Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: a Proteome profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA) probed with serum-free-conditioned medium (SF-CM) of C33a-FL-CA IX or C33a-NS-CA IX cells incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h. Spot doublets of corresponding proteins with differential intensities are framed and indicated by arrows. Medium pooled from five independent experiments was used for the array. Spot intensities representing b pro-oncogenic basigin, and c anti-angiogenic thrombospondin-1 were analysed in culture medium samples of C33a-FL-CA IX versus C33a-NS-CA IX cells. Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD. d Validation of the differential levels of basigin and thrombospondin-1, respectively, by Western blotting analysis of medium precipitates (MP) and cell lysates (CL) of normoxic (NO) and hypoxic (HY) cells. (* P < 0.05, ** P < 0.01, *** P < 0.005).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Incubation, Biomarker Discovery, Western Blot

Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: Analysis of the differential expression of a IGFBP-2 and b IGFBP-3 in SF-CM samples from C33a-FL-CA IX and C33a-NS-CA IX incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h and evaluated by Proteome Profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA). Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD of five independent samples. c Western blotting analysis of CA IX, IGFBP-2 and IGFBP-3 protein levels in medium precipitates (MP) of C33a-FL-CA IX and C33a-NS-CA IX cells. Impairment of shedding in NS-CA IX is associated with increased IGFBP-2 and reduced IGFBP-3 secretion to culture medium. Quantitative PCR analysis of relative mRNA levels of d IGFBP-2, e IGFBP-3 and f IGF1 in C33a-FL-CA IX versus C33a-NS-CA IX cells normalised to β-actin mRNA. (*** P < 0.005, ns non-significant).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Quantitative Proteomics, Incubation, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Science of the Total Environment

Article Title: Sampling methods and assays applied in SARS-CoV-2 exposure assessment

doi: 10.1016/j.scitotenv.2021.145903

Figure Lengend Snippet: Data obtained from the chosen articles.

Article Snippet: , 34. SARS-CoV-2 RNA contamination on surfaces of a COVID-19 ward in a hospital of Northern Italy: what risk of transmission? , Italy , No , Surface samples from ward in University Hospital of Ferrara , Sampling performed with sterile rayon swabs pre-moistened in sterile phosphate-buffered solution , Viral RNA extraction with Patho Gene-spin Extraction kit (Generon) RT-qPCR targeted the RNA-dependent RNA polymerase ( RdRp ) gene (Generon), and the orf1ab , spike ( S ), and nucleocapsid ( N ) genes (ThermoFisher) , • SARS-CoV-2 was only detected in 3 samples of two floors and one-bathroom sink. • Reported to persist for a longer duration on surfaces under controlled laboratory conditions. , ( ) .

Techniques: Sampling, Lysis, RNA Extraction, Environmental Monitoring, Virus, Multiplex Assay, Northern Blot, Marker, RNA Detection, Isolation, Membrane, Control, Environmental Sampling, Amplification, Transmission Assay, Aerosol, Diagnostic Assay, Infection, Sterility, Real-time Polymerase Chain Reaction, Nested PCR, Reverse Transcription, Extraction, Purification, Digital PCR, Preserving, Quantitative RT-PCR, cDNA Synthesis, Magnetic Beads, Incubation, Modification, One Step RT-PCR, Cell Culture, Sequencing

Journal: Nature Communications

Article Title: Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

doi: 10.1038/ncomms11150

Figure Lengend Snippet: ( a ) Transcriptome profiling of SKOV3 cells transfected with miR21 precursor or control miR. A heatmap shows the top 10 miR21 regulated genes related to chemoresistance and metastasis. ( b ) Pathway analysis showing chemoresistance-related genes that were upregulated (red) or downregulated (green) in miR21-transfected SKOV3 cells. ( c ) SKOV3 cells incubated with CAA- and CAF-derived exosomes showed lower APAF1 expression than did controls. The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001; two-tailed Student's t -test. ( d ) Immunolocalization of APAF1 on ovarian tumour tissue sections ( n =5) demonstrated lower APAF1 level in the stromal–epithelial interface compared with that in the centre of the tumour tissues. A representative serial section of the tumour tissue was stained with haematoxylin and eosin (right panel). Dotted line and arrowheads indicate the stromal–epithelial interface between CAF and tumour (T). Scale bar, 10 μm. ( e , f ) Ovarian cancer cells transfected with miR21 precursor had lower APAF1 mRNA and protein levels than did controls. The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001 and ** P <0.01; two-tailed Student's t -test. ( g ) The correlation between miR21 and APAF1 mRNA levels in microdissected ovarian cancer tissues was determined using a Spearman's correlation analysis. ( h ) Recurrent ovarian cancer PEA2 cells expressed lower APAF1 expression than did primary PEA1 cells. The results were the average from at least three independent experiments. Mean±s.d.; ** P <0.01; two-tailed Student's t -test. ( i ) A consensus miR21-binding site was identified within the coding sequence of APAF1. ( j ) Co-transfection of pre-miR21 and luciferase vector with APAF1 coding sequence decreased the luciferase expression in SKOV3 cells in a dose-dependent manner. The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001, ** P <0.01 and * P <0.05; two-tailed Student's t -test. ( k ) The luciferase activity was measured following co-transfection with either wild-type (WT) or mutated (Mut) APAF1 coding sequence vectors (mutated sequence shown in red in upper panel). The results were the average from at least three independent experiments. Mean±s.d.; *** P <0.001 and ** P <0.01; two-tailed Student's t -test.

Article Snippet: The expression of APAF1 was determined by multiplexing quantitative PCR (TaqMan Gene Expression Assay) using FAM-labelled APAF1 (Hs00559441_m1) together with VIC-labelled glyceraldehyde 3-phosphate dehydrogenase-specific TaqMan probes and primers.

Techniques: Transfection, Control, Incubation, Derivative Assay, Expressing, Two Tailed Test, Staining, Binding Assay, Sequencing, Cotransfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Nature Communications

Article Title: Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

doi: 10.1038/ncomms11150

Figure Lengend Snippet: ( a ) Overexpression of APAF1 by full-length transfection increased paclitaxel sensitivity in ovarian cancer SKOV3 and OVCA432 cells. The results were the average from at least three independent experiments. Mean±s.d.; ** P <0.01 and * P <0.05; two-tailed Student's t -test. ( b ) Co-transfection of miR21 precursor and full-length APAF1 decreased paclitaxel resistance compared with the co-transfection of pre-miR21 and the control vector in both SKOV3 and OVCA432 cells. The results were the average from at least three independent experiments. Mean±s.d.; ** P <0.01 and * P <0.05; two-tailed Student's t -test. ( c – e ) APAF1 overexpression enhanced the paclitaxel sensitivity of ovarian cancer cells in vivo . APAF1 stably overexpressing ovarian cancer OVCA432 cells were generated using the lentiviral transduction method and were intraperitoneally injected into female BALB/c athymic nude mice, followed by paclitaxel treatment. The tumour volumes were measured and quantified using the IVIS-Lumina XR in vivo imaging system after a 2-week 5 mg kg −1 paclitaxel treatment. ( c ) Box plot showing a significant decrease in luciferase activity in the APAF1 overexpression group ( n =7) compared with the control group ( n =7) after paclitaxel treatment ( P =0.038; Mann–Whitney U -test). ( d ) Representative images show a decrease in luminescence in the APAF1 overexpression group compared with the control group after paclitaxel treatment. ( e ) Immunolocalization of APAF1 on paraffinized sections of tumour tissues collected from mice demonstrated a higher APAF1 level in the APAF1 overexpression group ( n =7) compared with the control group ( n =7). Representative microscopic images were illustrated. Scale bar, 10 μm.

Article Snippet: The expression of APAF1 was determined by multiplexing quantitative PCR (TaqMan Gene Expression Assay) using FAM-labelled APAF1 (Hs00559441_m1) together with VIC-labelled glyceraldehyde 3-phosphate dehydrogenase-specific TaqMan probes and primers.

Techniques: Over Expression, Transfection, Two Tailed Test, Cotransfection, Control, Plasmid Preparation, In Vivo, Stable Transfection, Generated, Transduction, Injection, In Vivo Imaging, Luciferase, Activity Assay, MANN-WHITNEY