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: Genes and Immunity

Article Title: The epigenomic landscape of bronchial epithelial cells reveals the establishment of trained immunity

doi: 10.1038/s41435-025-00357-z

Figure Lengend Snippet: A On day -1 (D-1), BEAS-2B cells were cultured in 96-well plates (1.5 × 104 cells/well). On day (D0), the cells (2.8 × 104 cells/well) were prestimulated (F) (red arrow) or not (Ctl) (grey arrow) with 5 µg/mL ultrapure flagellin (InvivoGen) from Pseudomonas aeruginosa for 48 h. The cells were then washed twice with media on day 2 (D2 and D2F), followed by a resting period of 4 days, until they reached confluence (10.3 × 104 cells/well) on day 6 (D6 and D6F). RNA and DNA were extracted at days 0, 2, and 6 from BECs exposed (F) or not exposed (Ctl) to flagellin and then analysed via microarray and ATAC sequencing. In parallel experiments, to validate the trained response, BECs prestimulated with or without flagellin were infected with A. fumigatus (MOI of 2) for 15 h (dotted lines) on day 6. B Transcriptomic and epigenomic dynamics were analysed by differential analysis between days 2 and 6 vs . day 0 in BECs exposed to flagellin (F) or not exposed (Ctl). Accessible regions and genes undergoing significant expression changes were then jointly classified using an unsupervised machine learning approach to identify clusters with common regulatory profiles. The latter were functionally characterised by gene set enrichment analysis and transcription factor footprinting. D: day, F: P. aeruginosa flagellin, Ctl: control. Created with BioRender.com.

Article Snippet: Human BECs (BEAS-2B cell line; American Type Culture Collection, Rockville, MD, USA) were maintained at 37 °C and 5% CO 2 in F12 medium (Invitrogen, Waltham, MA, USA) supplemented with 10% foetal calf serum, 1% penicillin‒streptomycin, and 10 mM HEPES and stimulated in F12 without antibiotics.

Techniques: Cell Culture, Microarray, Sequencing, Infection, Expressing, Footprinting, Control

Journal: iScience

Article Title: Endothelial cell Nrf2 controls neuroinflammation following a systemic insult

doi: 10.1016/j.isci.2025.112630

Figure Lengend Snippet: Endothelial cell-specific knockout of Nrf2 impairs BEC homeostasis and reduces endothelial barrier strength (A) qRT-PCR confirms gene expression of exon 5 of Nrf2 is specifically deleted in BECs, but not in astrocytes, microglia or oligodendrocytes in Nfe2l2 ENDO mice. Data are represented as mean ± SE, from left to right p = 0.0007, 0.0995, 0.6903, and 0.7432, n = 4, unpaired t-test. (B) Mouse BECs isolated from Nfe2l2 ENDO mice and Nfe2l2 Flox mice were cultured in vitro and TEER was measured for 24hrs. ∗ p = 0.0005, One-way ANOVA plus Sidak post hoc (n = 8–9). Data are represented as mean ± SE. (C) Scatterplot of RNA-seq analysis showing the influence of Nrf2 specific KO in ECs on BEC transcriptome in basal conditions. Two weeks after tamoxifen-induced recombination, BECs were FACS-sorted from Nfe2l2 ENDO mice and their Nfe2l2 fl/fl littermate’s control mice. Scatterplot was generated for genes with average expression >0.1 FPKM across the datasets. Highlighted with red and blue crosses are the genes whose expression are significantly increased or decreased respectively ( DESeq2 P _adj < 0.05, n = 4–6). (D) Volcano plot of mass spectrometry analysis showing the influence of Nrf2 specific KO in ECs on BEC proteome in basal conditions. Two weeks after tamoxifen-induced recombination, BECs were FACS-sorted from Nfe2l2 ENDO mice and their Nfe2l2 fl/fl littermate’s control mice. Highlighted with red and blue dots are the proteins whose expression are significantly increased or decreased respectively ( p < 0.05, n = 4).

Article Snippet: Human Brain Microvascular Endothelial Cells (BECs) were purchase from Innoprot ( P10361 -IM, Elexalde Derio, Spain).

Techniques: Knock-Out, Quantitative RT-PCR, Gene Expression, Isolation, Cell Culture, In Vitro, RNA Sequencing, Control, Generated, Expressing, Mass Spectrometry

Journal: iScience

Article Title: Endothelial cell Nrf2 controls neuroinflammation following a systemic insult

doi: 10.1016/j.isci.2025.112630

Figure Lengend Snippet: Nrf2 activator RTA-404 modifies the BEC transcriptome under inflammatory conditions Mice were given i.p. injection of either saline or RTA-404 (n = 6–8) daily for 4 days, following which either saline or LPS was given for 24 h. The brain cells were isolated and then sorted with FACS for BECs, RNA extracted and RNA-seq performed. Scatterplots were generated for genes with average expression >0.1 FPKM across the datasets. Highlighted with red and blue crosses are the genes whose expression are significantly increased or decreased respectively ( DESeq2 P _adj < 0.05, n = 6–8). (A) Scatterplot of RNA-seq analysis showing the BEC transcriptome modified by peripheral LPS insult compared with saline in Nfe2l2 fl/fl mice (LPS vs. Saline). (B) Scatterplot of RNA-seq analysis showing the LPS-induced BEC transcriptome modified by pre-administration of RTA-404 in Nfe2l2 fl/fl mice (LPS+RTA-404 vs. LPS+saline). (C) IPA analysis identifying activated or inhibited pathways by pre-administration of RTA-404 under conditions of LPS exposure (LPS+RTA-404 vs. LPS+saline). Significant DEGs ( DESeq2 P _adj < 0.05, ∣Log 2 FC∣>1, n = 6–8) with the BEC transcriptome as reference dataset were analyzed to calculate the p-value of overlap and Z score of overall activation/inhibition states of individual pathways. The significant activated (p < 0.05, Z score >2) or inhibited (p < 0.05, Z score<−2) pathways are shown in the bar chart in orange and blue respectively. (D) Volcano plot of mass spectrometry analysis showing the LPS-induced BEC proteome modified by pre-administration of RTA-404 in Nfe2l2 fl/fl mice (LPS+RTA-404 vs. LPS+saline). (E) Scatterplot of RNA-seq analysis showing the LPS-induced BEC transcriptome modified by pre-administration of RTA-404 in Nfe2l2 ENDO mice.

Article Snippet: Human Brain Microvascular Endothelial Cells (BECs) were purchase from Innoprot ( P10361 -IM, Elexalde Derio, Spain).

Techniques: Injection, Saline, Isolation, RNA Sequencing, Generated, Expressing, Modification, Activation Assay, Inhibition, Mass Spectrometry