippet: Immunoprecipitation and immunoblotting As described in a previous report , the lysates (1–2 mg) of HeLa cells, HCT116 cells or MEFs were incubated with indicated antibodies (1–2 μg) at 37 °C overnight; 40 μl of protein G-sepharose (GE healthcare, Chicago, IL, USA) was then added.

    Techniques: Activation Assay, Immunoprecipitation, Western Blot

    Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. ( A ) U2OS or HeLa cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. ( B ) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.

    Journal: Nucleic Acids Research

    Article Title: Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP

    doi: 10.1093/nar/gkn1057

    Figure Lengend Snippet: Overexpression of p300 or CBP restores E1A 12S-repressed CR3 transactivation. ( A ) U2OS or HeLa cell extracts from cells transfected with empty vector, E1A 12S expression plasmid or E1A 13S expression plasmid were assayed for levels of endogenous p300 and E1A. ( B ) U2OS cells were co-transfected with plasmids expressing GAL4-CR3 (600 ng) and 600 ng of vectors expressing the indicated acetyltransferases, together with a GAL4-luciferase reporter plasmid (600 ng) and 60 ng of E1A 12S as indicated. Luciferase activity was assayed 24 h after transfection and the data was plotted versus CR3 alone (shown in grey), which was set to 1. Overexpressed p300, CBP, CBP AT- and pCAF were detected using anti-HA antibody for p300 and anti-FLAG M2 for CBP, CBP AT- and pCAF.

    Article Snippet: U2OS cells were used for the GAL4-CR3 experiments instead of HeLa cells because of poor activity of the GAL4-CR3 fusion in HeLa cells.

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

    Repression of E1A 13S- and CR3-mediated transactivation by E1A 12S is dose-dependent and independent of the method by which E1A is targeted to the promoter. ( A ) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E3-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 hours after transfection. TF, transcription factor. ( B ) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E4-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 h after transfection. ( C ) U2OS cells were co-transfected with plasmids expressing a GAL4 DNA-binding domain-CR3 (1 μg) fusion and E1A 12S (as indicated) together with a GAL4 responsive luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection.

    Journal: Nucleic Acids Research

    Article Title: Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP

    doi: 10.1093/nar/gkn1057

    Figure Lengend Snippet: Repression of E1A 13S- and CR3-mediated transactivation by E1A 12S is dose-dependent and independent of the method by which E1A is targeted to the promoter. ( A ) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E3-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 hours after transfection. TF, transcription factor. ( B ) HeLa cells were co-transfected with plasmids expressing E1A 13S (1.5 μg) and E1A 12S (as indicated) together with an adenovirus E4-luciferase reporter plasmid (0.5 μg). Luciferase activity was assayed 48 h after transfection. ( C ) U2OS cells were co-transfected with plasmids expressing a GAL4 DNA-binding domain-CR3 (1 μg) fusion and E1A 12S (as indicated) together with a GAL4 responsive luciferase reporter (1 μg). Luciferase activity was assayed 24 h after transfection.

    Article Snippet: U2OS cells were used for the GAL4-CR3 experiments instead of HeLa cells because of poor activity of the GAL4-CR3 fusion in HeLa cells.

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

    p300/CBP bind directly to E1A CR3. ( A ) Top panel: Full-length, purified FLAG-tagged CBP was incubated with either purified GST, GST-CR3 or GST-E1A-1-82. Protein complexes were then bound to Glutathione Sepharose 4B beads, washed and resolved on 4–12% gradient SDS–PAGE. Associated CBP was detected with anti-FLAG M5 monoclonal antibody, and input levels of GST-fusion proteins were determined by Ponceau S staining of the same membrane. Bottom panel: A549 total cell extract was incubated with either purified GST or GST-CR3. Protein complexes were then bound to Glutathione Sepharose 4B beads, washed and resolved on 4–12% gradient SDS–PAGE. The blot was probed for p300 using RW128 anti-p300 antibody and input levels of GST-fusion proteins was determined using Ponceau S staining of the same membrane. ( B ) HeLa cells were transfected with plasmids expressing either wild-type E1A 12S, 13S, 12SRG2 or 13SRG2 together with a plasmid expressing HA-tagged p300. Immunoprecipitations were carried out using M73 anti-E1A antibody and the proteins were resolved on a 4–12% gradient SDS–PAGE and western blots performed for p300 using the RW128 anti-p300 antibody. Input levels are shown. ( C ) HeLa cells were transfected with plasmids expressing either wild-type genomic E1A, E1A 12S dl 1101 or E1A 13S dl 1101, and immunoprecipitated with a mix of M73 and M58 anti-E1A antibodies. The immunoprecipitated proteins were resolved on a 3–8% gradient SDS–PAGE and Western blots performed for p300 using the RW128 anti-p300 antibody. Input levels are shown.

    Journal: Nucleic Acids Research

    Article Title: Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP

    doi: 10.1093/nar/gkn1057

    Figure Lengend Snippet: p300/CBP bind directly to E1A CR3. ( A ) Top panel: Full-length, purified FLAG-tagged CBP was incubated with either purified GST, GST-CR3 or GST-E1A-1-82. Protein complexes were then bound to Glutathione Sepharose 4B beads, washed and resolved on 4–12% gradient SDS–PAGE. Associated CBP was detected with anti-FLAG M5 monoclonal antibody, and input levels of GST-fusion proteins were determined by Ponceau S staining of the same membrane. Bottom panel: A549 total cell extract was incubated with either purified GST or GST-CR3. Protein complexes were then bound to Glutathione Sepharose 4B beads, washed and resolved on 4–12% gradient SDS–PAGE. The blot was probed for p300 using RW128 anti-p300 antibody and input levels of GST-fusion proteins was determined using Ponceau S staining of the same membrane. ( B ) HeLa cells were transfected with plasmids expressing either wild-type E1A 12S, 13S, 12SRG2 or 13SRG2 together with a plasmid expressing HA-tagged p300. Immunoprecipitations were carried out using M73 anti-E1A antibody and the proteins were resolved on a 4–12% gradient SDS–PAGE and western blots performed for p300 using the RW128 anti-p300 antibody. Input levels are shown. ( C ) HeLa cells were transfected with plasmids expressing either wild-type genomic E1A, E1A 12S dl 1101 or E1A 13S dl 1101, and immunoprecipitated with a mix of M73 and M58 anti-E1A antibodies. The immunoprecipitated proteins were resolved on a 3–8% gradient SDS–PAGE and Western blots performed for p300 using the RW128 anti-p300 antibody. Input levels are shown.

    Article Snippet: U2OS cells were used for the GAL4-CR3 experiments instead of HeLa cells because of poor activity of the GAL4-CR3 fusion in HeLa cells.

    Techniques: Purification, Incubation, SDS Page, Staining, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation

    Knockdown of p300 by siRNA impairs activation by E1A 13S and CR3. ( A ) HeLa cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing E1A 13S and 1 μg of E4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection. ( B ) U2OS cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing the GAL4-CR3 fusion and 1 μg of GAL4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection.

    Journal: Nucleic Acids Research

    Article Title: Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP

    doi: 10.1093/nar/gkn1057

    Figure Lengend Snippet: Knockdown of p300 by siRNA impairs activation by E1A 13S and CR3. ( A ) HeLa cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing E1A 13S and 1 μg of E4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection. ( B ) U2OS cells were transfected with siRNA for p300 or a negative control siRNA and subsequently with 1 μg of plasmid expressing the GAL4-CR3 fusion and 1 μg of GAL4-luciferase reporter. Luciferase activity was assayed 5 days after the initial siRNA transfection.

    Article Snippet: U2OS cells were used for the GAL4-CR3 experiments instead of HeLa cells because of poor activity of the GAL4-CR3 fusion in HeLa cells.

    Techniques: Activation Assay, Transfection, Negative Control, Plasmid Preparation, Expressing, Luciferase, Activity Assay

    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

    In vitro cell invasion is stimulated by the SDF-1/CXCR4 pathway independently from HPV status. The modulation of E6 expression was monitored using Western-blot in HPV-positive HeLa (A) and TC-1 (B) cells and in HPV-negative B16F10 (C) cells, after 3 and 6 days of incubation with Cidofovir (CDF). Modulation of P53 expression was assessed in HeLa cells after CDF incubation (A). Cell invasion was measured using a Matrigel assay in HeLa (A), TC-1 (B) and B16F10 (C) cells. Recombinant human CXCL12/SDF-1 (100 ng/mL; R D Systems) was used as chemoattractant and modulation of cell migration was recorded after treatment with CXCR4-blocking antibody or/and Cidofovir (CDF). The invasion rate was determined by counting crystal violet-stained cells. Invasion was stimulated by SDF-1α/CXCR4 independently from the HPV status of the cells but Cidofovir anti-invasive action was restricted to the two HPV-positive cell lines. Three independent experiments with three chambers each time were performed. ***P

    Journal: PLoS ONE

    Article Title: Novel Anti-Metastatic Action of Cidofovir Mediated by Inhibition of E6/E7, CXCR4 and Rho/ROCK Signaling in HPV+ Tumor Cells

    doi: 10.1371/journal.pone.0005018

    Figure Lengend Snippet: In vitro cell invasion is stimulated by the SDF-1/CXCR4 pathway independently from HPV status. The modulation of E6 expression was monitored using Western-blot in HPV-positive HeLa (A) and TC-1 (B) cells and in HPV-negative B16F10 (C) cells, after 3 and 6 days of incubation with Cidofovir (CDF). Modulation of P53 expression was assessed in HeLa cells after CDF incubation (A). Cell invasion was measured using a Matrigel assay in HeLa (A), TC-1 (B) and B16F10 (C) cells. Recombinant human CXCL12/SDF-1 (100 ng/mL; R D Systems) was used as chemoattractant and modulation of cell migration was recorded after treatment with CXCR4-blocking antibody or/and Cidofovir (CDF). The invasion rate was determined by counting crystal violet-stained cells. Invasion was stimulated by SDF-1α/CXCR4 independently from the HPV status of the cells but Cidofovir anti-invasive action was restricted to the two HPV-positive cell lines. Three independent experiments with three chambers each time were performed. ***P

    Article Snippet: In addition, E6/E7 expression was stably knocked-down in HeLa cells using the pEBVsiRNA as previously described .

    Techniques: In Vitro, Expressing, Western Blot, Incubation, Matrigel Assay, Recombinant, Migration, Blocking Assay, Staining

    Silencing of E6 and E7 showed contribution of the oncoproteins to the pro-migratory phenotype of HeLa cells. (A) Quantitative RT-PCR analysis of E6 and E7 mRNA level and E6 protein expression in knock-down HeLa cells lines. E6 and E7 expression were respectively knocked-down using the pEBVsiRNA in 100, and 102, 103. Inhibition was statically significant P

    Journal: PLoS ONE

    Article Title: Novel Anti-Metastatic Action of Cidofovir Mediated by Inhibition of E6/E7, CXCR4 and Rho/ROCK Signaling in HPV+ Tumor Cells

    doi: 10.1371/journal.pone.0005018

    Figure Lengend Snippet: Silencing of E6 and E7 showed contribution of the oncoproteins to the pro-migratory phenotype of HeLa cells. (A) Quantitative RT-PCR analysis of E6 and E7 mRNA level and E6 protein expression in knock-down HeLa cells lines. E6 and E7 expression were respectively knocked-down using the pEBVsiRNA in 100, and 102, 103. Inhibition was statically significant P

    Article Snippet: In addition, E6/E7 expression was stably knocked-down in HeLa cells using the pEBVsiRNA as previously described .

    Techniques: Quantitative RT-PCR, Expressing, Inhibition

    In vitro MMR. ( A ) HeLa nuclear extracts from cells carrying empty vector (vector), wild type Polδ, PolδD316A;E318A or PolδD515V were analyzed by western blot. Western blot data are presented for MSH2, MLH1 and PCNA. ( B) Mutation frequency in HCT116 (MLH1-/-) cells overexpressing PolδD316A;E318A or PolδD515V. (C) In vitro MMR assay using 2 nt indel heteroduplex DNA substrates Δ2–3′ or Δ2–5′ (see Materials and Methods). (D) As in B except using chromosome 3 complemented HCT116 (HCT116+Chr3) cells. The data are the mean ± SD of three independent experiments. * P

    Journal: Nucleic Acids Research

    Article Title: Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

    doi: 10.1093/nar/gkx611

    Figure Lengend Snippet: In vitro MMR. ( A ) HeLa nuclear extracts from cells carrying empty vector (vector), wild type Polδ, PolδD316A;E318A or PolδD515V were analyzed by western blot. Western blot data are presented for MSH2, MLH1 and PCNA. ( B) Mutation frequency in HCT116 (MLH1-/-) cells overexpressing PolδD316A;E318A or PolδD515V. (C) In vitro MMR assay using 2 nt indel heteroduplex DNA substrates Δ2–3′ or Δ2–5′ (see Materials and Methods). (D) As in B except using chromosome 3 complemented HCT116 (HCT116+Chr3) cells. The data are the mean ± SD of three independent experiments. * P

    Article Snippet: To obtain the nuclear extracts from HeLa cells treated with siEXO1 and overexpressing Polδ or Polδ mutant proteins, approximately 1.25 × 106 cells were seeded in T75 flasks.

    Techniques: In Vitro, Plasmid Preparation, Western Blot, Mutagenesis

    Mismatch-provoked excision in HeLa cells expressing PolδD316A;E318A. (A) Mismatch provoked excision assay was performed as described in Materials and Methods using the nuclear extracts indicated. The extent of DNA excision was estimated by measuring susceptibility/resistance to cleavage by Eco RI, whose recognition sequence lies in between the DNA excision initiation site and the mismatch (schematic diagram right). Reaction products were digested with Eco RI and Bam HI, separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. The intensity of each band was quantified using ImageJ, and the relative excision capacity was calculated as the intensity of the slowest migrating (largest) reaction product per lane /total intensity per lane × 100. Nuclear extracts from MSH2-deficient LoVo cells were used as the negative control. (B) Western blot of pS 33 -RPA32 was performed to monitor amount of ssDNA generated during in vitro MMR. Briefly, MMR assay was performed as described in Materials and Methods with no substrate (NO), Δ2–3′ (3′) or Δ2–5′ (5′) substrate in the reaction. The MMR was terminated by adding SDS containing loading buffer and boiling at 95°C for 10 min. Western blot was performed as described in Materials and Methods. Aphidicolin was included and dNTPs were omitted as indicated for inhibition of polymerase synthesis of Polδ. The total RPA32 was used as control.

    Journal: Nucleic Acids Research

    Article Title: Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

    doi: 10.1093/nar/gkx611

    Figure Lengend Snippet: Mismatch-provoked excision in HeLa cells expressing PolδD316A;E318A. (A) Mismatch provoked excision assay was performed as described in Materials and Methods using the nuclear extracts indicated. The extent of DNA excision was estimated by measuring susceptibility/resistance to cleavage by Eco RI, whose recognition sequence lies in between the DNA excision initiation site and the mismatch (schematic diagram right). Reaction products were digested with Eco RI and Bam HI, separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. The intensity of each band was quantified using ImageJ, and the relative excision capacity was calculated as the intensity of the slowest migrating (largest) reaction product per lane /total intensity per lane × 100. Nuclear extracts from MSH2-deficient LoVo cells were used as the negative control. (B) Western blot of pS 33 -RPA32 was performed to monitor amount of ssDNA generated during in vitro MMR. Briefly, MMR assay was performed as described in Materials and Methods with no substrate (NO), Δ2–3′ (3′) or Δ2–5′ (5′) substrate in the reaction. The MMR was terminated by adding SDS containing loading buffer and boiling at 95°C for 10 min. Western blot was performed as described in Materials and Methods. Aphidicolin was included and dNTPs were omitted as indicated for inhibition of polymerase synthesis of Polδ. The total RPA32 was used as control.

    Article Snippet: To obtain the nuclear extracts from HeLa cells treated with siEXO1 and overexpressing Polδ or Polδ mutant proteins, approximately 1.25 × 106 cells were seeded in T75 flasks.

    Techniques: Expressing, Excision Assay, Sequencing, Agarose Gel Electrophoresis, Staining, Negative Control, Western Blot, Generated, In Vitro, Inhibition

    Effect of PolδD316A;E318A on susceptibility to killing by MNNG. ( A and B) HeLa cells were transfected and seeded as described in Materials and Methods. After one day, cells were treated with 10 μM O 6 -Benzylguanine and MNNG at the concentration indicated. Colonies were counted after 8 days after crystal violet staining. The percentage of survival for each drug concentration was calculated as number of colonies after MNNG treatment / number of colonies without MNNG treatment. The asterisk above the error bar indicates the P value between group siEXO1+Polδ and group siEXO1+PolδD316A;E318A. ( C and D ) Cells were seeded into T 75 flasks one day after transfection. After 24 hours, cells were treated with 10 μM O 6 -Benzylguanine and 0.2 μM MNNG and incubated for another two days before subject to FACS analysis. The data represent the mean ± SD of three independent experiments. * P ≤ 0.05.

    Journal: Nucleic Acids Research

    Article Title: Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

    doi: 10.1093/nar/gkx611

    Figure Lengend Snippet: Effect of PolδD316A;E318A on susceptibility to killing by MNNG. ( A and B) HeLa cells were transfected and seeded as described in Materials and Methods. After one day, cells were treated with 10 μM O 6 -Benzylguanine and MNNG at the concentration indicated. Colonies were counted after 8 days after crystal violet staining. The percentage of survival for each drug concentration was calculated as number of colonies after MNNG treatment / number of colonies without MNNG treatment. The asterisk above the error bar indicates the P value between group siEXO1+Polδ and group siEXO1+PolδD316A;E318A. ( C and D ) Cells were seeded into T 75 flasks one day after transfection. After 24 hours, cells were treated with 10 μM O 6 -Benzylguanine and 0.2 μM MNNG and incubated for another two days before subject to FACS analysis. The data represent the mean ± SD of three independent experiments. * P ≤ 0.05.

    Article Snippet: To obtain the nuclear extracts from HeLa cells treated with siEXO1 and overexpressing Polδ or Polδ mutant proteins, approximately 1.25 × 106 cells were seeded in T75 flasks.

    Techniques: Transfection, Concentration Assay, Staining, Incubation, FACS

    Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. HeLa cells were transfected with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. ( A ) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; ( B ) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; ( C ) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and ( D ) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.

    Journal: International Journal of Molecular Sciences

    Article Title: Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo

    doi: 10.3390/ijms160612243

    Figure Lengend Snippet: Synergistic effect of E6/E7-specific siRNA pools in combination with radiation on cervical cancer cells. HeLa cells were transfected with 20 nM of each selected siRNA derivative or siRNA pools (SP; 10 nM siRNA each) or in combination with γ-irradiation. Similarly, SiHa cells were transfected with 20 nM of each selected siRNA derivative or SP (7 nM siRNA each) or in combination with γ-irradiation. The number of cancer cells was determined by siRNAs alone (non-IR) or siRNAs in combination with γ-irradiation. Untransfected and control siRNA served as controls. ( A ) Trypan blue assay showing the number of viable cells transfected with siRNA pools, siRNA alone and in combination with γ-irradiation; ( B ) Western blotting analyses showing the expression of TP53 and HPV E7 proteins. M, mock; C, control siRNA; SP, siRNA pool; ( C ) Annexin-V and PI binding assay showing the percentages of apoptotic cells transfected with selected siRNA derivatives; and ( D ) The effect of HPV E6/E7-specific siRNAs on cellular senescence is also shown.

    Article Snippet: HeLa-Luc, a stably-transfected and bioluminescent human tumor cell line derived from HeLa cells, was obtained from Xenogen Corp. (Alameda, CA, USA).

    Techniques: Transfection, Irradiation, Western Blot, Expressing, Binding Assay

    Determining the stability and silencing activities of chemically-modified derivatives of HPV16- and 18 E6/E7-specific lead siRNAs. ( A ) Trypan blue assay showing the number of viable HeLa cells transfected with 2ʹ-OMe-modified derivatives of siRNA 426 and SiHa cells transfected with 2ʹ-OMe modified derivatives of siRNA 497. GFP-specific siRNA (control siRNA) served as controls; ( B ) Silencing efficiency of 2ʹ-OMe-modified siRNA derivatives on E7 expression and changes in TP53 expression were also analyzed by Western blotting. β-actin was used as a loading control; ( C ) E6 and ( D ) CDKN1A mRNA expression as determined by qRT-PCR; and ( E ) Gel electrophoresis analysis showing the serum stability of 2ʹ-OMe-modified siRNA derivatives. Unmodified (Lane 0) and modified siRNA 426 or 497 derivatives were analyzed by electrophoresis on 15% native polyacrylamide gels.

    Journal: International Journal of Molecular Sciences

    Article Title: Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo

    doi: 10.3390/ijms160612243

    Figure Lengend Snippet: Determining the stability and silencing activities of chemically-modified derivatives of HPV16- and 18 E6/E7-specific lead siRNAs. ( A ) Trypan blue assay showing the number of viable HeLa cells transfected with 2ʹ-OMe-modified derivatives of siRNA 426 and SiHa cells transfected with 2ʹ-OMe modified derivatives of siRNA 497. GFP-specific siRNA (control siRNA) served as controls; ( B ) Silencing efficiency of 2ʹ-OMe-modified siRNA derivatives on E7 expression and changes in TP53 expression were also analyzed by Western blotting. β-actin was used as a loading control; ( C ) E6 and ( D ) CDKN1A mRNA expression as determined by qRT-PCR; and ( E ) Gel electrophoresis analysis showing the serum stability of 2ʹ-OMe-modified siRNA derivatives. Unmodified (Lane 0) and modified siRNA 426 or 497 derivatives were analyzed by electrophoresis on 15% native polyacrylamide gels.

    Article Snippet: HeLa-Luc, a stably-transfected and bioluminescent human tumor cell line derived from HeLa cells, was obtained from Xenogen Corp. (Alameda, CA, USA).

    Techniques: Modification, Transfection, Expressing, Western Blot, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Electrophoresis

    Screening and systematic analysis of HPV18 E6/E7-specific siRNA in combination with radiation. ( A ) Trypan blue assay showing the number of viable HeLa cells transfected with library siRNAs (103, 426, 450, 456 and 458). In these studies, HeLa cells were transfected with 5 or 25 nM of each siRNA. The number of cells was compared to reagent alone without siRNAs (mock); ( B ) Changes in TP53 and HPV18 E7 expression levels in HeLa cells following transfection with HPV18 E6/E7-specific library siRNAs were detected by Western blotting. β-actin was used as a loading control; ( C ) Annexin-V binding assay showing the percentage of apoptotic HeLa cells transfected with siRNA 426 or 450 for 28 h or siRNAs in combination with γ-irradiation; ( D ) The effects of E6/E7-specific siRNA 426 or 450 in combination with γ-irradiation on cell viability and morphology are shown. Scale bar: all are 200 μm; and ( E ) The effect of HPV E6/E7-specific siRNAs alone or combined with γ-irradiation (IR) on cellular senescence, Scale bar: all are 200 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo

    doi: 10.3390/ijms160612243

    Figure Lengend Snippet: Screening and systematic analysis of HPV18 E6/E7-specific siRNA in combination with radiation. ( A ) Trypan blue assay showing the number of viable HeLa cells transfected with library siRNAs (103, 426, 450, 456 and 458). In these studies, HeLa cells were transfected with 5 or 25 nM of each siRNA. The number of cells was compared to reagent alone without siRNAs (mock); ( B ) Changes in TP53 and HPV18 E7 expression levels in HeLa cells following transfection with HPV18 E6/E7-specific library siRNAs were detected by Western blotting. β-actin was used as a loading control; ( C ) Annexin-V binding assay showing the percentage of apoptotic HeLa cells transfected with siRNA 426 or 450 for 28 h or siRNAs in combination with γ-irradiation; ( D ) The effects of E6/E7-specific siRNA 426 or 450 in combination with γ-irradiation on cell viability and morphology are shown. Scale bar: all are 200 μm; and ( E ) The effect of HPV E6/E7-specific siRNAs alone or combined with γ-irradiation (IR) on cellular senescence, Scale bar: all are 200 μm.

    Article Snippet: HeLa-Luc, a stably-transfected and bioluminescent human tumor cell line derived from HeLa cells, was obtained from Xenogen Corp. (Alameda, CA, USA).

    Techniques: Transfection, Expressing, Western Blot, Binding Assay, Irradiation

    A statistical analysis of the level of FITC fluorescence in HeLa cells transfected with nanoparticles, in the presence of Lipofectamine, GeneJuice or Matra, compared to untransfected HeLa cells. HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence or the presence of transfection reagents. The FITC fluorescence in the cells was quantified as described in the methodology, and the levels of fluorescence in nanoparticle-transfected cells compared to those in untransfected cells. This multi-level statistical analysis takes into account local variations within the cell population. The bars represent the region where there is a 95% probability that the mean fluorescence increase lies within it. The dot represents the mean calculated effect size. Bars not crossing the 1 line show significant evidence for an effect following transfection. The p-value indicates the probability that there was no difference in expression levels between the control and treatment samples. Hence, a lower p-value indicates a greater likelihood that there was a difference between the transfected and the untransfected cells.

    Journal: PLoS ONE

    Article Title: Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    doi: 10.1371/journal.pone.0099458

    Figure Lengend Snippet: A statistical analysis of the level of FITC fluorescence in HeLa cells transfected with nanoparticles, in the presence of Lipofectamine, GeneJuice or Matra, compared to untransfected HeLa cells. HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence or the presence of transfection reagents. The FITC fluorescence in the cells was quantified as described in the methodology, and the levels of fluorescence in nanoparticle-transfected cells compared to those in untransfected cells. This multi-level statistical analysis takes into account local variations within the cell population. The bars represent the region where there is a 95% probability that the mean fluorescence increase lies within it. The dot represents the mean calculated effect size. Bars not crossing the 1 line show significant evidence for an effect following transfection. The p-value indicates the probability that there was no difference in expression levels between the control and treatment samples. Hence, a lower p-value indicates a greater likelihood that there was a difference between the transfected and the untransfected cells.

    Article Snippet: Transfection and treatment of HeLa cells for hMT-IIa and hMT-IIA knockdown studies For each transfection, the required concentration of siRNA, ssRNA or ssDNA (Eurofins MWG, ) or ssDNA-functionalized nanoparticles were transfected into 2×105 HeLa cells using GeneJuice (Novagen) as recommended by the manufacturer's instructions.

    Techniques: Fluorescence, Transfection, HMT Assay, Sequencing, Expressing

    Fluoresence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in live HeLa cells. HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence (NanoParticles column) or the presence of the transfection reagents Matra, Lipofectamine 2000 or GeneJuice. Control cells were treated with neither nanoparticles nor transfection reagents. The same signal intensity range was used for all FITC images. The scale bar represents 50 microns.

    Journal: PLoS ONE

    Article Title: Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    doi: 10.1371/journal.pone.0099458

    Figure Lengend Snippet: Fluoresence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in live HeLa cells. HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence (NanoParticles column) or the presence of the transfection reagents Matra, Lipofectamine 2000 or GeneJuice. Control cells were treated with neither nanoparticles nor transfection reagents. The same signal intensity range was used for all FITC images. The scale bar represents 50 microns.

    Article Snippet: Transfection and treatment of HeLa cells for hMT-IIa and hMT-IIA knockdown studies For each transfection, the required concentration of siRNA, ssRNA or ssDNA (Eurofins MWG, ) or ssDNA-functionalized nanoparticles were transfected into 2×105 HeLa cells using GeneJuice (Novagen) as recommended by the manufacturer's instructions.

    Techniques: Microscopy, HMT Assay, Sequencing, Transfection

    Levels of hMT-IIa in HeLa cells transfected with increasing concentrations of hMT-IIa or control ssDNA. Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. All samples were induced samples with 12.5 µM CdCl 2 . Total hMT-IIa activity relative to B2M in cells transfected with varying levels of ssDNA was normalized to hMT-IIa- B2M expression in cells transfected with 0 nM ssDNA giving us a value for hMT-IIa activity in the presence of x nM ssDNA. The levels of hMT-IIa transcript in cells transfected with control ssDNA (C) was set to 1.0. The level of hMT-IIa gene expression (MT) in cells transfected with hMT-IIa ssDNA was compared to the levels of hMT-IIa in cells transfected with control ssDNA at each concentration providing a measure of the reduction in hMT-IIa activity in hMT-IIa ssDNA transfected compared to those transfected with control ssDNA. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. All samples were induced with 12.5 µM CdCl 2 . HeLa cells were transfected with hMT-IIa-specific ssDNA (ssDNA). Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in Table S2B .

    Journal: PLoS ONE

    Article Title: Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    doi: 10.1371/journal.pone.0099458

    Figure Lengend Snippet: Levels of hMT-IIa in HeLa cells transfected with increasing concentrations of hMT-IIa or control ssDNA. Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. All samples were induced samples with 12.5 µM CdCl 2 . Total hMT-IIa activity relative to B2M in cells transfected with varying levels of ssDNA was normalized to hMT-IIa- B2M expression in cells transfected with 0 nM ssDNA giving us a value for hMT-IIa activity in the presence of x nM ssDNA. The levels of hMT-IIa transcript in cells transfected with control ssDNA (C) was set to 1.0. The level of hMT-IIa gene expression (MT) in cells transfected with hMT-IIa ssDNA was compared to the levels of hMT-IIa in cells transfected with control ssDNA at each concentration providing a measure of the reduction in hMT-IIa activity in hMT-IIa ssDNA transfected compared to those transfected with control ssDNA. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. All samples were induced with 12.5 µM CdCl 2 . HeLa cells were transfected with hMT-IIa-specific ssDNA (ssDNA). Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in Table S2B .

    Article Snippet: Transfection and treatment of HeLa cells for hMT-IIa and hMT-IIA knockdown studies For each transfection, the required concentration of siRNA, ssRNA or ssDNA (Eurofins MWG, ) or ssDNA-functionalized nanoparticles were transfected into 2×105 HeLa cells using GeneJuice (Novagen) as recommended by the manufacturer's instructions.

    Techniques: HMT Assay, Transfection, Activity Assay, Expressing, Concentration Assay, Western Blot

    Levels of hMTIIa in HeLa cells transfected with gold nanoparticles. Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. Samples were treated with 12.5 µM CdCl 2 . The level of hMTIIa gene expression (normalized to B2M ) in untransfected and induced HeLa cells was defined as 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. Induced samples were treated with 12.5 µM CdCl 2 (+Cd). HeLa cells were transfected with unfunctionalized (−), control (C) or hMT-IIa (MT) ssDNA-functionalized nanoparticles. Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in Table S2C .

    Journal: PLoS ONE

    Article Title: Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    doi: 10.1371/journal.pone.0099458

    Figure Lengend Snippet: Levels of hMTIIa in HeLa cells transfected with gold nanoparticles. Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. Samples were treated with 12.5 µM CdCl 2 . The level of hMTIIa gene expression (normalized to B2M ) in untransfected and induced HeLa cells was defined as 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. Induced samples were treated with 12.5 µM CdCl 2 (+Cd). HeLa cells were transfected with unfunctionalized (−), control (C) or hMT-IIa (MT) ssDNA-functionalized nanoparticles. Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in Table S2C .

    Article Snippet: Transfection and treatment of HeLa cells for hMT-IIa and hMT-IIA knockdown studies For each transfection, the required concentration of siRNA, ssRNA or ssDNA (Eurofins MWG, ) or ssDNA-functionalized nanoparticles were transfected into 2×105 HeLa cells using GeneJuice (Novagen) as recommended by the manufacturer's instructions.

    Techniques: Transfection, HMT Assay, Expressing, Western Blot

    Confocal fluorescence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in fixed HeLa cells. HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the presence of Lipofectamine. The overall FITC signal was reduced so that the level of fluorescence from the observed circular bodies was not saturating. The distribution of the LAMP1 protein extends through the majority of the cytoplasmic area thus provides an impression of the cell. In the overlayed image, the magenta regions indicate the presence of cellular bodies in which the FITC signal from the nanoparticles overlaps with the signal for the LC3A/B autophagosomal protein marker. The scale bar represents 25 microns and the images shown are all from a single z-plane (a 0.7 micron section).

    Journal: PLoS ONE

    Article Title: Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    doi: 10.1371/journal.pone.0099458

    Figure Lengend Snippet: Confocal fluorescence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in fixed HeLa cells. HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the presence of Lipofectamine. The overall FITC signal was reduced so that the level of fluorescence from the observed circular bodies was not saturating. The distribution of the LAMP1 protein extends through the majority of the cytoplasmic area thus provides an impression of the cell. In the overlayed image, the magenta regions indicate the presence of cellular bodies in which the FITC signal from the nanoparticles overlaps with the signal for the LC3A/B autophagosomal protein marker. The scale bar represents 25 microns and the images shown are all from a single z-plane (a 0.7 micron section).

    Article Snippet: Transfection and treatment of HeLa cells for hMT-IIa and hMT-IIA knockdown studies For each transfection, the required concentration of siRNA, ssRNA or ssDNA (Eurofins MWG, ) or ssDNA-functionalized nanoparticles were transfected into 2×105 HeLa cells using GeneJuice (Novagen) as recommended by the manufacturer's instructions.

    Techniques: Fluorescence, Microscopy, HMT Assay, Sequencing, Transfection, Marker

    CUX1, YY1, EAP1, and TTF1 are recruited to different regions of the human EAP1 promoter in Hela cells, as assessed by ChIP assays Putative binding sites for three transcription factors (TTF1, CUX1, and YY1) implicated in the control of puberty were identified in silico (TESS, Vector NTI) and are shown by horizontal wide arrows. The direction of each arrow indicates the presence of each binding site on either the sense or antisense DNA strand. Potential EAP1 binding sites are not depicted, because it is currently unknown if EAP1 binds directly to DNA. Endogenous CUX1, as well as transfected HA-tagged EAP1 and untagged TTF1, strongly associates with the proximal promoter region of the EAP1 gene (Amplicon II). No association of YY1 to this region was detected. In contrast to the proximal promoter, an upstream region (Amplicon I) showed CUX1 and YY1 binding, but no EAP1 or TTF1 association. The endogenous proteins (CUX1 and YY1) were immunoprecipitated using specific monoclonal antibodies (see Material and Methods) and mouse IgGs as a negative control for the immunoprecipitation. To immunoprecipitate proteins produced by transfected expression vectors (EAP1-HA and TTF1), we used a mouse monoclonal antibody against HA (for EAP) and rabbit polyclonal antibodies (against TTF1). Chromatin prepared from cells transfected with the empty expression vector was used as the negative control. Inp: input DNA, chromatin precleared with protein A beads before immunoprecipitation. Transf = transfected.

    Journal: Molecular and Cellular Endocrinology

    Article Title: Transcription of the human EAP1 gene is regulated by upstream components of a puberty-controlling Tumor Suppressor Gene network

    doi: 10.1016/j.mce.2011.12.004

    Figure Lengend Snippet: CUX1, YY1, EAP1, and TTF1 are recruited to different regions of the human EAP1 promoter in Hela cells, as assessed by ChIP assays Putative binding sites for three transcription factors (TTF1, CUX1, and YY1) implicated in the control of puberty were identified in silico (TESS, Vector NTI) and are shown by horizontal wide arrows. The direction of each arrow indicates the presence of each binding site on either the sense or antisense DNA strand. Potential EAP1 binding sites are not depicted, because it is currently unknown if EAP1 binds directly to DNA. Endogenous CUX1, as well as transfected HA-tagged EAP1 and untagged TTF1, strongly associates with the proximal promoter region of the EAP1 gene (Amplicon II). No association of YY1 to this region was detected. In contrast to the proximal promoter, an upstream region (Amplicon I) showed CUX1 and YY1 binding, but no EAP1 or TTF1 association. The endogenous proteins (CUX1 and YY1) were immunoprecipitated using specific monoclonal antibodies (see Material and Methods) and mouse IgGs as a negative control for the immunoprecipitation. To immunoprecipitate proteins produced by transfected expression vectors (EAP1-HA and TTF1), we used a mouse monoclonal antibody against HA (for EAP) and rabbit polyclonal antibodies (against TTF1). Chromatin prepared from cells transfected with the empty expression vector was used as the negative control. Inp: input DNA, chromatin precleared with protein A beads before immunoprecipitation. Transf = transfected.

    Article Snippet: To detect CUX1, EAP1, YY1, and TTF1 binding to the 5′-flanking region of the human EAP1 gene in Hela cells we PCR-amplified two DNA fragments using two different sets of primers.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, In Silico, Plasmid Preparation, Transfection, Amplification, Immunoprecipitation, Negative Control, Produced, Expressing

    Neither low protein high carbohydrate diet ( LPHC ) nor high protein low carbohydrate ( HPLC ) diet altered neither skeletal muscle glycogen content nor cellular signaling. (A) Tibialis anterior ( TA ) muscle glycogen content. The color legend in panel A is shared with panel B. (B) TA muscle cellular signaling determined by western blotting. The signal intensities are presented relative to chow (dashed line). Representative blots and a coomassie loading control are shown to the left. For p70S6K T389 and GCN 2 T899, 25 μ g of total protein were loaded, and the positive controls were HEK 293 and 100 nmol/L Calyculin A‐treated HeLa cell lysates, respectively. Data are expressed as mean ± SEM . N = 7 for chow and n = 8 for LPHC /HPLC.

    Journal: Physiological Reports

    Article Title: Low‐ and high‐protein diets do not alter ex vivo insulin action in skeletal muscle. Low‐ and high‐protein diets do not alter ex vivo insulin action in skeletal muscle

    doi: 10.14814/phy2.13798

    Figure Lengend Snippet: Neither low protein high carbohydrate diet ( LPHC ) nor high protein low carbohydrate ( HPLC ) diet altered neither skeletal muscle glycogen content nor cellular signaling. (A) Tibialis anterior ( TA ) muscle glycogen content. The color legend in panel A is shared with panel B. (B) TA muscle cellular signaling determined by western blotting. The signal intensities are presented relative to chow (dashed line). Representative blots and a coomassie loading control are shown to the left. For p70S6K T389 and GCN 2 T899, 25 μ g of total protein were loaded, and the positive controls were HEK 293 and 100 nmol/L Calyculin A‐treated HeLa cell lysates, respectively. Data are expressed as mean ± SEM . N = 7 for chow and n = 8 for LPHC /HPLC.

    Article Snippet: For the cell culture positive control lysates, confluent 6‐well dishes with fed HEK293 cells were lysed in 100 μ L of lysis buffer, and HeLa cells lysates stimulated with 100 nmol/L calyculin A for 30 min was a gift from Abcam.

    Techniques: High Performance Liquid Chromatography, Western Blot

    Evaluation of NAMAs self-assembled on GONPs for target miR-21 imaging in single cells (HepG2, MCF-7 cells, Hela and L-02 cells).

    Journal: Chemical Science

    Article Title: Self-assembly of nucleic acid molecular aggregates catalyzed by a triple-helix probe for miRNA detection and single cell imaging of nucleic acid molecular aggregates catalyzed by a triple-helix probe for miRNA detection and single cell imaging †Electronic supplementary information (ESI) available: DNA and miRNA sequences, TEM images of CMFs, and the adsorption selectivity of GONPs for different types of nucleic acids. See DOI: 10.1039/c6sc00694a

    doi: 10.1039/c6sc00694a

    Figure Lengend Snippet: Evaluation of NAMAs self-assembled on GONPs for target miR-21 imaging in single cells (HepG2, MCF-7 cells, Hela and L-02 cells).

    Article Snippet: Reagents and materials MCF-7 and HeLa cells were purchased from the KeyGEN biotechnology Company (Nanjing, China).

    Techniques: Imaging

    Targeting labeling of cells using CPV-VLPs-QDs. DAPI-labeled nucleus (blue), QDs (red), and FITC-labeled goat anti-mouse secondary antibody (green). Anti-CPV mouse monoclonal antibody was used as the primary antibody. Hela and F81 cells show high uptake (red), whereas no obvious fluorescence signal was detected in BHK-21 cells.

    Journal: PLoS ONE

    Article Title: Quantum Dots Encapsulated with Canine Parvovirus-Like Particles Improving the Cellular Targeted Labeling

    doi: 10.1371/journal.pone.0138883

    Figure Lengend Snippet: Targeting labeling of cells using CPV-VLPs-QDs. DAPI-labeled nucleus (blue), QDs (red), and FITC-labeled goat anti-mouse secondary antibody (green). Anti-CPV mouse monoclonal antibody was used as the primary antibody. Hela and F81 cells show high uptake (red), whereas no obvious fluorescence signal was detected in BHK-21 cells.

    Article Snippet: When the concentration exceeded a threshold for imaging (60 μmol/mL), the cytotoxicity of CPV-VLPs-QDs on F81 and Hela cells was stronger than that on BHK-21 cells.

    Techniques: Labeling, Fluorescence

    Cytotoxicity of the CPV-VLPs-QDs and MPA-QDs in BHK-21(a), F81 (b), and Hela (c) cells. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated as [ A ] sample /[ A ] control × 100%, where [ A ] Sample is the absorbance of the test sample and [A] control is the absorbance of control sample. To determine 50% inhibitory concentration, namely, IC50, concentration—response curves were generated relative to the negative control. IC50 values were calculated from the non-linear regression analyses. In comparison with MPA-QDs, the encapsulated QDs, i.e., CPV-VLPs-QDs with low cytotoxicity ( d ), show that CPV-VLPs can be employed as safe delivery carriers. *p value

    Journal: PLoS ONE

    Article Title: Quantum Dots Encapsulated with Canine Parvovirus-Like Particles Improving the Cellular Targeted Labeling

    doi: 10.1371/journal.pone.0138883

    Figure Lengend Snippet: Cytotoxicity of the CPV-VLPs-QDs and MPA-QDs in BHK-21(a), F81 (b), and Hela (c) cells. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated as [ A ] sample /[ A ] control × 100%, where [ A ] Sample is the absorbance of the test sample and [A] control is the absorbance of control sample. To determine 50% inhibitory concentration, namely, IC50, concentration—response curves were generated relative to the negative control. IC50 values were calculated from the non-linear regression analyses. In comparison with MPA-QDs, the encapsulated QDs, i.e., CPV-VLPs-QDs with low cytotoxicity ( d ), show that CPV-VLPs can be employed as safe delivery carriers. *p value

    Article Snippet: When the concentration exceeded a threshold for imaging (60 μmol/mL), the cytotoxicity of CPV-VLPs-QDs on F81 and Hela cells was stronger than that on BHK-21 cells.

    Techniques: Cell Culture, Concentration Assay, Generated, Negative Control

    DNA laddering: apoptotic activity was detected by agarose gel electrophoretic separation of DNA fragmentation after exposure to rStx, A and B subunits. Concentration of A and B subunits was two times higher than rStx. DNA was isolated and analyzed by ethidium bromide/agarose gel electrophoresis. DNA fragmentation in a dose-dependent manner was shown after 16 h treatment with rStx ( AB ), A and B subunits. a DNA laddering in HeLa cell. b DNA laddering in Vero cell

    Journal: Cytotechnology

    Article Title: Study on induction of apoptosis on HeLa and Vero cells by recombinant shiga toxin and its subunits

    doi: 10.1007/s10616-009-9220-1

    Figure Lengend Snippet: DNA laddering: apoptotic activity was detected by agarose gel electrophoretic separation of DNA fragmentation after exposure to rStx, A and B subunits. Concentration of A and B subunits was two times higher than rStx. DNA was isolated and analyzed by ethidium bromide/agarose gel electrophoresis. DNA fragmentation in a dose-dependent manner was shown after 16 h treatment with rStx ( AB ), A and B subunits. a DNA laddering in HeLa cell. b DNA laddering in Vero cell

    Article Snippet: Vero and HeLa cells (National Cell Bank, Pasteur Institute of Iran) were grown in RPMI 1640 medium (Biosera), pH 7.4, supplemented with 5% heat-inactivated fetal bovine serum, (FBS) 1% (w/v) penicillin, and 1% (w/v) streptomycin at 37 °C in a 5% CO2 atmosphere.

    Techniques: DNA Laddering, Activity Assay, Agarose Gel Electrophoresis, Concentration Assay, Isolation

    Girdin regulates cell proliferation and apoptosis in HeLa cells. (A) Western blot analysis indicated that Girdin was suppressed in the Girdin shRNA group but not in the control after 48 h of transfection. (B) Cell proliferation detected by trypan blue staining indicated that Girdin shRNA caused significant attenuation of proliferation in HeLa cells at 72 h (P

    Journal: Oncology Letters

    Article Title: Girdin expression in cervical carcinoma and its role in the malignant properties of HeLa cells

    doi: 10.3892/ol.2016.4250

    Figure Lengend Snippet: Girdin regulates cell proliferation and apoptosis in HeLa cells. (A) Western blot analysis indicated that Girdin was suppressed in the Girdin shRNA group but not in the control after 48 h of transfection. (B) Cell proliferation detected by trypan blue staining indicated that Girdin shRNA caused significant attenuation of proliferation in HeLa cells at 72 h (P

    Article Snippet: shRNA methodology was used to examine the potential role of endogenous Girdin expression in HeLa cells, as previously described by Jiang et al ( ).

    Techniques: Western Blot, shRNA, Transfection, Staining

    Screening of candidate human PRE/TREs by dual luciferase reporter assay. ( A ) Overview of the screening strategy. Two plasmids were common for all samples: 2FRT-YY1pLuc and pGL4.74. Each of the candidate human PRE/TREs was inserted between the two FRT sequences in 2FRT-YY1pLuc to yield 2FRT-PRE/TRE-YY1pLuc. pGL4.74 contains a Renilla luciferase gene under the control of an upstream TK promoter. This allowed the signal of firefly luciferase to be normalized against that of Renilla luciferase from the same sample, yielding relative luciferase activity (RLU), to account for any variations in transfection and expression. In FLP(+) samples, an additional plasmid harboring the wild-type FLPe recombinase gene was co-transfected with the two luciferase vectors to allow for partial excision of candidate PRE/TRE at the two FRT sites. In FLP(m), a plasmid containing a C-terminally truncated, inactive FLPe (shown with an asterisk in the FLPe gene) was used in the transfection to serve as a negative control. The RLU values of the FLP(m) and FLP(+) samples were normalized against that of the FLP(-) for the same DNA insert to give rise to Normalized RLU. As PREs or TREs were supposed to repress or activate gene expression, respectively, the partial excision of inserted DNA fragments by FLPe in FLP(+) samples should increase the firefly luciferase signals for PREs, and decrease the firefly luciferase signals for TREs. ( B ) Results of dual luciferase assay in HeLa cells. The data were represented as mean ± SD (standard deviations) from at least three biological replicates that were performed on different samples on different days. The mean signal of the Gene Desert FLP(+) sample and its plus and minus 3*SD were shown as black dashed line or red or blue solid line, respectively. Candidate PRE/TREs were selected as PRE-like or TRE-like if the Normalized RLU values of their FLP(+) samples were above the red line or below the blue line, respectively.

    Journal: Nucleic Acids Research

    Article Title: Three classes of response elements for human PRC2 and MLL1/2–Trithorax complexes

    doi: 10.1093/nar/gky595

    Figure Lengend Snippet: Screening of candidate human PRE/TREs by dual luciferase reporter assay. ( A ) Overview of the screening strategy. Two plasmids were common for all samples: 2FRT-YY1pLuc and pGL4.74. Each of the candidate human PRE/TREs was inserted between the two FRT sequences in 2FRT-YY1pLuc to yield 2FRT-PRE/TRE-YY1pLuc. pGL4.74 contains a Renilla luciferase gene under the control of an upstream TK promoter. This allowed the signal of firefly luciferase to be normalized against that of Renilla luciferase from the same sample, yielding relative luciferase activity (RLU), to account for any variations in transfection and expression. In FLP(+) samples, an additional plasmid harboring the wild-type FLPe recombinase gene was co-transfected with the two luciferase vectors to allow for partial excision of candidate PRE/TRE at the two FRT sites. In FLP(m), a plasmid containing a C-terminally truncated, inactive FLPe (shown with an asterisk in the FLPe gene) was used in the transfection to serve as a negative control. The RLU values of the FLP(m) and FLP(+) samples were normalized against that of the FLP(-) for the same DNA insert to give rise to Normalized RLU. As PREs or TREs were supposed to repress or activate gene expression, respectively, the partial excision of inserted DNA fragments by FLPe in FLP(+) samples should increase the firefly luciferase signals for PREs, and decrease the firefly luciferase signals for TREs. ( B ) Results of dual luciferase assay in HeLa cells. The data were represented as mean ± SD (standard deviations) from at least three biological replicates that were performed on different samples on different days. The mean signal of the Gene Desert FLP(+) sample and its plus and minus 3*SD were shown as black dashed line or red or blue solid line, respectively. Candidate PRE/TREs were selected as PRE-like or TRE-like if the Normalized RLU values of their FLP(+) samples were above the red line or below the blue line, respectively.

    Article Snippet: Before transfection, HeLa cells were seeded in a 96-well plate at 20 000 cells/well with 100 μl/well DMEM medium (Lonza) and 10% FBS (HyClone), and incubated at 37°C with 5% CO2 .

    Techniques: Luciferase, Reporter Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Negative Control

    Immunoprecipitation of xDYRK1B . A , C-terminal sequences of human and Xenopus DYRK1A and DYRK1B. Amino acids identical with the immunogenic peptide used to raise the DYRK1B antibody are highlighted in bold. B , Immunoprecipitation of xDYRK1B. HeLa cells were transiently transfected with expression plasmids for xDYRK1B, GFP-DYRK1B or the empty vector ( control ). After immunoprecipitation with DYRK1B-specific antiserum ( αD1B ) or preimmune serum ( Pre ), bound proteins were subjected to Western blot analysis with a phosphotyrosine-specific antibody ( P-Tyr ) and the DYRK1B antiserum as indicated. Detection of IgG is shown as a loading control. The asterisk marks an unspecific band. C , Specificity of the DYRK1B antiserum. COS7 cells were transiently transfected with expression plasmids for the GFP fusion proteins of mammalian DYRK1A or DYRK1B as indicated on top of the lanes. Aliquots of the whole cell lysates ( input ) or proteins eluted from the immunoprecipitates ( αD1B, Pre ) were subjected to Western blot analysis with anti GFP and anti DYRK1B antibodies as indicated. Detection of IgG is shown as a loading control.

    Journal: BMC Biochemistry

    Article Title: Development of a sensitive non-radioactive protein kinase assay and its application for detecting DYRK activity in Xenopus laevis oocytes

    doi: 10.1186/1471-2091-11-20

    Figure Lengend Snippet: Immunoprecipitation of xDYRK1B . A , C-terminal sequences of human and Xenopus DYRK1A and DYRK1B. Amino acids identical with the immunogenic peptide used to raise the DYRK1B antibody are highlighted in bold. B , Immunoprecipitation of xDYRK1B. HeLa cells were transiently transfected with expression plasmids for xDYRK1B, GFP-DYRK1B or the empty vector ( control ). After immunoprecipitation with DYRK1B-specific antiserum ( αD1B ) or preimmune serum ( Pre ), bound proteins were subjected to Western blot analysis with a phosphotyrosine-specific antibody ( P-Tyr ) and the DYRK1B antiserum as indicated. Detection of IgG is shown as a loading control. The asterisk marks an unspecific band. C , Specificity of the DYRK1B antiserum. COS7 cells were transiently transfected with expression plasmids for the GFP fusion proteins of mammalian DYRK1A or DYRK1B as indicated on top of the lanes. Aliquots of the whole cell lysates ( input ) or proteins eluted from the immunoprecipitates ( αD1B, Pre ) were subjected to Western blot analysis with anti GFP and anti DYRK1B antibodies as indicated. Detection of IgG is shown as a loading control.

    Article Snippet: Cell culture and transfection HeLa cells were cultured in Quantum 101 Medium for HeLa cells (PAA Laboratories, Pasching, Austria) and COS7 cells in DMEM medium supplemented with 10% fetal calf serum (PAA) at 37°C in a humidified 5% CO2 atmosphere.

    Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Western Blot

    NleC inhibits NFκB-dependent protein expression and IL-8 secretion. A , relative change in NFκB-dependent luciferase activity in pEGFP (empty vector control)-transfected HeLa cells following treatment with NFκB activation inhibitor (Calbiochem product no. 481406). B , fold increase in NFκB-dependent luciferase activity. C , representative immunoblot showing similar expression levels of eGFP and eGFP-NleC in transfected cells. D , IL-8 secretion levels in untreated or TNFα-treated HeLa cells transfected with pEGFP-NleC or pEGFP vectors. Transfection with pEGFP-NleC inhibits luciferase activity and IL-8 secretion. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. **, p ≤ 0.01; ***, p ≤ 0.005 as compared with empty vector control.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: NleC inhibits NFκB-dependent protein expression and IL-8 secretion. A , relative change in NFκB-dependent luciferase activity in pEGFP (empty vector control)-transfected HeLa cells following treatment with NFκB activation inhibitor (Calbiochem product no. 481406). B , fold increase in NFκB-dependent luciferase activity. C , representative immunoblot showing similar expression levels of eGFP and eGFP-NleC in transfected cells. D , IL-8 secretion levels in untreated or TNFα-treated HeLa cells transfected with pEGFP-NleC or pEGFP vectors. Transfection with pEGFP-NleC inhibits luciferase activity and IL-8 secretion. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. **, p ≤ 0.01; ***, p ≤ 0.005 as compared with empty vector control.

    Article Snippet: Luciferase and IL-8 Assays Approximately 24 h post-transfection, HeLa cells were either left untreated or treated with TNFα (final concentration, 50 ng/ml) for 30 min prior to addition of luciferase cell culture lysis buffer (Stratagene) following the manufacturer's protocol.

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

    NleC inhibits NFκB function by targeting is constituents. Fold increase in NFκB-dependent luciferase activity of Hela cells co-transfected with vectors encoding pEGFP or pEGFP-NleC and NFκB pathway components TRAF2, IKKα, or IKKβ ( A ) and p65 ( C ). Transfection with pEGFP-NleC inhibits luciferase activity associated with plasmid expression of all interrogated IKK pathway components. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. ***, p ≤ 0.005 as compared with empty vector control. B , representative immunoblot probing for actin, IKK (α and β), p65, and p50 demonstrates similar levels of IKK and loading control, actin, with decreased cellular levels of p65 and p50 in pEGFP-NleC-transfected cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: NleC inhibits NFκB function by targeting is constituents. Fold increase in NFκB-dependent luciferase activity of Hela cells co-transfected with vectors encoding pEGFP or pEGFP-NleC and NFκB pathway components TRAF2, IKKα, or IKKβ ( A ) and p65 ( C ). Transfection with pEGFP-NleC inhibits luciferase activity associated with plasmid expression of all interrogated IKK pathway components. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. ***, p ≤ 0.005 as compared with empty vector control. B , representative immunoblot probing for actin, IKK (α and β), p65, and p50 demonstrates similar levels of IKK and loading control, actin, with decreased cellular levels of p65 and p50 in pEGFP-NleC-transfected cells.

    Article Snippet: Luciferase and IL-8 Assays Approximately 24 h post-transfection, HeLa cells were either left untreated or treated with TNFα (final concentration, 50 ng/ml) for 30 min prior to addition of luciferase cell culture lysis buffer (Stratagene) following the manufacturer's protocol.

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

    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