Journal: Scientific Reports

Article Title: Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics

doi: 10.1038/s41598-018-34467-8

Figure Lengend Snippet: TEM analyses of HRNR-treated E . coli and P . aeruginosa . ( a , b ) Transmission electron microscopy (TEM) analyses of 6.25 × 10 /ml E . coli ATCC 11775 in 10 mM NaP, pH 5.5, 1 h treatment with 312.5 µg/ml rHRNR 2591–2684 . ( c ) E . coli control. Note the absence of membrane perturbation and the presence of intracytoplasmic electron-dense aggregates in rHRNR 2591–2684 -treated bacteria ( a , b ). The hyperhydrated looking periplasmic space of many cells in the control (a), is similar as seen for E . coli treated with low ion strength and acidic buffers . TEM of 6.25 × 10 /ml P . aeruginosa ATCC 10145, in 10 mM NaP, pH 5.5, 1 h treatment with 312.5 µg/ml rHRNR 2591–2684 ( d , e ). ( f ) P . aeruginosa control. Note condensation of electron-dense cytoplasmic material and blebs of the outer membrane with an occasional ballooning ( d , e ). 1 h treatment of 6.25 × 10 /ml P . aeruginosa ATCC 10145 with 469 µg/ml rSUMO3-HRNR 2591–2684 in 10 mM NaP, pH 5.5 revealed widespread peeling of the outer membrane ( g , h ). ( i ) P . aeruginosa control. Images are representative of two independent experiments, sampling on average 10 images per condition and species in each experiment.

Article Snippet: TEM analyses of 6.25 × 10 7 /ml C . albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR 2591–2684 in 10 mM NaP, pH 5.5 ( a – d ). ( e , f ) Control.

Techniques: Transmission Assay, Electron Microscopy, Sampling

Journal: Scientific Reports

Article Title: Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics

doi: 10.1038/s41598-018-34467-8

Figure Lengend Snippet: Ultrastructural analyses of rHRNR 2591–2684 -treated S . aureus . TEM analyses of 6.25 × 10 7 /ml S . aureus ATCC 6538, in 10 mM NaP, pH 5.5, treated with 312.5 µg/ml rHRNR 2591–2684 for 2 h. Note the condensation of electron-dense cytoplasmic material and formation of membrane blebs ( a – c ). Occasionally ballooning ( c ) and aggregated cells ( d ), connected via electron-dense contacts, were found upon rHRNR 2591–2684 -treatment. ( e , f ) Control. Images are representative of two independent experiments, sampling on average 10 images.

Article Snippet: TEM analyses of 6.25 × 10 7 /ml C . albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR 2591–2684 in 10 mM NaP, pH 5.5 ( a – d ). ( e , f ) Control.

Techniques: Sampling

Journal: Scientific Reports

Article Title: Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics

doi: 10.1038/s41598-018-34467-8

Figure Lengend Snippet: Ultrastructural analyses of HRNR-treated C . albicans . TEM analyses of 6.25 × 10 7 /ml C . albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR 2591–2684 in 10 mM NaP, pH 5.5 ( a – d ). ( e , f ) Control. Note the release of electron dense vesicles ( a – c ) and marked changes of the intracytoplasmic morphology. Images are representative of two independent experiments, sampling on average 10 images in each experiment.

Article Snippet: TEM analyses of 6.25 × 10 7 /ml C . albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR 2591–2684 in 10 mM NaP, pH 5.5 ( a – d ). ( e , f ) Control.

Techniques: Sampling

Journal: Scientific Reports

Article Title: Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics

doi: 10.1038/s41598-018-34467-8

Figure Lengend Snippet: rHRNR 2591–2684 is a non-permeabilizing, energy-dependently translocating CIDAMP. ( a ) rHRNR 2591–2684 does not permeabilize the bacterial membrane. Lysis by 50 μg/mL lysozyme (expressed as OD 595 against time ± s. e. m., n = 3) of chloramphenicol-treated P . aeruginosa PAO1 cells (gray line) in the presence of polymyxin B (PMB, 10 μg/mL; dotted line) or rHRNR 2591–2684 (5 μg/mL; black line). ( b ) rHRNR 2591–2684 is translocated into bacterial cytosol. HRNR-Western blot of fractionated, rhHRNR 2591–2684 -treated PAO1, Ctrl: control (untreated PAO1). S: sample supernatant, OM: outer membrane-, P: periplasmic-, IM: inner membrane-, C: cytoplasmic fraction, HR: Hornerin fragment rHRNR 2591–2684 . ( c ) rHRNR 2591–2684 translocation is energy-dependent. PAO1 was treated with rHRNR 2591–2684 in the presence of NaN 3 , fractionated and analyzed by HRNR-Western blot.

Article Snippet: TEM analyses of 6.25 × 10 7 /ml C . albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR 2591–2684 in 10 mM NaP, pH 5.5 ( a – d ). ( e , f ) Control.

Techniques: Lysis, Western Blot, Translocation Assay

Journal: Scientific Reports

Article Title: Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics

doi: 10.1038/s41598-018-34467-8

Figure Lengend Snippet: HRNR binds to intracytoplasmic aggregates in P . aeruginosa . P . aeruginosa ATCC 10145 was treated for 5 min with rHRNR 2591–2684 (45 µg/ml 10 mM NaP, pH 5.5), and then cellular localization of this CIDAMP was analyzed by immunocytochemistry with a HRNR 2591–2684 -specific polyclonal antibody, followed by incubation with a gold-conjugated secondary antibody ( a ). Bacteria, treated with rHRNR 2591–2684 ( b ) or buffer ( c ), followed by incubation with the gold-conjugated secondary antibody, served as controls. Note accumulation of intracytoplasmic immuno-gold ( a ), which is corresponding to electron dense cytoplasmic aggregates seen upon TEM-analyses of CIDAMP-treated P . aeruginosa (Fig. ). Images are representative of two independent experiments, sampling on average 10 images in each experiment.

Article Snippet: TEM analyses of 6.25 × 10 7 /ml C . albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR 2591–2684 in 10 mM NaP, pH 5.5 ( a – d ). ( e , f ) Control.

Techniques: Immunocytochemistry, Incubation, Sampling

Journal: Scientific Reports

Article Title: Hornerin contains a Linked Series of Ribosome-Targeting Peptide Antibiotics

doi: 10.1038/s41598-018-34467-8

Figure Lengend Snippet: Distinct HRNR-derived CIDAMPs show similar ribosomal protein binding patterns. ( a ) SulfoLink®–column-bound proteins of an E . coli -extract were separated on a Jupiter® C18 RP-HPLC column with a Prp-gradient. HPLC fractions containing UV-absorbing peaks (C1–D10) were divided into five aliquots and adjusted in parallel to five PAGE-gels and separated. ( b ) Silver-stained proteins. ( c ) HRNR-Far-Western blot for probing with biotinylated HR1-18 (HRNR 2556–2677 ) using Strep - Tactin ®, ( d ) HRNR-Far-Western blot for probing with biotinylated rSumo3-HRNR 2591–2684 using Strep - Tactin ®, ( e ) HRNR-Far-Western blot for probing with rHRNR 2591–2684 using anti-HRNR 2591–2684 antibodies, ( f ) HRNR-Far-Western blot for probing with rHRNR 1075–1172 using anti-HRNR 1075–1172 antibodies. Note similarities of the staining patterns, irrespective the CIDAMP AA-sequence or biotin-labeling and irrespective whether a Strep - Tactin ®- or antibody-detectable CIDAMP was used to probe and detect the target protein on the membrane. Note the presence of 70 kDa bands upon HRNR-Far-Western blot analyses in most of the investigated HPLC fractions with highest intensity for rHRNR 1075–1172 binding ( f ). The most intensive band, corresponding to a 37 kDa protein in fraction number C13, was identified as E . coli ribosomal protein L2.

Article Snippet: TEM analyses of 6.25 × 10 7 /ml C . albicans ATCC 244433, treated for 2 h with 312.5 µg/ml rHRNR 2591–2684 in 10 mM NaP, pH 5.5 ( a – d ). ( e , f ) Control.

Techniques: Derivative Assay, Protein Binding, Staining, Far Western Blot, Sequencing, Labeling, Binding Assay

Journal: Oncogene

Article Title: B591, a novel specific pan-PI3K inhibitor, preferentially targets cancer stem cells

doi: 10.1038/s41388-018-0674-5

Figure Lengend Snippet: B591 inhibits PI3K/Akt pathway in multiple cancer cell lines. a Representative images of Akt1 redistribution in IGF-1-treated cells in the absence or presence of B591. Scale bar represents 10 μm. Arrows indicate IGF-1-mediated membrane translocation detected by the image analysis algorithm. b Inhibition of mTORC1 and mTORC2 signaling in multiple cancer cell lines, exposed for 5 h to increasing concentrations of B591. c HepG2 cells were treated with DMSO or 20 μM of B591 for 5 h. Immunofluorescence (red) intensity reflects eIF4E subcellular distribution. Cells were visualized with high content reader. Representative images are shown, and scale bar represents 50 μm. d Nuclear-to-cytoplasmic ratio of eIF4E in five cancer cell lines was determined via high content reader. e The phosphorylation of AKT and ERK was determined by immunoblotting in RD cells in the presence of 10 μM of B591 after indicated time of exposure. f Inhibition of the phosphorylation of AKT and ERK in RD cells treated with B591, PI-103 or Rapamycin for 24 h was determined by immunoblotting

Article Snippet: Rapamycin, PI-103 and paclitaxel were purchased from Selleckchem (Houston, TX, USA).

Techniques: Translocation Assay, Inhibition, Immunofluorescence, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tumor promoter role of miR-647 in gastric cancer via repression of TP73

doi: 10.3892/mmr.2018.9358

Figure Lengend Snippet: miR-647 reduces the apoptosis of MGC-803 cells. Flow cytometry was performed to detect cell apoptosis. MGC-803 cells were transfected with miR-647 mimics, miR-647 inhibitor or NC. A total of 24 h following transfection, cells that stained positive for FITC-Annexin V and negative for PI were scored as exhibiting early apoptosis. Experiments were performed in triplicate. *P<0.05 vs. NC. miR, microRNA; PI, propidium iodide; NC, negative control.

Article Snippet: Cells were then labeled with 50 µl/ml Annexin V-FITC and propidium iodide (PI; Cell Signaling Technology Inc.) according to the manufacturer's protocol.

Techniques: Flow Cytometry, Transfection, Staining, Negative Control