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, Knockdown, Cell Analysis

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, Knockdown, Immunofluorescence, Staining, Software, Two Tailed Test