Journal: Nanomaterials

Article Title: Hydrocarbon-Stapled Peptide Based-Nanoparticles for siRNA Delivery

doi: 10.3390/nano10122334

Figure Lengend Snippet: Cytotoxicity and cellular uptake studies of FITC-stapled peptides ( a ) Cytotoxicity study in human breast adenocarcinoma MDA-MB-231 cells incubated with increasing concentrations (from 0 to 20 µM) of the stapled peptides for 72 h. ( b ) Uptake of FITC-stapled peptides at 5 µM final concentration in MDA-MB-231 cells after 4 h of incubation represented as relative mean fluorescence. The values are normalized with respect to penetratin. Results are presented as means ± standard deviations of three independent experiments performed in triplicate.

Article Snippet: MDA-MB-231-Luc-RFP stable cell line was obtained from AMSBIO (SC041, Abingdon, UK).

Techniques: Incubation, Concentration Assay, Fluorescence

Journal: Nanomaterials

Article Title: Hydrocarbon-Stapled Peptide Based-Nanoparticles for siRNA Delivery

doi: 10.3390/nano10122334

Figure Lengend Snippet: Luciferase activity assay showing the transfection of a 21-mer small interfering RNAs (siRNAs) targeting the expression of luciferase inside MDA-MB-231-Luc-RFP cells. The experiments were carried out with increasing amounts of siLuc (from 50 to 200 nM) complexed with the stapled peptides. The corresponding concentrations of the peptides are from 1.05 µM to 4.2 µM for JMV6580, from 1.4 µM to 5.6 µM for JMV6583 and from 5.25 µM to 21 µM for JMV6582. For lipofectamine transfection, the siRNA concentration used is 50 nM. In parallel, cell viability was measured for each condition. Results are expressed as mean ± standard deviation (n = 3). Statistically different, the level of significance was defined ** p < 0.01, and *** p < 0.001.

Article Snippet: MDA-MB-231-Luc-RFP stable cell line was obtained from AMSBIO (SC041, Abingdon, UK).

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Concentration Assay, Standard Deviation

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: The 9 bp sequence in the 5′-untranslated region (UTR) of Far2 binds NKX3-2. A: electrophoretic mobility shift assay (EMSA) performed with nuclear extract from mouse mesangial cells incubated with biotin-labeled oligonucleotide probes corresponding to the Far2 5′-UTR sequences with (ins-probe) and without (del-probe) the 9 bp sequence (lanes 2 and 4, respectively). B: EMSA performed with purified NKX3-2 protein (lane 2). A supershift assay was performed with an antibody against NKX3-2 (lane 3).

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Sequencing, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Purification

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: Far2 is an enzyme involved in de novo platelet-activating factor (PAF) production. A: PAF concentration measured in both culture media and cell lysates between Far2-overexpressing (gray, n = 3) and wild-type (black, n = 3) mesangial cells. Labeled fatty alcohol (hexadecanol) (B) and PAF (C) were detected through mass spectrometry after a mixture of 13C- and 12C-palmitate was added to Far2-overexpressing cells. D: proposed pathway highlighting the role of FAR2 in de novo synthesis of PAF.

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Concentration Assay, Labeling, Mass Spectrometry

Journal: Physiological Genomics

Article Title: FAR2 is associated with kidney disease in mice and humans

doi: 10.1152/physiolgenomics.00118.2017

Figure Lengend Snippet: Generation and characterization of B6N(Cg)-Far2tm2a(KOMP)Wtsi/2J. A: a simplified scheme showing the wild-type Far2 allele and knock-in allele Far2tm2a(KOMP)Wtsi. The lacZ and neomycin expression cassette was inserted between exon 4 and exon 5 and is flanked by FRT (Flippase Recognition Target) sites. Exons 5–8 are flanked by loxP sites. B: quantitative PCR for Far2 on brain and eyelid of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) knockout mice (n = 3/genotype) levels were calculated as relative fold change (RFC) compared with wild type. *P < 0.01 compared with +/+; #P < 0.01 compared with +/−. C: quantification of mesangial matrix expansion (MME) in knockout mice vs. wild-type mice at 12 and 18 mo of age relative to 6 mo measurements. MME is reported as percentage of the glomerular area occupied by matrix for 50 glomeruli per mouse and 10 mice per group. P = 1.27 × 10−6. D: glomerular filtration rate (GFR) data in knockout mice (gray) vs. wild-type mice (black) at 6, 12, and 18 mo of age. n = 10 per group. *P = 0.0118.

Article Snippet: A stable Far2 -overexpressing mesangial cell line was established by transfecting MES13 cells with the pCMV6 plasmid (OriGene) containing the mouse Far2 cDNA clone {"type":"entrez-nucleotide","attrs":{"text":"NM_178797","term_id":"1331036860","term_text":"NM_178797"}} NM_178797 with a Myc/DDK-tag (MR225671, OriGene) using 350 ng FuGENE and 100 ng of plasmid DNA.

Techniques: Knock-In, Expressing, Real-time Polymerase Chain Reaction, Knock-Out, Filtration

Journal: British Journal of Cancer

Article Title: Evaluation of the activity of a chemo-ablative, thermoresponsive hydrogel in a murine xenograft model of lung cancer

doi: 10.1038/s41416-020-0904-9

Figure Lengend Snippet: a Relative viability of A549 cells treated with 0–30 µL of TGel for 24 and 48 h ( n = 3). Significance was determined using a two-way ANOVA, with Tukey's multiple comparisons test. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. b Representative images of Live/Dead staining of A549 cells at 24 and 48 h with 0,10, 20 and 30 µL of TGel (i–iv, respectively). Live cells stained green, dead cells stained red. Magnification, ×10. Scale bar, 200 µm. c Flow cytometric analysis of apoptosis of Medium (left, top) and TGel (right, top) treated A549 cells for 24 h in vitro. Proportion of live, early apoptotic, late apoptotic/necrotic cells treated with Medium or TGel (bottom). Q 1 = early apoptotic cells, Q 2 and Q 4 = late apoptotic/necrotic cells and Q 3 = live cells ( n = 2). Significance within treatment groups was determined using an unpaired t test. * = p < 0.05, ** = p < 0.01. d Relative viability of Balb/c 3T3 clone A31 cells treated with TGel for 24 and 48 h ( n = 3). Significance was determined using a two-way ANOVA, with Tukey's multiple comparisons test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: An A549 cell line expressing firefly luc with puromycin resistance (SC043-luc) was purchased from AMS Biotechnology (Abingdon, UK).

Techniques: Staining, In Vitro

Journal: British Journal of Cancer

Article Title: Evaluation of the activity of a chemo-ablative, thermoresponsive hydrogel in a murine xenograft model of lung cancer

doi: 10.1038/s41416-020-0904-9

Figure Lengend Snippet: a Representative overlay images of bioluminescent A549-luc cells post intratumoural administration of 100 µL of fluorescently tagged TGel at Day 0. Blue represents bioluminescent signal from A549-luc cells, Yellow represents fluorescent signal from TGel. b Representative fluorescent images at Day 0 (top) and Day 14 (bottom) post intratumoural administration of saline (left) or TGel (right). c Representative images (greyscale photograph [left], fluorescent signal [centre] and overlay [right]) of saline (top) or TGel (bottom) treated tumours excised on Day 14 post intratumoural administration.

Article Snippet: An A549 cell line expressing firefly luc with puromycin resistance (SC043-luc) was purchased from AMS Biotechnology (Abingdon, UK).

Techniques:

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

doi: 10.1002/ijc.28890

Figure Lengend Snippet: Effect of β1 over-expression on breast tumour growth in vivo . MDA-MB-231 (“Control”) and MDA-MB-231-β1 (“β1”) cells were implanted into the inguinal mammary fat pads of female Rag2 −/− Il2rg −/− mice. ( a ) Representative bioluminescent images of mice bearing control and β1 tumours, 4 weeks after implantation. ( b ) Bioluminescence measured from primary tumours on the indicated days post-implantation ( n ≥ 12). Data are mean ± SEM; ** p < 0.01. ( c ) Calculated volume derived from calliper measurement of primary tumours over the same period ( n ≥ 12). ( d ) Percentage of mice whose primary tumour burden reached 10% of starting body weight within the 5-week tumour growth period is shown for control and β1 tumours. ( e ) Kaplan–Meier analysis comparing overall survival of mice bearing control and β1 tumours ( n = 13). ( f ) Images of control and β1 tumour tissue sections stained with H&E showing (i) mammary fat pad and (ii) skeletal muscle invasion. Arrows, infiltration of tumour cells (T) into fibroadipose tissue (F) or skeletal muscle fibers (M). Scale bar, 100 µm. Insets, higher magnification images of invading tumour cells, scale bar, 50 µm. (G) Invasion of control MDA-MB-231 and MDA-MB-231-β1 cells ± TTX (30 µM) for 48 hr ( n = 12; * p < 0.05; Neuman–Keuls test).

Article Snippet: MDA-MB-231-GFP and MDA-MB-231 β1-GFP cells were stably transduced with recombinant lentivirus for firefly luciferase (AMS Biotechnology).

Techniques: Over Expression, In Vivo, Derivative Assay, Staining

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

doi: 10.1002/ijc.28890

Figure Lengend Snippet: Effect of β1 upregulation on proliferation, apoptosis and angiogenesis. ( a ) Control and β1 tumour sections stained with anti-Ki67 (red) and DAPI (blue). Scale bar, 20 µm. ( b ) Ki67-positive nuclei per field of view for control and β1 tumours ( n = 30). ( c ) Control and β1 tumour sections stained with anti-activated caspase-3 (red) and DAPI (blue). Scale bar, 20 µm. ( d ) Activated caspase-3-positive cells per field of view for control and β1 tumours ( n = 30). ( e ) Images of control and MDA-MB-231-β1 cells treated for 24 hr with/without 0.5 µ M staurosporine, analyzed by TUNEL assay (red), counterstained with DAPI (blue). Scale bar, 100 µm. ( f ) Proportion (%) of TUNEL-positive nuclei per field of view ( n = 60). ( g ) Blood vessels stained with endothelial marker CD31 (red) and DAPI (blue) in control and β1 tumour sections. Scale bar, 20 µm. ( h ) CD31-positive blood vessels per field of view for control and β1 tumours ( n = 30). ( i ) VEGF content of culture medium of control and MDA-MB-231-β1 cells 1–3 days after plating ( n = 6). Data are mean ± SEM; ** p < 0.01; *** p < 0.001.

Article Snippet: MDA-MB-231-GFP and MDA-MB-231 β1-GFP cells were stably transduced with recombinant lentivirus for firefly luciferase (AMS Biotechnology).

Techniques: Staining, TUNEL Assay, Marker

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

doi: 10.1002/ijc.28890

Figure Lengend Snippet: Effect of β1 on process outgrowth in breast cancer cells. ( a ) Regions of skeletal muscle infiltration in control (i,iii,v) and β1 (ii,iv,vi) tumour sections showing GFP signal (green) and skeletal fast myosin (red). Arrows indicate cells in β1 tumours that have more elongate processes. Scale bar, 50 µm. Insets, higher magnification images of tumour cells showing processes. Inset scale bar, 10 µm. ( b ) Process length (µm) of control MDA-MB-231 and MDA-MB-231-β1 cells in tumours ( n ≥ 134 cells/each). ( c ) Length (µm) of muscle fibers in control and β1 tumours ( n ≥ 79). ( d ) Images of MDA-MB-231 and MDA-MB-231-β1 cells grown on control or β1-expressing CHL fibroblast monolayers, and stained with anti-GFP antibody. Scale bar, 20 µm. ( e ) Process length (µm) of MDA-MB-231 and MDA-MB-231-β1 cells grown on control or β1-expressing CHL monolayers ( n = 300). ( f ) Process length (µm) of MDA-MB-231-β1Δ 40–124 and MDA-MB-231-β1 cells grown on control or β1-expressing CHL monolayers ( n = 300). ( g ) Process length (µm) of MDA-MB-231 cells grown on control or β1-expressing CHL monolayers ± 30 µ M TTX ( n ≥ 144). ( h ) Process length (µm) of MDA-MB-231-β1 cells grown on control or β1-expressing CHL monolayers ± 30 µM TTX ( n = 150). Bars are mean + SEM; *** p < 0.001.

Article Snippet: MDA-MB-231-GFP and MDA-MB-231 β1-GFP cells were stably transduced with recombinant lentivirus for firefly luciferase (AMS Biotechnology).

Techniques: Expressing, Staining

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

doi: 10.1002/ijc.28890

Figure Lengend Snippet: A mechanism for β1-mediated process outgrowth and migration in BCa cells. ( a ) Images of MDA-MB-231 and MDA-MB-231-β1 cells. Green: anti-β1 for parental MDA-MB-231 (with Alexa-488-conjugated secondary antibody) and GFP signal for MDA-MB-231-β1; red: anti-fyn; magenta: phalloidin to label the actin cytoskeleton; blue: DAPI to label nucleus. White boxes indicate locations of inset zoomed images. Phalloidin is omitted from merged image for clarity. Scale bar, 10 µm. ( b ) Intensity correlation-quotients (ICQ) for β1 and fyn in MDA-MB-231 and MDA-MB-231-β1 cells ( n = 20/each). ( c ) Process length (µm) of MDA-MB-231 cells grown on control or β1-expressing CHL monolayers ± 5 µM PP2 ( n ≥ 147). ( d ) Process length (µm) of MDA-MB-231-β1 cells grown on control or β1-expressing CHL monolayers ± 5 µ M PP2 ( n ≥ 223). ( e ) Process length (µm) of MDA-MB-231 cells grown on control or β1-expressing CHL monolayers ± fyn siRNA ( n = 150). ( f ) Process length (µm) of MDA-MB-231-β1 cells grown on control or β1-expressing CHL monolayers ± fyn siRNA ( n = 150). ( g ) A model of possible signalling mechanism underlying β1-mediated process outgrowth in BCa cells. β1 from an adjacent fibroblast or cancer cell interacts in trans with β1 on the BCa cell, initiating a signaling cascade via fyn kinase, leading to process outgrowth. Na + conductance through the pore-forming α subunit is also required. Figure was produced using Science Slides software. Bars are mean + SEM; *** p < 0.001.

Article Snippet: MDA-MB-231-GFP and MDA-MB-231 β1-GFP cells were stably transduced with recombinant lentivirus for firefly luciferase (AMS Biotechnology).

Techniques: Migration, Expressing, Produced, Software

Journal: Science Advances

Article Title: Identification of the NRF2 transcriptional network as a therapeutic target for trigeminal neuropathic pain

doi: 10.1126/sciadv.abo5633

Figure Lengend Snippet: Key resources table. N/A, not available.

Article Snippet: NRF2/ARE Luciferase Reporter HEK-293 Stable Cell Line , Signosis Inc. , Cat# SL-0042-NP.

Techniques: Recombinant, Modification, Protease Inhibitor, Transfection, Peroxidation Assay, Enzyme-linked Immunosorbent Assay, Luciferase, RNA Sequencing, Gene Expression, Stable Transfection, Sequencing, Cloning, Construct, Plasmid Preparation, Software