Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: cAMP increases Na+/K+-ATPase activity, protein abundance at the plasma membrane and the distance traveled by the Na+/K+-ATPase-containing vesicles in A549-GFPα1 cells. (A) A549-GFPα1 cells were incubated in the absence (CT) or presence of 50 μM forskolin (FSK) for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three experiments. (B) A549-GFPα1 cells were incubated as in A, and the Na+/K+-ATPase abundance at the basolateral plasma membrane was determined by western blot of the BLM fraction using a specific antibody against GFP. E-cadherin was used as a loading control. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (C) The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Vesicles were randomly selected from those that showed plus-end-directed displacement. Left panel shows a representative image of A549-GFPα1 cells. Arrowhead indicates the vesicle whose trajectory is shown in the right panel before (CT) and after FSK treatment (FSK). (D) Average contour length traveled by the vesicles as a function of time. The black line represents control vesicles; at 60 seconds, upon addition of FSK (red line), the vesicles move at a faster rate. The average contour length is determined by averaging over many trajectories as described in the Materials and Methods. **P<0.01; ***P<0.001. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Activity Assay, Incubation, Western Blot, Labeling, Single-particle Tracking, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The three isoforms of myosin-V are expressed in A549 cells. (A) RT-PCR using mRNA obtained from A549 and HeLa cells. Primers used for the amplification are described in supplementary material Table S6. (B) Cell lysates from A549 and HeLa cells were obtained and analyzed by western blot with specific antibodies against the three myosin-V isoforms. A representative western blot is shown. (C) The particulate fraction (100,000 g pellet) of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. Rab5 and Rab7 are used as markers of early and late endosomes, respectively. (D) Gradients obtained in C were scanned and the marker content was digitally quantified as indicated. Results are expressed as percentage of the total amount of protein.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Marker

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and myosin-Vc colocalize with Na+/K+-ATPase. (A) A549-GFPα1 cells were incubated in the absence or presence of 50 μM FSK for 10 minutes, basolateral membranes (BLM) and intracellular compartments (IC) were isolated and the Na+/K+-ATPase abundance was determined by western blot using a specific antibody against GFP. E-cadherin and actin were used as loading controls for the BLM and IC fractions, respectively. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (B) The IC fraction of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. C+, positive control.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Incubation, Isolation, Western Blot, Positive Control

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a myosin-Va stalk-tail. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Va that has a m-cherry-tag (red) (m-cherry-DN-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (B) Average contour length traveled by the vesicles in A as a function of time. The black line represents the control vesicles; FSK was added at time 60 seconds and is represented as a red line. (C) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Vc that has a m-cherry-tag (red) (m-cherry-DN-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (D) Average contour length traveled by the vesicles in C as a function of time. The black line represents control vesicles; FSK was added at 60 seconds and is represented as the red line. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, Imaging, Transfection, Dominant Negative Mutation, Labeling

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a shRNA against myosin-Va. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Va that has a m-cherry-tag (red) (m-cherry-sh-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions. (B) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Vc that has a m-cherry-tag (red) (m-cherry-shRNA-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions (C). Graph represents the average contour length traveled by the vesicles as a function of time, calculated as described in methods. The black line represents the m-cherry-sh-Va vesicles and the red line, the m-cherry-sh-Vc vesicles. (D) A549-GFPα1 cells were transfected with a shRNA against myosin-Va or myosin-Vc, cell lysates were isolated and the myosin-Va (left panel) or myosin-Vc (right panel) abundance was determined by western blot using specific antibodies. E-cadherin and tubulin were used as loading controls. Scale bars: 10 μm and 4 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, shRNA, Imaging, Transfection, Labeling, Isolation, Western Blot

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Dominant-negative myosin-Va mimics cAMP-mediated Na+/K+-ATPase increased activity and recruitment to the plasma membrane in A549-GFPα1 cells. (A) Stable clones expressing myosin-Va tail (DN-Va) and myosin-Vc tail (DN-Vc) were generated as described. Expression of the constructs in the permanent clones was analyzed by western blotting using and antibody against the V5 tag. A representative western blot is shown. (B) A549-GFPα1 cells (CT) and A549-GFPα1 cells permanently transfected with DN-Va and DN-Vc were incubated in the absence or presence of 50 μM FSK for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three different experiments. (C) Control (CT), DN-Va and DN-Vc cells were incubated in the absence or presence of 50 μM FSK for 10 minutes and western blots of the basolateral membrane fraction were performed using a specific antibody against GFP. E-cadherin was used as loading control. A representative western blot is shown. *P<0.05; **P<0.01; n.s., not significant; u.s., unstimulated.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Dominant Negative Mutation, Activity Assay, Clone Assay, Expressing, Generated, Construct, Western Blot, Transfection, Incubation

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and the Na+/K+-ATPase-containing vesicles colocalize. A549-GFPα1 cells were fixed, permeabilized and blocked. Myosin-Va was visualized by using an anti-myosin-Va antibody and a secondary antibody labeled with Alexa Fluor 568. GFP was directly visualized. Cellular distribution of Na+/K+-ATPase-GFPα1 and myosin-Va was analyzed using a Zeiss LSM 510 laser-scanning confocal microscope and colocalization (blue) was determined using the LSM 510 Meta software.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Labeling, Microscopy, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Microtubules and actin filaments are involved in Na+/K+-ATPase traffic. (A) Live imaging of A549 cells incubated with 10 μM nocodazole for 3 hours. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the microtubule cytoskeleton in control (left) and nocodazole (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and nocodazole (right) conditions. (B) Live imaging of A549 cells incubated with 5 μM cytochalasin D (Cyto D) for 1 hour. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the actin cytoskeleton under control (left) and cytochalasin D (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and cytochalasin D (right) conditions. (C) Average contour length traveled by the vesicles as a function of time. The blue line represents the control vesicles; the black line, cells treated with cytochalasin D and the red line, cells treated with nocodazole. Scale bars: 10 μm and 2 μm (inset images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Imaging, Incubation, Labeling, Single-particle Tracking, Software, Immunofluorescence

Journal: Molecular Biology of the Cell

Article Title: Structure and functional studies of N-terminal Cx43 mutants linked to oculodentodigital dysplasia

doi: 10.1091/mbc.E12-02-0128

Figure Lengend Snippet: N-terminal–linked Cx43 mutants do not make functional intercellular channels and exhibit dominant-negative properties on coexpressed endogenous Cx43. HeLa (A) or NRK (B) cells expressing Cx43 or mutants were pressure microinjected with 5% Lucifer yellow, and the incidence of dye transfer to neighboring cells was assessed ( n > 20 for each group). (C) N2A cells expressing wild-type or Cx43 mutants were patch-clamped to measure electrical coupling conductance. In all cases, the mutants failed to form functional gap junction channels (A, C) and exhibited dominant-negative properties on coexpressed Cx43 (B). WT, untransfected wild-type cells; control in C, untransfected N2A cells.

Article Snippet: NRK, mouse N2A, and human cervical tumor (HeLa) cells obtained from the American Type Culture Collection (Manassas, VA) were grown in high-glucose (4500 mg glucose/l) DMEM (Invitrogen, Burlington, Canada) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l -glutamine (except N2A cells).

Techniques: Functional Assay, Dominant Negative Mutation, Expressing, Control

Journal: Molecular Biology of the Cell

Article Title: Structure and functional studies of N-terminal Cx43 mutants linked to oculodentodigital dysplasia

doi: 10.1091/mbc.E12-02-0128

Figure Lengend Snippet: The G2S, but not the G2V or W4A, mutant forms functional gap junctions. (A) NRK and HeLa cells were untreated (WT) or engineered to express W4A-GFP or G2S-GFP before microinjection with 5% Lucifer yellow and assessment of dye transfer. (B) Gap junctional intercellular communication–deficient N2A cells were engineered to express Cx43-GFP, W4A-GFP, G2V-GFP, or G2S-GFP. Patch-clamp analysis was used to assess gap junction coupling conductance. Whereas the W4A and G2V mutants did not form functional gap junction channels, the G2S mutant exhibited functional conductance that even exceeded that of Cx43. The numbers above each column depict the number of cell pairs recorded.

Article Snippet: NRK, mouse N2A, and human cervical tumor (HeLa) cells obtained from the American Type Culture Collection (Manassas, VA) were grown in high-glucose (4500 mg glucose/l) DMEM (Invitrogen, Burlington, Canada) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l -glutamine (except N2A cells).

Techniques: Mutagenesis, Functional Assay, Microinjection, Patch Clamp

Journal: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 1. Combined treatment for 4 h with EPA-FFA, EGCG and GS in CRC cells had effects on mTOR pathway. (A) HCT116 cells were treated with EPA- FFA (0–150 µM), EGCG (0–175 µM), GS (0–15 µM) and the number of viable cells compared with the control (%) was assessed with MTT assay (ANOVA P = 0.0016, **P < 0.01 Dunnett’s test, n = 3) (left). mTOR downstream targets P-p70S6K, p70S6K, P-4EBP1, 4EBP1 were detected by western blotting on cells treated with compounds alone or in combinations or with Rapamycin. Statistical significance was tested only on HCT116 cells treated with EPA-FFA 150 µM, EGCG 175 µM, GS 15 µM or Rapamycin compared with the control (untreated cells) using one-way ANOVA (P = 0.0285 for P-p70S6K on logarithmic transformed data and P = 0.0347 for P-4EBP1) followed by Tukey’s test, n = 4 (right). (B) SW480 cells were treated with EPA-FFA, EGCG and GS and the number of viable cells compared with the control was evaluated with MTT assay (ANOVA P = 0.0004, *P < 0.05; ***P < 0.001 Dunnett’s test, n = 3) (left). mTOR downstream targets P-p70S6K, p70S6K, P-4EBP1, 4EBP1 were assessed upon treatment. Statistical significance was tested on SW480 cells treated with EPA-FFA 150 µM, EGCG 175 µM, GS 15 µM or Rapamycin 20 nM compared with the control (untreated cells) using one-way ANOVA (P = 0.0294 for P-p70S6K on logarithmic transformed data and P = 0.0008 for P-4EBP1) followed by Tukey’s test, n = 4 (right).

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Control, MTT Assay, Western Blot, Transformation Assay

Journal: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 2. Effect of EPA-FFA, EGCG and GS on mRNA translation in HCT116 cells. (A) Polysomal profiles on control and treated cells are shown. Statistical significance was assessed using unpaired t-test (P = n.s., n = 2) (B) mRNA levels of L5, L11, L13, c-Myc and cyclin D1 were assessed by qRT-PCR both in total RNA and in polysomal associated RNA. Statistical significance was assessed using one sample t-test, n = 2, using 100 (control) as the reference value.

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Control, Quantitative RT-PCR

Journal: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 3. Effect of EPA-FFA, EGCG and GS on c-Myc and cyclin D1. c-Myc and cyclin D1 proteins levels were assessed in cell extracts treated with EPA- FFA+EGCG+GS or Rapamycin from (A) HCT116 or (B) SW480 cells. Analyses were performed on logarithmic transformed data for HCT116. After the ANOVA global test (P = 0.0190, n = 4, HCT116; P = n.s., n = 4), Tukey’s post hoc test was used for pairwise comparisons.

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Transformation Assay

Journal: Carcinogenesis

Article Title: A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

doi: 10.1093/carcin/bgu173

Figure Lengend Snippet: Fig. 4. Effect of EPA-FFA, EGCG and GS on cell proliferation and apoptosis in HCT116 and SW480 cells. (A) Clonogenic assay on HCT116 and SW480 cells treated with EPA-FFA+EGCG+GS or Rapamycin (ANOVA P = 0.0012 and P = 0.0017 for HCT116 and SW480, respectively; Tukey’s test was applied as post hoc test, n = 3). (B) Cell cycle analysis (ANOVA P = 0.0059 for HCT116 and P = n.s. for SW480, n = 3, Dunnett’s test) (C) Tunnel assay: representative pictures and quantification. Statistical significance was tested using one-way ANOVA (P = 0.0447 for HCT116 and P = 0.0011 for SW480, followed by Dunnett’s test for comparison with the control cells, n = 2). Ctrl = control; Comb = Combination; Rapa = Rapamycin.

Article Snippet: The human CRC cell lines HCT116 (PIK3CA mutant, Rapamycin sensitive, p53 wild-type) and SW480 (PIK3CA wild-type, Rapamycin resistant, p53 mutant) were obtained from ATCC (Manassas, VA) and cultured in IMDM Abbreviations: CRC, colorectal cancer; EPA-FFA, eicosapentaenoic acidfree fatty acid; EGCG, epigallocatechin-3-gallate; GS, grape seed; mRNA, messenger RNA; mTOR, mammalian target of rapamycin.

Techniques: Clonogenic Assay, Cell Cycle Assay, Comparison, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: LAI-1 and lysosomes subcellular colocalization in A549 cell line. Live cell imaging fluorescence microscopy was performed. ( A ) Differential interference contrast of A549 cells. ( B ) Lysotracker Green labelling lysosomes (green channel). ( C ) A total of 10 µM of LAI-1 treatment after 3 h (red channel). ( D ) Merged channel showing lysosomes and LAI-1 colocalization (orange). Images are representative of three independent experiments. Scale bar 30 µm. Colocalization between Lysotracker and LAI-1 was quantified using Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC).

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Live Cell Imaging, Fluorescence, Microscopy

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Cell viability dose–response curves, evaluated with MTT assay, for all cell lines (A549, SW900 and DMS53) after 24 h incubation with chloroquine (CQ), 3-Methyladenine (3-MA) and LAI-1 treatment at different concentrations. Each point represents the mean value ± SD.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: MTT Assay, Incubation

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Inhibitory concentration (IC); IC 25 , IC 50 and IC 75 values after LAI-1 treatment for A549, SW900 and DMS53 cell lines. Data show mean value ± SD.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Concentration Assay

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Autophagy markers after treatment with different autophagy inhibitors. ( A ) Western blot showing autophagy-related proteins LC3 and p62/SQSTM1 after 24 h with chloroquine (CQ, 150 µM), 3-Methyladenine (3-MA, 10 mM) and LAI-1 (10 µM) treatment in A549 cells. ( B ) LC3-I and LC3-II protein levels and their LC3-II/I ratios. ( C ) p62/SQSTM1 protein expression. Protein expression was normalized using GAPDH as loading control. Fold induction against control group (CT) was calculated. Figure shows mean ± SEM. Statistical differences against CT are shown as *** p < 0.001, ** p < 0.01 and * p < 0.05. The whole western blot figures are showed in .

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Western Blot, Expressing, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Autophagy modulation after LAI-1 treatment. ( A ) Dose–response Western blot showing autophagy-related proteins LC3 and p62/SQSTM1 after 24 h of LAI-1 treatment at different concentrations (10, 15 and 20 µM) in A549, SW900 and DMS53 cell lines. ( B ) LC3-I and LC3-II protein levels and their LC3-II/I ratios after dose–response assay. ( C ) p62/SQSTM1 protein expression after dose–response experiment. ( D ) Time-course Western blot showing autophagy-related proteins LC3 and p62/SQSTM1 after treating A549 cells with LAI-1 (10 µM) at different time points (0, 4, 8, 16, 24, 48 h). ( E ) LC3-I and LC3-II protein levels and their LC3-II/I ratios after time-course experiment. ( F ) p62/SQSTM1 protein expression after time-course assay. ( G ) Time-course Western blot showing autophagy activation proteins Akt and mTOR after treating A549 cells with LAI-1 (10 µM) at different time points (0, 4, 8 h). ( H ) Akt and phospho-Akt at S473 (pAkt) protein levels after time-course experiment. ( I ) mTOR and phospho-mTOR at S2778 (pmTOR) protein expression after time-course assay. Protein expression was normalized using GAPDH as loading control. Fold induction against control group (CT) was calculated. Figure shows mean ± SEM. Statistical differences against CT are shown as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The whole western blot figures are showed in .

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Western Blot, Expressing, Activation Assay, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Subcellular localization of LC3 and LAMP-1. A549 cells were harvested for 24 h on coverslips and then treated with LAI-1 (15 µM) and chloroquine (CQ, 50 µM) for different times. The fusion between autophagosomes, marked with LC3 (green), and lysosomes, marked with LAMP-1 (red), was studied in merged images. Zoomed images were used to better observe this process. The localization and intensity of LC3 staining were also analyzed. DAPI (blue) staining was used for nuclear localization. Images are representative of three independent experiments. Scale bar 30 µm.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Staining

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Lysosomal and intracellular pH modifications using LAI-1 treatment. ( A ) A549 cells were treated for 1 h with LAI-1 or chloroquine (CQ) at different concentrations. After that, acridine orange staining (5 µg/mL) was performed for 30 min at room temperature. Images are representative of three independent experiments. Scale bar 50 µm. ( B ) Intracellular pH measurement in A549 cells treated with different LAI-1 concentrations for 1 h. Staining with pH Rodo Red AM staining kit and a calibration curve was conducted to quantify intracellular pH. Figure shows mean ± SEM. Statistical differences against control group (CT) are shown as ** p < 0.01.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Staining, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Cell death characterization using flow cytometry. The different cell lines (A549, SW900 and DMS53) were treated for 24 h with different LAI-1 concentrations. Cells were stained with Annexin-V APC/ Sytox Green kit. Early apoptotic cells were defined as Annexin-positive and Sytox-negative, whereas late apoptotic or necrotic cells were defined as Annexin- and Sytox-positive. ( A ) Representative plots. ( B ) Quantification of % of cells in early apoptosis and in late apoptosis or necrosis after different treatment. Statistical differences against control group (CT) are shown as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Flow Cytometry, Staining, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Expression of apoptotic and cell-cycle related proteins. The different cell lines (A549, SW900 and DMS53) were treated for 24 h with different LAI-1 concentrations and several proteins were assessed with Western blot ( A ). Quantification of procaspase 3 ( B ), PARP ( C ), cleaved-PARP ( D ), p21 ( E ) and p53 ( F ) protein expression after Western blot analysis was performed using GAPDH expression as loading control. Fold induction against control group (CT) was calculated. Figures show mean ± SEM. Statistical differences against CT are shown as * p < 0.05, ** p < 0.01 and *** p < 0.001. The whole western blot figures are showed in .

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: Expressing, Western Blot, Control

Journal: Cancers

Article Title: A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer

doi: 10.3390/cancers14143387

Figure Lengend Snippet: Cell viability, evaluated with MTT assay, for A549, SW900 and DMS53 lung cancer cell lines after 24 h incubation with cisplatin (CisPt, 40 µM), LAI-1 (15 µM) and the combination. Figure shows mean ± SEM. Statistical differences are shown as **** p < 0.0001 ** p < 0.01 and * p < 0.05.

Article Snippet: Human lung cancer cell lines A549 (adenocarcinoma, KRAS-mutated), DMS53 (small cell carcinoma, p53-mutated) and SW900 (squamous carcinoma, KRAS- and p53-mutated) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and maintained in DMEM (A549) or RPMI (DMS53 and SW900) medium (Biological Industries, Beit Haemek, Israel).

Techniques: MTT Assay, Incubation

Journal: PLoS ONE

Article Title: Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

doi: 10.1371/journal.pone.0158698

Figure Lengend Snippet: BRAF mutation status determined by HRM using DNA treated with BSA or purified using the NucleoSpin ® Kit.

Article Snippet: The human melanin-free 1676 melanoma cell line and the BRAF -wildtype LNCaP and the BRAF -mutated WiDR cell lines were obtained from the American Type Culture Collection (Manassas, USA) and cultured as recommended.

Techniques: Mutagenesis, Purification

Journal: PLoS ONE

Article Title: Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

doi: 10.1371/journal.pone.0158698

Figure Lengend Snippet: Comparison of BSA and NucleoSpin ® Kit procedures for routine detection of BRAF mutations.

Article Snippet: The human melanin-free 1676 melanoma cell line and the BRAF -wildtype LNCaP and the BRAF -mutated WiDR cell lines were obtained from the American Type Culture Collection (Manassas, USA) and cultured as recommended.

Techniques: Comparison, Mutagenesis, Amplification

Journal: PLoS ONE

Article Title: Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

doi: 10.1371/journal.pone.0158698

Figure Lengend Snippet: Combinaison of the Nucleospin ® Kit and BSA treatment for routine detection of BRAF mutation in highly pigmented samples.

Article Snippet: The human melanin-free 1676 melanoma cell line and the BRAF -wildtype LNCaP and the BRAF -mutated WiDR cell lines were obtained from the American Type Culture Collection (Manassas, USA) and cultured as recommended.

Techniques: Mutagenesis

Journal: The Journal of biological chemistry

Article Title: c-Myb trans-activates the human DNA topoisomerase IIalpha gene promoter.

doi: 10.1074/jbc.272.10.6278

Figure Lengend Snippet: FIG. 6. A dominant negative c-Myb peptide abrogates endoge- nous and exogenous c-Myb-mediated trans-activation of the hu- man topo IIa promoter. A, HL-60 cells were co-transfected with 2562TOP2LUC (20 mg), CMV-b-gal reference plasmid (1 mg), and 1 mg of either the empty CMV vector, CMV-myb, or CMV-mybDNRD. Sam- ples in each group also received either 1 mg of CMV vector, 1 mg of pSCDMS/MenT (a CMV-driven expression vector encoding the c-Myb DBD fused to the D. engrailed transcriptional repressor (34)) or 5 mg of pCMV-mybDBD (a passive dominant negative effector encoding the c-Myb DBD alone (34)). Cells were harvested at 24 h and processed for measurement of each reporter enzyme activity. Data is represented as -fold luciferase activity relative to the basal reporter activity in the absence of any effector plasmids. B, HL-60 and HeLa cells were co- transfected with 2562TOP2LUC (20 mg), CMV-b-gal reference plasmid (1 mg), and the indicated amounts of pSCDMS/MenT. Controls received 1 mg (HL-60) or 2 mg (HeLa) of empty CMV vector. Cells were processed for measurement of corrected reporter activity as in panel A.

Article Snippet: Cell Culture—HL-60 human promyelocytic leukemia cells (ATCC CCL 240), U937 histiocytic leukemia cells (ATCC CRL 1593), CCRFCEM lymphoblastic leukemia cells (ATCC 119), and HeLa S3 human cervical carcinoma cells (ATCC CCL 2.2) were obtained from the American Type Culture Collection (Rockville, MD).

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

Journal: The Journal of biological chemistry

Article Title: c-Myb trans-activates the human DNA topoisomerase IIalpha gene promoter.

doi: 10.1074/jbc.272.10.6278

Figure Lengend Snippet: FIG. 7. c-Myb trans-activation of 2562TOP2LUC in leukemia and epithelial cell lines. The T-cell leukemia (CCRF-CEM), promonocytic leukemia (U937), and cervical epithelial carcinoma (HeLa) lines were independently co-transfected with 2562TOP2LUC (20 mg), CMV-b-gal reference plasmid (1 mg), and the indicated amounts of CMV-myb expression plasmid. The control group in each case (0 mg of CMV-myb) received 2 mg of the empty CMV vector. Cells were harvested at 24 h and processed for measurement of each reporter enzyme activity. Numbers above each bar represent the -fold increase over each respective CMV only control.

Article Snippet: Cell Culture—HL-60 human promyelocytic leukemia cells (ATCC CCL 240), U937 histiocytic leukemia cells (ATCC CRL 1593), CCRFCEM lymphoblastic leukemia cells (ATCC 119), and HeLa S3 human cervical carcinoma cells (ATCC CCL 2.2) were obtained from the American Type Culture Collection (Rockville, MD).

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

Journal: The Journal of biological chemistry

Article Title: c-Myb trans-activates the human DNA topoisomerase IIalpha gene promoter.

doi: 10.1074/jbc.272.10.6278

Figure Lengend Snippet: FIG. 8. Topo IIa promoter trans-activation by B-Myb. HL-60 and HeLa cells were independently co-transfected with 2562TOP2LUC (20 mg), RSV-b-gal reference plasmid (1 mg), and the indicated amounts of a CMV-B-myb expression plasmid (pKCB-myb). A b-gal internal control driven by the RSV promoter was required since the standard CMV-b-gal control was activated by B-Myb. The control group in each case received 15 mg of the empty CMV vector and each B-Myb group received an amount of CMV vector to bring the total to 15 mg. Cells were harvested at 24 h and processed for measurement of each reporter enzyme activity. Numbers above each bar represent the -fold increase over each respective CMV only control.

Article Snippet: Cell Culture—HL-60 human promyelocytic leukemia cells (ATCC CCL 240), U937 histiocytic leukemia cells (ATCC CRL 1593), CCRFCEM lymphoblastic leukemia cells (ATCC 119), and HeLa S3 human cervical carcinoma cells (ATCC CCL 2.2) were obtained from the American Type Culture Collection (Rockville, MD).

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

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: ( A ) Schematic representation of Crk proteins under investigation. ( B ) WB of HeLa cell extracts using anti-CrkI/II MoAb and anti-CrkL Ab and the Odyssey imaging system to show expression levels of the corresponding proteins, or ( C ) to show decreased Crk expression in infected cells pretreated with siRNA. As a loading control the blots were probed with anti-actin monoclonal Ab (MoAb) or anti-tubulin Ab (upper bands). ( D ) Confocal fluorescence images of HeLa cells pretreated with siRNA against CrkI/II and CrkL or control siRNA and infected with preactivated EPEC for 2 h at an MOI of 3. Actin was stained with TRITC-phalloidin (red), while bacteria were stained with DAPI (blue). Arrows point at pedestals and bacteria. The scale bar represents 20 μm. ( E ) Quantitation of the number of pedestals on infected HeLa cells pretreated using siRNA with two oligonucleotides against CrkI/II and CrkL (black bar) compared to control oligonucleotide treated cells (white bar). Quantitation was done by counting the number of pedestals on 100 cells. Data in the graph show mean ± standard deviation (SD) for three independent experiments. The difference between groups was statistically significant based on Students t -test analysis; **, p<0.01.

Article Snippet: HeLa human epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Imaging, Expressing, Infection, Control, Fluorescence, Staining, Bacteria, Quantitation Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: ( A ) Expression of Crk in transfectants was assessed by WB with anti-Myc MoAb. Blots were probed with anti-actin Ab as a loading control. ( B ) Immunofluorescence images of HeLa cells transfected with Wild-Type (WT) CrkII, a CrkII dominant-negative mutant (R38V), or transfection reagent alone (mock; data not shown), then infected with preactivated EPEC for 2 h at an MOI of 3. Myc-tagged Crk constructs were visualized in green using anti-Myc MoAb, followed by anti-mouse Alexa-488 secondary Ab. Actin was stained in red using TRITC-phalloidin, while bacteria were stained blue using DAPI. The merged images shown were generated using AxioVision software. Insets are 4× digital zoom images. ( C ) Quantitation of the number of pedestals on mock-treated HeLa cells (white bar), cells overexpressing CrkII (CrkII, black bar) or cells overexpressing the dominant-negative R38V CrkII mutant (R38V, grey bar). Quantitation was done by counting the number of pedestals on 100 cells. The graph shows mean ± standard deviation (SD) for three independent experiments. The differences among the groups were not statistically significant based on Student's t -test analysis.

Article Snippet: HeLa human epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Control, Immunofluorescence, Transfection, Dominant Negative Mutation, Infection, Construct, Staining, Bacteria, Generated, Software, Quantitation Assay, Mutagenesis, Standard Deviation

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: ( A ) Expression of GFP alone (GFP) or GFP-tagged CrkII SH2 domain (SH2-GFP) in transfectants was assessed by WB with anti-GFP Ab. Blots were probed with anti-actin MoAb as a loading control. ( B ) Fluorescence images of HeLa cells transfected with GFP or SH2-GFP and then infected with preactivated EPEC for 2 h at an MOI of 3. GFP-tagged constructs appear green; actin, red (TRITC-phalloidin) and EPEC, blue (DAPI). The merged images shown were generated using AxioVision software. Insets are 4× digital zoom images. ( C ) Quantitation of the number of pedestals on cells expressing GFP or SH-GFP. Quantitation was done by counting the number of pedestals on cells expressing the constructs in six experiments. ( D, E ) HeLa cells were transfected with Myc-tagged empty vector or with plasmids encoding Myc-CrkII-Y221F or CrkL-Y207F phosphorylation-deficient mutants, and then infected with preactivated EPEC at an MOI of 3. Pedestal number was compared in cells expressing Myc and Myc-CrkII-Y221F or between Mock-transfected cells and CrkL-Y207F transfected cells in three different experiments. ( F ) WB of Nck1/2-deficient MEFs transfected with Flag-Nck2 plasmid as well as a plasmid encoding either GFP alone or GFP-tagged CrkII SH2. ( G ) Immunofluorescence staining of transfected Nck1/2-deficient MEFs after infection with EPEC at an MOI of 225. Cells were stained with anti-Flag MoAb followed by Alexa 568-conjugated goat anti-mouse Ab. F-actin was visualized with Alexa 350 Phalloidin. GFP expression is shown in green. ( H ) The number of cells in three different experiments showing Flag staining at pedestals was counted among cells expressing empty GFP or GFP-tagged CrkII SH2 and normalized to 100. The graph shows mean ± standard deviation (SD) for three independent experiments. The differences among the groups were statistically significant based on Student's t -test; *, p<0.05, **, p<0.01, ***, p<0.001.

Article Snippet: HeLa human epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Control, Fluorescence, Transfection, Infection, Construct, Generated, Software, Quantitation Assay, Plasmid Preparation, Phospho-proteomics, Immunofluorescence, Staining, Standard Deviation

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: HeLa cells were serum-starved for 16 h prior to infection with preactivated EPEC for 1, 2 or 3 h at an MOI of 45. The basal level of phosphorylation of Crk proteins was visualized in uninfected cells (- EPEC). ( A ) CrkII or IgG isotype control immunoprecipitates were probed by WB with a phosphospecific Ab against phospho-Tyr221 in CrkII (phospho-CrkII) to show induction of phosphorylation. The blot was also probed with anti-CrkI/II MoAb to show total levels of CrkII. The ratio of phospho-CrkII to non-phosphorylated CrkII in a representative experiment is shown. ( B ) Statistical analysis of the ratio of phospho-CrkII to CrkII signals with respect to the basal level in uninfected cells using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. ***, p<0.001. ( C ) WBs with a phosphospecific Ab against phospho-Tyr207 in CrkL to show the induction of phosphorylation, and with anti-CrkL Ab to show total levels of CrkL. The ratio of the phospho-CrkL to CrkL in a representative experiment is shown. ( D ) Statistical analysis of the ratio of phospho-CrkL to CrkL levels with respect to the basal level at 0 h using one-way ANOVA with Dunnett test. The graph shows mean ± SD for four independent experiments. a.u.: arbitrary units. *, p<0.05. ( E ) Immunofluorescence images of HeLa cells that were starved for 16 h prior to infection with preactivated EPEC for 3 h at an MOI of 15. Immunofluorescence staining was done using phosphospecific Abs against Tyr221 in CrkII (phospho-CrkII) or Tyr207 in CrkL (phospho-CrkL) followed by Alexa 488-conjugated goat anti-rabbit secondary Ab (green). Actin was stained red using TRITC-phalloidin; bacteria were stained blue using DAPI. Pictures were taken on a confocal microscope and images from one section are shown, together with 4× digital zoom images (insets). Images were merged using Leica software.

Article Snippet: HeLa human epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Infection, Phospho-proteomics, Control, Immunofluorescence, Staining, Bacteria, Microscopy, Software

Journal: PLoS Pathogens

Article Title: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

doi: 10.1371/journal.ppat.1004022

Figure Lengend Snippet: For pull-down experiments, total cell lysates were mixed with the following GST fusion proteins: GST alone, GST-SH2-Nck, GST-SH2-CrkII or GST-SH2-CrkL. As a control, 1:20 of the total amount of cell lysate used for each pull-down assay was analyzed (Lys). The GST fusion proteins used in the pull-down assays were analyzed by Coomassie blue staining; the amounts were 1:20 the amounts used in the pull-down experiments. These experiments were performed at least three times. ( A ) HeLa cells were left uninfected (- EPEC) or infected with preactivated EPEC for 1 or 2 h at an MOI of 180. Pull-down experiments were analyzed by WB using an anti-Tir MoAb. ( B ) HeLa cells were left uninfected (- EPEC) or infected with preactivated EPEC for 2 h at an MOI of 180. Pull-down experiments were analyzed by WB using an anti-Tir polyclonal Ab and a generic anti-phophotyrosine MoAb. ( C ) HeLa cells were left uninfected (- EPEC) or infected for 2 h at an MOI of 150 with preactivated Δ tir mutant EPEC complemented with a low-copy-number plasmid expressing WT Tir (p tir ) or Tir with a Tyr474Phe substitution (p tir Y474F). Pull-down experiments were analyzed by WB using an anti-Tir polyclonal Ab. As a control, infections with WT EPEC were performed in parallel. ( D ) Binding of recombinant CrkII SH2 domain to Tir competes with the binding of the SH2 domain of Nck. Recombinant CrkII SH2 domain was excised using PreScission protease from the GST-SH2 domain coupled to GSH beads. The indicated amounts of soluble SH2 domain were added to infected HeLa cell lysates. After incubation, pull-downs were performed by adding empty GST or GST-SH2 Nck, and then visualized by WB with sequential blotting with anti-Tir and anti-GST MoAbs.

Article Snippet: HeLa human epithelial cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Control, Pull Down Assay, Staining, Infection, Mutagenesis, Low Copy Number, Plasmid Preparation, Expressing, Binding Assay, Recombinant, Incubation

Journal: The Journal of Experimental Medicine

Article Title: Control of Lytic Function by Mitogen-activated Protein Kinase/Extracellular Regulatory Kinase 2 (ERK2) in a Human Natural Killer Cell Line: Identification of Perforin and Granzyme B Mobilization by Functional ERK2

doi:

Figure Lengend Snippet: Detection of tyrosine-phosphorylated p42/p44 MAPK protein in Raji-activated YT effector cells. ( A ) YT cells were cultured alone or with Raji cells at a 1:1 ratio for 0–15 min at 37°C. YT cells were also pretreated with 100 μM of PD098059 for 1 h at 37°C before incubation for 5 min at 37°C with Raji tumor cells. The cells were then lysed and immunoprecipitated ( IP ) with monoclonal antiphosphotyrosine, 4G10. Immunoprecipitation of YT cells, which had been preincubated with Raji tumor cells for 5 min at 37°C, with isotype-matched IgG was also performed as a control. Raji cells alone were included to check for background phosphorylation. The immunoprecipitates were then probed with 4G10 by Western blotting ( WB ). ( B ) YT cells were untreated or pretreated for 1 h at 37°C with 100 μM of PD098059 or an equivalent amount of DMSO used to dilute PD098059. The cells were then mixed with Raji tumor target cells for 0–5 min at 37°C and lysed. The lysates were immunoprecipitated with antiphosphotyrosine, 4G10, or control isotype-matched IgG and then probed with anti-panERK.

Article Snippet: Its ability to serve as an NK effector cell against a human B lymphoma cell line, Raji (American Type Culture Collection, Rockville, MD), was exploited in this study.

Techniques: Cell Culture, Incubation, Immunoprecipitation, Control, Phospho-proteomics, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Control of Lytic Function by Mitogen-activated Protein Kinase/Extracellular Regulatory Kinase 2 (ERK2) in a Human Natural Killer Cell Line: Identification of Perforin and Granzyme B Mobilization by Functional ERK2

doi:

Figure Lengend Snippet: Detection of the activated form of MAPK in Raji-triggered YT effector cells. ( A ) YT cells untreated or treated with 100 μM of PD098059 for 1 h at 37°C were mixed with equal numbers of Raji tumor cells for 0–15 min at 37°C. Whole cell lysates were then prepared and analyzed by Western blotting ( WB ) with anti-AMAPK (α-AMAPK) that was generated against the phosphorylated TEY epitope of MAPK ( top ). The blots were then stripped and reprobed with anti-panERK to show equal loading of all the lanes ( bottom ). ( B ) YT cells were mixed with equal numbers of either Jurkat or HL60 tumor cells, both of which are NK-resistant, or with the NK-sensitive Raji tumor cells, for 0 or 5 min at 37°C. Whole cell lysates were prepared and analyzed by Western blotting with anti-AMAPK ( top ). The blots were then stripped and reprobed with anti-panERK ( bottom ). ( C ) YT cells were pretreated with medium ( MED ), DMSO, or 10–200 μM of PD098059 for 1 h at 37°C before addition of Raji tumor cells for 0 or 5 min at 37°C. Cell lysates were then prepared and probed with anti-AMAPK ( top ) and consecutively restripped and reprobed with anti-ERK2 ( middle ) and anti-ERK1 ( bottom ).

Article Snippet: Its ability to serve as an NK effector cell against a human B lymphoma cell line, Raji (American Type Culture Collection, Rockville, MD), was exploited in this study.

Techniques: Western Blot, Generated

Journal: The Journal of Experimental Medicine

Article Title: Control of Lytic Function by Mitogen-activated Protein Kinase/Extracellular Regulatory Kinase 2 (ERK2) in a Human Natural Killer Cell Line: Identification of Perforin and Granzyme B Mobilization by Functional ERK2

doi:

Figure Lengend Snippet: Analysis of kinase function in p42/44 MAPK from Raji-activated YT effector cells. YT cells untreated or treated for 1 h at 37°C with 100 μM of PD098059 or an equal concentration of the diluent, DMSO, were mixed with Raji tumor cells at a 1:1 ratio for 0–5 min at 37°C. The cells were then lysed and immunoprecipitated ( IP ) with anti-panERK. The immunoprecipitates were incubated with [α- 32 P]ATP and the 18-kD MBP as a substrate for the in vitro kinase assay in a 15% SDS gel ( top ). The filter was then probed with anti-panERK to show equal loading in all the lanes ( bottom ). WB , Western blotting. MED , Medium.

Article Snippet: Its ability to serve as an NK effector cell against a human B lymphoma cell line, Raji (American Type Culture Collection, Rockville, MD), was exploited in this study.

Techniques: Concentration Assay, Immunoprecipitation, Incubation, In Vitro, Kinase Assay, SDS-Gel, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Control of Lytic Function by Mitogen-activated Protein Kinase/Extracellular Regulatory Kinase 2 (ERK2) in a Human Natural Killer Cell Line: Identification of Perforin and Granzyme B Mobilization by Functional ERK2

doi:

Figure Lengend Snippet: Identification of ERK2 as the activated MAPK isoform in Raji-triggered YT effector cells. ( A ) YT cells untreated or pretreated 1 h at 37°C with 100 μM of PD098059 or an equivalent amount of the diluent, DMSO, were mixed with Raji tumor cells at a 1:1 ratio for 0–5 min at 37°C. The cells were then lysed and immunoprecipitated ( IP ) with antiphosphotyrosine, 4G10, followed by Western blot analysis ( WB ) with anti-ERK2. MED , Medium. ( B ) YT cells similarly treated as in A were immunoprecipitated with anti-AMAPK (α-AMAPK) and probed with the same antibody to locate the activated phosphorylated form of MAPK. The filter was then stripped and reprobed with anti-ERK2 to identify the activated MAPK isoform as ERK2. NRS , Normal rabbit serum.

Article Snippet: Its ability to serve as an NK effector cell against a human B lymphoma cell line, Raji (American Type Culture Collection, Rockville, MD), was exploited in this study.

Techniques: Immunoprecipitation, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Control of Lytic Function by Mitogen-activated Protein Kinase/Extracellular Regulatory Kinase 2 (ERK2) in a Human Natural Killer Cell Line: Identification of Perforin and Granzyme B Mobilization by Functional ERK2

doi:

Figure Lengend Snippet: Inhibition of NK function by transient transfection with dominant-negative MAPK/ERK2 in YT effector cells. Equal aliquots of YT cells were left untransfected or transiently transfected with KD MAPK/ERK2 in which K52R substitution was constructed, or with mutant ERK2 where Thr and Tyr residues in the TEY motif were replaced with glutamic acid (TEYE) or with alanine and phenylalanine (TAYF). The wild-type ERK2 plasmid ( WT ) was also used to transfect YT cells. After 24 h at 37°C, untransfected and transfected YT cells were assessed for viability and adjusted to the appropriate concentrations of viable cells before testing for lysis of 51 Cr-labeled Raji tumor cells at the indicated E/T ratios. The SEM of each mean percent cytotoxicity was <5%, and was not shown.

Article Snippet: Its ability to serve as an NK effector cell against a human B lymphoma cell line, Raji (American Type Culture Collection, Rockville, MD), was exploited in this study.

Techniques: Inhibition, Transfection, Dominant Negative Mutation, Construct, Mutagenesis, Plasmid Preparation, Lysis, Labeling