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: Oncology Letters

Article Title: Analysis of PKC-ζ protein levels in normal and malignant breast tissue subtypes

doi: 10.3892/ol.2018.9792

Figure Lengend Snippet: PKC-ζ knockdown retards invasion of MDA-MB-231 breast cancer cells. MDA-MB-231 breast cancer cells were placed in the upper chamber of Transwell plate coated with 0.5X BME and serum containing media was placed in the lower chamber as a chemoattractant. Following transfection with siPRKCZ and scrambled RNA, cells that invaded through the BME and migrated into the lower chamber were stained with crystal violet and (A) observed under a microscope at magnification, ×10; scale bar, 1 mm. Untreated cells and cells treated with the transfection reagent (Si-Tran) were also used to establish the effect on invasion. (B) The cells were counted for each treatment image ( N =3), averaged and analyzed with the standard deviation, a Student's t-test (*P<0.05) and a one-way ANOVA was also used to evaluate the data with the post-hoc Tukey's HSD test (P<0.01), Scheffé multiple comparison (P<0.01), Bonferroni (P<0.01) and Holm (P<0.01). All statistical tests were used to determine the statistical significance of the invasion data. (C) The cells were further investigated by western blot to analyze the Rac1/RhoA pathway. This data was quantified and averaged for the graph with standard error of the mean represented. PKC-ζ, protein kinase C-ζ; BME, basement membrane extract; Rac1, Ras-related C3 botulinum toxin substrate 1; RhoA, Ras homolog gene family member A.

Article Snippet: Human breast cancer cells MDA-MB-231 were grown in 100 mm plates and transfected with 20 nM of scrambled RNA and siPRKCZ (5′-GCAUGAUGACGAGGAUAUUGACUGG-3′, SR303747A; Origene, Rockville, MD, USA) for 48 h. Cells were lysed as previously described and the lysates were run on western blots.

Techniques: Transfection, Staining, Microscopy, Standard Deviation, Western Blot

Journal: Oncology Letters

Article Title: Analysis of PKC-ζ protein levels in normal and malignant breast tissue subtypes

doi: 10.3892/ol.2018.9792

Figure Lengend Snippet: Organization of F-actin upon the knockdown of PKC-ζ. MDA-MB-231 breast cancer cells were treated with SiTran, scrambled RNA and siPRKC-ζ for 24 h. The cells that received no treatment were used as the control. These cells were then stained with 1X Phalloidin-iFluor 594 and counterstained with DAPI. Scale bar, 100 µm. Original magnification, ×20. DAPI, 4′,6-diamidino-2-phenylindole; F-actin, filamentous actin; PKC-ζ, protein kinase C-ζ.

Article Snippet: Human breast cancer cells MDA-MB-231 were grown in 100 mm plates and transfected with 20 nM of scrambled RNA and siPRKCZ (5′-GCAUGAUGACGAGGAUAUUGACUGG-3′, SR303747A; Origene, Rockville, MD, USA) for 48 h. Cells were lysed as previously described and the lysates were run on western blots.

Techniques: 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 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