Journal: bioRxiv

Article Title: A scalable long-term in vitro model of human colonic epithelium with continuous barrier function

doi: 10.1101/2025.09.24.677447

Figure Lengend Snippet: A. Hourly TEER graphed as mean ± SEM of each group with the following technical replicates: Control (n = 14), 5 ng/mL IFN-γ (n = 9), 5 ng/mL TNF-α (n = 9), 5 ng/mL IFN-γ and 5 ng/mL TNF-α (n = 9), 2 mM EDTA (n = 9), and 10 ng/mL TcdB (n=9). Treatments were applied to three different cell lines using Colon 81 (left), Colon 83 (middle), and Colon 88 (right). Treatment was added to respective groups at time indicated by dotted lines within gray shaded area of each graph. B. Daily mean percent change in TEER from control ± SEM of each group per cell line. Bi. Bii. Biii. Statistical significance of percent change of mean daily TEER from control was determined using p values calculated by Welch’s t -test. A significant increase (blue p value) or decrease (red p value) in the percent change in TEER from control is shown. C. Representative bright field images of Colon 81 LT monolayer treatment groups at day 12. Images were taken at 10X with an Olympus IX73 microscope. Scale bars represent 100 μm.

Article Snippet: The addition of cytokines for barrier challenge occurred on day 8 and included recombinant human TNF-α (5 ng/mL, InvivoGen rcyc-htnfa) and/or recombinant human IFN-γ (5 ng/mL, InvivoGen rcyec-hifng) diluted in the basolateral medium where the Clostridium difficile Toxin B (TcdB; 10 ng/mL, Sigma-Aldrich SML1153) and EDTA (2mM) were diluted in the apical medium.

Techniques: Control, Microscopy

Journal: Cancers

Article Title: Bone Marrow CX3CL1/Fractalkine is a New Player of the Pro-Angiogenic Microenvironment in Multiple Myeloma Patients

doi: 10.3390/cancers11030321

Figure Lengend Snippet: MM cells increase the angiogenic activity of ECs through CX3CL1 production. HUVEC ( A ) and HBMESC ( B ) were treated for 24 h with TNFα (50 ng/ml) and IFNγ (10 ng/ml), as positive control, and with CM of HMCLs (JJN3, OPM2, and MM1S) in presence or absence of anti-TNFα neutralizing antibody (2.7 µg/ml). At the end of the experiments, CX3CL1 levels were evaluated by enzyme-linked immunosorbent assay (ELISA) in the supernatants. The histograms represent the mean ± SD of CX3CL1 levels in two different experiments. (ECs + CM HMCLs vs. Cnt (HUVEC or HBMESC alone): # P < 0.05). # P values were calculated by two-tailed Student’s t -test. HUVEC—human umbilical vein endothelial cells; TNFα—with tumor necrosis factor alpha; IFNγ—interferon gamma.

Article Snippet: In collagen-coated six-well plates, HUVEC and HBMESC were incubated in the presence or absence of diluted CM (1:3) of OPM2, JJN3, and MM1S for 24 h and with or without recombinant TNFα (50 ng/ml) (OriGene; Rockville, MD, USA) and/or neutralizing anti-TNFα mAb (2.7 μg/ml able to neutralize 50 ng/ml) (R&D systems Minneapolis, MN, USA) for 24 h. HUVEC and HBMESC treated with TNFα (50 ng/ml) and IFNγ (10 ng/ml) (Sigma–Aldrich, Italia, Milan, Italy) for 24 h were used as a positive control.

Techniques: Activity Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: βIII-Tubulin is a Brake on Extrinsic Cell-Death in Pancreatic Cancer

doi: 10.1101/2022.09.29.510034

Figure Lengend Snippet: (A-D) Apoptosis measured using flow cytometry for Annexin V/DAPI in PDAC cells transfected with non-silencing (ns) or βIII-tubulin (βIII-Tub) siRNA and treated ± TNFα in MiaPaCa2 (n=3) (A) , PANC1 (n=5) (B) , TKCC5 (n=3) (C) and TKCC10 (n=3) (D) cells. (E) Cell proliferation analysis in MiaPaCa2 cells (n=4) with βIII-tubulin knockdown ±TNFα, measured on IncuCyte® S3 Live-Cell Analysis. (F-G) Apoptosis measured in MiaPaCa2 (n=3) (F) and TKCC10 (n=4) (G) cells transfected with ns-siRNA or βIII-tubulin siRNA and treated ± FasL. Bars represent mean of n≥3 independent experiments (data points shown from independent experiments) ± SEM. Asterisks indicate significance as assessed by one-way ANOVA, Bonferroni’s multiple comparisons test (*p≤0.05, **p≤0.01, ***p≤0.001, ****p ≤0.0001, n.s.; non-significant).

Article Snippet: Similarly, cells were treated with human recombinant TNFα (abcam, cat. ab9642) 48 hours post transfection, and apoptosis measured 24 hours after treatment with TNFα (MiaPaCa2: 10 ng/mL; PANC1: 10 ng/mL; TKCC5: 20 ng/mL; TKCC10: 20 ng/mL).

Techniques: Flow Cytometry, Transfection

Journal: bioRxiv

Article Title: βIII-Tubulin is a Brake on Extrinsic Cell-Death in Pancreatic Cancer

doi: 10.1101/2022.09.29.510034

Figure Lengend Snippet: (A) Live cell proliferation measured in GFP-MiaPaCa2 cells with non-silencing (ns) or βIII-tubulin (βIII-Tub) siRNA and co-cultured with cancer-associated fibroblasts (CAFs) (n=3 independent experiments with CAFs from 3 PDAC patients). (B) Apoptosis was measured using flow cytometry for AnnexinV/DAPI in GFP-MiaPaCa2 cells with βIII-tubulin knockdown and co-cultured with CAFs (n=3). (C) Immunofluorescence was used to measure the percentage of cleaved caspase 8 positive cells in cytokeratin positive PDAC cells when cultured alone or in the presence of CAFs. Cells were treated with or without TNFα-neutralising antibody (2 μg/mL). Representative images show immunofluorescence staining in MiaPaCa2 cells. All scale bars represent 100 μm. Equal number of total cells (MiaPaCa2 + CAFs) were seeded across each well. (D-E) QuPath software was used to measure the percentage of cleaved caspase 8 positive cells in cytokeratin positive MiaPaCa2 (D) or TKCC10 (E) cells (n=3). Bars represent mean of n≥3 independent experiments (data points shown from independent experiments) ± SEM. Asterisks indicate significance as assessed by one-way ANOVA, Bonferroni’s multiple comparisons test or two-tailed paired t-test (*p≤0.05, **p≤0.01, ***p≤0.001, ****p ≤0.0001, n.s.; non-significant).

Article Snippet: Similarly, cells were treated with human recombinant TNFα (abcam, cat. ab9642) 48 hours post transfection, and apoptosis measured 24 hours after treatment with TNFα (MiaPaCa2: 10 ng/mL; PANC1: 10 ng/mL; TKCC5: 20 ng/mL; TKCC10: 20 ng/mL).

Techniques: Cell Culture, Flow Cytometry, Immunofluorescence, Staining, Software, Two Tailed Test