Journal: Molecular Microbiology

Article Title: Mycoplasma pneumoniae Community Acquired Respiratory Distress Syndrome toxin expression reveals growth phase and infection-dependent regulation

doi: 10.1111/j.1365-2958.2010.07092.x

Figure Lengend Snippet: Transcription of cards during in vitro growth of M. pneumoniae in SP-4 broth. A. Expression of cards along with other genes was analysed by DNA slot blot. M. pneumoniae gene-specific PCR products were blotted onto Zeta probe membranes. M. pneumoniae S1 cells were grown and harvested at early-log (24 h), mid-log (48 h), late-log (72 h) and stationary phases (120 h). 32 P-labelled cDNAs generated by reverse transcription of isolated total RNA were used as hybridization probes. Experiments were repeated three times. B. Expression of cards by relative real-time quantitative RT-PCR (qRT-PCR). M. pneumoniae S1 cells were grown and RNA isolated at specific growth phases. Real-time qRT-PCR was performed using SYBR green chemistry as detailed in Experimental procedures . Experiments were repeated two times. The average fold differences in expression levels of cards mRNA at various time points (when compared with the stationary phase) and standard deviations (SD) are presented.

Article Snippet: All generated amplicons were purified by gel extraction, and 200 ng of each specific PCR product was blotted in triplicate on Zeta probe membranes by using Bio-Dot SF microfiltration apparatus as suggested by the manufacturer (Bio-Rad Laboratories, Hercules, CA).

Techniques: In Vitro, Expressing, Dot Blot, Generated, Reverse Transcription, Isolation, Hybridization, Quantitative RT-PCR, SYBR Green Assay

Journal: Nanotechnology, Science and Applications

Article Title: EGF Receptor-Targeting Cancer Therapy Using CD47-Engineered Cell-Derived Nanoplatforms

doi: 10.2147/NSA.S352038

Figure Lengend Snippet: Preparation and characterization of CD47+ iCDNs-DOX. ( A ) The process of CD47 + iCDNs-DOX preparation is illustrated. ( B ) The prepared CD47 + CDNs, CD47 + CDNs-DOX, CD47 + iCDNs, and CD47 + iCDNs-DOX (from left to right) were negatively stained with 2% uranyl acetate and then examined with a transmission electron microscope (×80 (K)). ( C ) The size and surface charge of the nanoparticles were analyzed three times with a ζ-sizer. Each diameter (nm) and zeta-potential represents the mean ± S.D. for three independent experiments. ( D ) The change of CD47 + CDN size during storage at 4℃ was monitored for 10 days. Each error bar represents the mean ± S.D. for three independent experiments.

Article Snippet: The mouse CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP), was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Staining, Transmission Assay, Microscopy, Zeta Potential Analyzer

Journal: Nanotechnology, Science and Applications

Article Title: EGF Receptor-Targeting Cancer Therapy Using CD47-Engineered Cell-Derived Nanoplatforms

doi: 10.2147/NSA.S352038

Figure Lengend Snippet: Interaction of CDNs with macrophages. ( A ) The expression of CD47-GFP fusion protein on the cell surface was identified using a confocal microscope (×800). ( B ) Western blotting of transfected cell lysates and CDNs verified the CD47 presence. ( C ) RAW264.7 cells were treated with DiD-labeled CD47 − CDNs or CD47 + CDNs for 1 or 4 h, and then subjected to flow cytometry. ( D ) The macrophages incubated with CDNs for 4 h were also examined with a confocal microscope (×800). ***, p<0.001 vs cells treated with CDNs. Each error bar represents the mean ± S.D. for three independent experiments.

Article Snippet: The mouse CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP), was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Microscopy, Western Blot, Transfection, Labeling, Flow Cytometry, Incubation

Journal: Nanotechnology, Science and Applications

Article Title: EGF Receptor-Targeting Cancer Therapy Using CD47-Engineered Cell-Derived Nanoplatforms

doi: 10.2147/NSA.S352038

Figure Lengend Snippet: EGFR-specific cell biding of CD47+ iCDNs and tumor targeting and cytotoxicity of CD47+ iCDNs-DOX. ( A ) MDA-MB-231 and MDA-MB-453 cells were treated with Alexa fluor 488-labeled CD47 + iCDNs and then subjected to FACS analysis (upper). ( B ) MDA-MB-231 and MDA-MB-453 cells were treated with CD47 + iCDNs-DOX and stained with DAPI. The treated cells were examined with a confocal microscope at various time points. PMT, photomultiplier tube. ( C ) The tumor cells were also treated with various concentrations of DOX in a free form, CD47 + CDNs, or CD47 + iCDNs for 48 h, and the cell viability was quantified using the CCK-8 assay. The mean fluorescence of the treated cells was calculated (n=5) (bottom). ***, p<0.001 vs cells treated with CDNs. Each error bar represents the mean ± S.D. for three separate experiments.

Article Snippet: The mouse CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP), was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Labeling, Staining, Microscopy, CCK-8 Assay, Fluorescence

Journal: Nanotechnology, Science and Applications

Article Title: EGF Receptor-Targeting Cancer Therapy Using CD47-Engineered Cell-Derived Nanoplatforms

doi: 10.2147/NSA.S352038

Figure Lengend Snippet: Biodistribution of CD47+ iCDNs in MDA-MB-231-xenografted mice. ( A ) The mice bearing MDA-MB-231 tumors were intravenously injected with DiO-labeled CD47 + CDNs or CD47 + iCDNs. ( B ) The whole-body fluorescence images were taken with an in vivo imaging system Then, major organs were removed, and the fluorescence images of the organs were also taken. ( C ) The relative fluorescence intensities of the organs at each time point were calculated by dividing the measured fluorescence intensity by area. ( D ) Tumor-to-liver ratios of CD47 + CDNs and CD47 + iCDNs were compared to each other at the same time point. Each error bar represents the mean ± S.D. of three measurements.

Article Snippet: The mouse CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP), was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Injection, Labeling, Fluorescence, In Vivo Imaging

Journal: Nanotechnology, Science and Applications

Article Title: EGF Receptor-Targeting Cancer Therapy Using CD47-Engineered Cell-Derived Nanoplatforms

doi: 10.2147/NSA.S352038

Figure Lengend Snippet: Suppression of in vivo tumor growth by a series of CDNs-DOX. ( A ) MDA-MB-231 cells were subcutaneously implanted into mice, which were then intravenously injected with saline, free DOX, CD47 − CDNs-DOX, CD47 + CDNs-DOX, or CD47 + iCDNs-DOX at days 1, 4, and 7. ( B ) Tumor volumes were measured once every 3–4 days. ( C and D ) On day 39, the tumors were excised and weighed. ( E ) The body weights of the mice were also measured on the measurement days. Each error bar represents the mean ± S.D. (n=4). *, p<0.05 and **, p<0.01 vs CD47 − CDNs-DOX-injected group.

Article Snippet: The mouse CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP), was purchased from Sino Biological Inc. (Beijing, China).

Techniques: In Vivo, Injection, Saline

Journal: Nanotechnology, Science and Applications

Article Title: EGF Receptor-Targeting Cancer Therapy Using CD47-Engineered Cell-Derived Nanoplatforms

doi: 10.2147/NSA.S352038

Figure Lengend Snippet: Histopathology of the major organs of mice treated with a series of CDNs-DOX. Tumors and major organs including the heart, kidneys, liver, and lungs were removed and dissected on day 39 post-injection of saline, free DOX, CD47 − CDNs-DOX, CD47 + CDNs-DOX, or CD47 + iCDNs-DOX. The organs were embedded in paraffin, sliced, and then hematoxylin and eosin (H&E)-stained. The black dotted lines in the tumor pictures show the necrosis area (bright pink).

Article Snippet: The mouse CD47 ORF cDNA clone expression plasmid, C-GFPSpark tag (pCD47-GFP), was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Histopathology, Injection, Saline, Staining