Journal: bioRxiv

Article Title: C-type lectin-like receptor 2 (CLEC-2)-dependent DC migration is controlled by tetraspanin CD37

doi: 10.1101/227918

Figure Lengend Snippet: (A) Adherence of WT (left) and Cd37-/- (right) DCs on TNFα-stimulated lymphatic endothelial cells (LEC). One representative image per genotype is shown. Scale bar = 50μm. (B) Adhesion (% of total cells added per well) of WT (black line) and Cd37-/- (grey line) DCs on LECs for the duration of the experiment. Data shown from one representative experiment. Experiments were repeated two times yielding similar results. (C) Migration velocity (μm/min) of WT (white box) and Cd37-/- (grey box) DCs over LECs. Bars represent median with interquartile range from two independent experiments, n=107-115 cells per genotype, respectively. Non-parametric Mann Whitney test, two-tailed, ****p<0.0001. (D) Left: zoom of field of view shown in (A) with individual cell tracks. Tracking paths of each cell of one representative experiment are shown in Supplementary Movies 1A-B. Upper image= WT, lower image = Cd37-/-. Scale bar = 25μm. Right: Mean square displacement (MSD, in μm 2 ) of WT (black line) and Cd37-/- (grey line) DCs on LEC. Data are shown as mean±SEM from two independent experiments. Two-way ANOVA with Sidak’s multiple comparisons, ****p<0.0001 at t=5 min.

Article Snippet: Primary human dermal lymphatic endothelial cells (LEC) were purchased from PromoCell and cultured in manufacturer’s recommended medium (Endothelial Cell Growth Medium MV2, PromoCell, Heidelberg, Germany) supplemented with 35μg/mL gentamicin (Gibco, via Fisher Scientific - UK Ltd, Loughborough, UK).

Techniques: Migration, MANN-WHITNEY, Two Tailed Test

Journal: Cancer Science

Article Title: Suppression of lymph node and lung metastases of endometrial cancer by muscle‐mediated expression of soluble vascular endothelial growth factor receptor‐3

doi: 10.1111/cas.12184

Figure Lengend Snippet: Suppression of vascular endothelial growth factor (VEGF)‐C‐driven lymphatic endothelial cell (LEC) proliferation by conditioned medium of soluble vascular endothelial growth factor receptor‐3 (sVEGFR‐3)‐expressing cells. Cells were plated at 5 × 103 cells/well in 96‐well plates, and 50% sVEGFR‐3‐conditioned medium or luciferase‐conditioned medium was added with 100 ng/mL recombinant human VEGF‐C. The number of LEC with 50% sVEGFR‐3‐conditioned medium (b) was clearly smaller than that with control (a). The cells were counted by colorimetric assay 48 h after plating. Each bar represents the mean ± SD. (*P < 0.01) (c).

Article Snippet: Human neonatal dermal lymphatic endothelial cells (LEC) were purchased from AngioBio (Del Mar, CA, USA) and maintained in EGM‐MV2 BulletKit (Cambrex, East Rutherford, NJ, USA) supplemented with 10% inactivated FCS at 37°C in a 5% CO 2 atmosphere.

Techniques: Expressing, Luciferase, Recombinant, Colorimetric Assay

Journal: PLoS Pathogens

Article Title: Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs

doi: 10.1371/journal.ppat.1006524

Figure Lengend Snippet: (A) Schematic illustration of the procedure used in this study to infect educated cells. 10 5 non-infected cells were incubated for total of 48 hours when 2.5x10 9 exosomes were added every 24 hours, and infected with BAC16-derived WT KSHV. Cells were analysed 48 hours’ post KSHV infection. (B) Percentage of infected cells after education using LEC or KLEC exosomes. Cells were analysed using flow cytometry. (C) KSHV genome copy number in LEC educated using KLEC and ΔmiR-KLEC exosomes, 48 hours post KSHV infection. qPCR was carried out as previously described . (D) Expression of HIF1α in LEC expressing the HIF1α P402A/P564A mutant. Lysates from cell infected with empty vector (EV), mutant or WT HIF1α were separated by SDS/PAGE and analysed by immunoblot for expression of HIF1α. (E) LEC expressing WT and mut-HIF1α were infected with BAC16-derived WT KSHV and examined for KSHV genome copy number using qPCR. (F) Schematic illustration of the procedure used in this study to co-culture KLEC with educated cells. 10 5 HUVEC cells were incubated for total of 48 hours when 2.5x10 9 exosomes were added every 24 hours. 10 5 of the educated cells were washed, trypsinized and then co-cultured with 10 5 KLEC in Transwell plates for 2 days. (G) KLEC growth in the presence of educated HUVEC. KLEC were trypsinised and each condition was counted using both hemocytometer and the Millipore Scepter 2.0 Handheld Automated Cell Counter. (H) KLEC growth in the presence of HUVEC over-expressing HIF1α. The indicated cells were co-cultured and counted as described in (G). (I) KLEC media was supplemented with 1.5 or 10mM sodium L-Lactate. Cell number was measured using the CellTiter-Fluor assay (promega) after 24 and 48 hours. All bar graphs present the mean and standard deviation of 3 biological repeats. Statistical significance was denoted by *P < .05, **P < .01, ***P < .001.

Article Snippet: HUVEC and LEC were purchased from Promocell and grown in endothelial growth medium 2 and MV2 (Promocell) respectively.

Techniques: Infection, Incubation, Derivative Assay, Flow Cytometry, Expressing, Mutagenesis, Plasmid Preparation, SDS Page, Western Blot, Co-Culture Assay, Cell Culture, Standard Deviation

Journal: PLoS Pathogens

Article Title: Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs

doi: 10.1371/journal.ppat.1006524

Figure Lengend Snippet: (A-B) 10 5 LEC were educated using 2.5x10 9 LEC, KLEC or ΔmiR-KLEC—derived exosomes. Angiogenic potential was measured using an endothelial cell tube-formation assay. (A) Representative images of tubes formed on matrigel. (B) Average number of tubes per field acquired. The graph presents the mean and standard deviation of 3 biological repeats. Statistical significance was denoted by *P < .05, **P < .01, ***P < .001. (C-D) Cell migration of educated HUVEC was measured using wound healing assay (scratch assay). Educated HUVEC were seeded at 80% confluence in a 24-well plate and allowed to equilibrate overnight. Once scratched, plates were immediately placed in Nikon Biostation CT or Zeiss Cell Observer and Images were acquired for 16 hours. (C) representative images of educated HUVEC at 0, 3, 6 and 9 hours’ post scratch. (D) Relative scratch area over time as analysed using ImageJ.

Article Snippet: HUVEC and LEC were purchased from Promocell and grown in endothelial growth medium 2 and MV2 (Promocell) respectively.

Techniques: Derivative Assay, Endothelial Cell Tube Formation Assay, Standard Deviation, Migration, Wound Healing Assay