Journal: Cancers

Article Title: Niclosamide and Palbociclib Act Synergistically to Reduce Cholangiocarcinoma Cell Viability In Vitro and Inhibit Tumour Growth in a Mouse Model

doi: 10.3390/cancers17223721

Figure Lengend Snippet: Drug screening and validation of Niclosamide using CCA cell lines and primary biliary epithelial cells. ( A ) A library of 104 off-patent drugs was screened using the CCLP CCA cell line. CCLP cells were plated at 1 × 10 4 cells per well in a 96 well plate and treated with each drug at its clinically relevant peak serum concentration for 96 h. Cell viability was then determined using an MTT assay and normalised to cells treated with the corresponding vehicle control for each drug. N = 3 biological repeats with a paired, two-tailed T-test followed by Benjamini–Hochberg multiple corrections. The inhibition of viability (%) is plotted against the reciprocal of the p value for each drug and Niclosamide is labelled. ( B – E ) Niclosamide dose–response curves for CCA cell lines (CCLP, RBE, and KKU-M055) and primary biliary epithelial cells (BECs) plated as in ( A ) and treated with Niclosamide [10 nM–100 μM] or vehicle control for 72 h. Cell viability was measured using an MTT assay and normalised to vehicle control. N = 3 biological repeats each performed in triplicate. Error bars represent SEM. When error bars cannot be seen they are smaller than the symbols. ( F ) The graph shows the relative EC50 values for each cell type calculated using GraphPad Prism. Error bars represent SEM (* = p < 0.05 unpaired t -test). The inserted Western blot shows PRH levels in the CCA cell lines prior to treatment and with β-actin as a loading control.

Article Snippet: Primary Biliary Epithelial Cells (BECs) obtained from healthy human liver tissue (Innoprot, Elexalde Derio, Spain) were grown in Epithelial Cell Media (Innoprot, P60106 , Elexalde Derio, Spain) supplemented with 2% FBS and 1% Epithelial Growth Supplement, as per the supplier’s protocol for a maximum of ten passages.

Techniques: Drug discovery, Biomarker Discovery, Concentration Assay, MTT Assay, Control, Two Tailed Test, Inhibition, Western Blot

Journal: Cancers

Article Title: Niclosamide and Palbociclib Act Synergistically to Reduce Cholangiocarcinoma Cell Viability In Vitro and Inhibit Tumour Growth in a Mouse Model

doi: 10.3390/cancers17223721

Figure Lengend Snippet: Palbociclib decreases the viability of CCA cells but has less effect on primary biliary epithelial cells. CCA cell lines (CCLP ( A ), RBE ( B ), KKU-M055 ( C ), or primary BECs ( D )) were plated at 1 × 10 4 cells per well in a 96-well plate and treated with increasing concentrations of Palbociclib for 96 h. Cell viability was then measured using an MTT assay and normalised to the vehicle control. Three biological repeats (five for CCLP cells) each performed in triplicate. Error bars represent SEM. When error bars cannot be seen, they are smaller than the symbols. ( E ) The graph shows the relative EC50 values calculated using GraphPad Prism. Error bars represent SEM (** = p < 0.01 *** = p < 0.001 unpaired t -test).

Article Snippet: Primary Biliary Epithelial Cells (BECs) obtained from healthy human liver tissue (Innoprot, Elexalde Derio, Spain) were grown in Epithelial Cell Media (Innoprot, P60106 , Elexalde Derio, Spain) supplemented with 2% FBS and 1% Epithelial Growth Supplement, as per the supplier’s protocol for a maximum of ten passages.

Techniques: MTT Assay, Control

Journal: Cancers

Article Title: Niclosamide and Palbociclib Act Synergistically to Reduce Cholangiocarcinoma Cell Viability In Vitro and Inhibit Tumour Growth in a Mouse Model

doi: 10.3390/cancers17223721

Figure Lengend Snippet: Niclosamide and Pablociclib act synergistically to reduce CCA cell viability. ( A ) CCLP cells were plated at 1 × 10 4 cells per well in a 96-well plate before treatment with Niclosamide (NIC), Palbociclib (PAL), or both drugs in combination (COM) at the concentrations shown and for 72 h. Cell viability was then measured using an MTT assay. N = 3 biological experiments each performed in triplicate. Error bars represent SEM (* = p < 0.05, ** = p < 0.01, ns = not significant) two-tailed paired t -test. ( B ) From the data shown in ( A ) combination index (CI), values were calculated and plotted against the corresponding response (Fa) values in a CI plot. CI < 1 indicates synergism. ( C ) CCLP cells were treated with 0.5 μM Niclosamide (NIC), 1 μM Palbociclib (PAL), or both drugs in combination (COM) for 24 h, then proteins were extracted for Western blot. ( D ) CCLP cells and BECs were grown as spheroids for 5 days before treatment with 0.5 μM Niclosamide, 1 μM Palbociclib, or both drugs in combination for a further 72 h. Images were taken using a Nikon brightfield microscope, and spheroid size was calculated using ImageJ. N = 3 biological repeats. Error bars represent SEM (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) two-tailed paired t -test. ( E ) Cell viability was measured in the spheroids from ( D ) using a PrestoBlue assay. Original western blots are presented in .

Article Snippet: Primary Biliary Epithelial Cells (BECs) obtained from healthy human liver tissue (Innoprot, Elexalde Derio, Spain) were grown in Epithelial Cell Media (Innoprot, P60106 , Elexalde Derio, Spain) supplemented with 2% FBS and 1% Epithelial Growth Supplement, as per the supplier’s protocol for a maximum of ten passages.

Techniques: MTT Assay, Two Tailed Test, Western Blot, Microscopy, Prestoblue Assay

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) A schematic of an organotypic 3D lymphatic vessel model (LV-on-chip). Prox-1 (green) and CD31 (red) expression confirms lymphatic endothelial identity and cell morphology in the channel. (B) Morphologic changes in human dermal microvascular blood endothelial cells (BECs) with lymphatic endothelial cells (LECs) after one day of cell seeding. BECs become more contractile than LECs, forming a smaller vessel diameter compared to LECs. (C) BVs and LVs observed in mouse ear tissues. mLYVE-1, anti-mouse LYVE-1 antibody; mCD31, anti-mouse CD31 antibody. (D) Phalloidin (red) and anti-VE-cad (VE-cadherin) antibody (green) staining to visualize F-actin and adherens junctions. (E) Lymphatic and blood vessel barrier function. 70 kDa dextran was introduced into the vessel lumens and dextran diffusion was observed in real time under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (F) Quantification of the permeability of BEC-generated engineered BVs and LEC-generated LVs. ** p = 0.0016, two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M.

Article Snippet: Cell culture Primary human dermal microvascular lymphatic endothelial cells (LECs, neonatal) were purchased from Lonza.

Techniques: Expressing, Staining, Diffusion-based Assay, Microscopy, Permeability, Generated, Two Tailed Test