Journal: Clinical and Experimental Immunology

Article Title: Tolerogenic dendritic cells generated with dexamethasone and vitamin D3 regulate rheumatoid arthritis CD4 + T cells partly via transforming growth factor‐ β 1

doi: 10.1111/cei.12870

Figure Lengend Snippet: Transforming growth factor (TGF)‐β1 diminishes the ability of tolerogenic dendritic cells (tolDC) to stimulate healthy control (HC) T cells and is involved in their regulatory function. (a–d) Mature lipopolysaccharide (LPS)‐activated dendritic cells (matDC) or tolDC (1 × 104 cells/well) were co‐cultured with allogeneic HC CD4+ T cells (1 × 105 cells/well) in the absence or presence of increasing concentrations of SB‐505124, a small molecule inhibitor of TGF‐βRI (a) or 1 μM SB‐505124 (b,c) or increasing concentrations of recombinant latency‐associated peptide (LAP) (d). Proliferation (a,b,d, left panels), measured using [3H]‐thymidine uptake, and interferon (IFN)‐γ production (a,b,d, right panels), measured using enzyme‐linked immunosorbent assay (ELISA), were assessed at day 6. (c) Additional cytokines were detected in day 6 culture supernatants from tolDC‐CD4+ T‐cell cultures using an ELISA [interleukin (IL)−17A] or Meso Scale Discovery (MSD) immunoassay (all others). The fold change in cytokine production following inhibition of TGF‐β1 signalling was calculated by: concentration of cytokine produced in the presence of 1μM SB‐505124 ÷ concentration of cytokine produced in the absence of 1 μM SB‐505124. IL‐17A was undetectable in one experiment. Error bars in (a) and (d) represent standard error of the mean (s.e.m.) of triplicates (proliferation) or duplicates (IFN‐γ production). Horizontal lines in (b) and (c) represent median values, (b) left panel (proliferation) n = 7; (b) right panel (IFN‐γ production) n = 15; (c) (cytokines) n = 3. (e) Allogeneic healthy control (HC) CD4+ T cells were primed with DC (10 : 1) for 6 days and rested for 4 days with 10 IU/ml IL‐2. T cell lines primed by matDC (Tmat), tolDC (Ttol) and tolDC + SB‐505124 (Ttol‐SB) were restimulated with matDC and IFN‐γ (left panel) and IL‐10 (right panel) production, measured using ELISA, was assessed on day 3. Results are depicted as the percentage cytokine production of Tmat cell lines. Cytokine concentrations range: IFN‐γ in Ttol = 0.9–20·6 ng/ml and in Ttol‐SB = 4·4–80·7 ng/ml; IL‐10 in Ttol = 0·4–5·1 ng/ml and in Ttol‐SB = 0·9–31·7 ng/ml. Horizontal lines represent median values, n (IFN‐γ production) = 7; n (IL‐10 production) = 6. *P < 0·05 and ***P < 0·0001 calculated with Wilcoxon signed‐rank test. #Significant differences (P < 0·05) between Ttol and Tmat cells.

Article Snippet: TGF‐βRI (ALK5) inhibitor (SB‐505124; Sigma) or recombinant human LAP (R&D Systems) was added where indicated.

Techniques: Control, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Inhibition, Concentration Assay, Produced

Journal: Anticancer research

Article Title: Intraductal Adaptation of the 4T1 Mouse Model of Breast Cancer Reveals Effects of the Epithelial Microenvironment on Tumor Progression and Metastasis

doi: 10.21873/anticanres.13344

Figure Lengend Snippet: Tumor sphere formation and drug sensitivity. A: Representative image of 4T1 tumor sphere (scale bar=100 μm). B: Numbers of primary and secondary 4T1 tumor spheres (n=3). C: Lapatinib reduced 4T1 tumor sphere formation. D: Inhibitor of activin-like kinase 4/5/7 receptor signaling SB-505124 had no effect on 4T1 tumor sphere formation. Data are presented as the mean±standard error. DMSO: Dimethyl sulfoxide; SFE: Sphere-forming efficiency.

Article Snippet: Lapatinib (Santa Cruz Dallas, TX, USA) and SB-505124 (Sigma) were diluted in dimethyl sulfoxide (DMSO).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method

doi: 10.1186/s13287-017-0738-4

Figure Lengend Snippet: Schematic illustration of in vivo development and in vitro differentiation of RPE and LESCs. a At early stages of embryonic eye development, surface ectoderm thickens and invaginates together with the underlying neuroepithelium of the optic vesicle. The bilayered optic cup gives rise to the neural retina and the retinal pigment epithelium ( RPE ), while the lens and corneal epithelium develop from the surface ectoderm . Some of the known signaling pathways affecting the cell fate choice during differentiation, such as bone morphogenetic protein ( BMP ) for surface ectoderm, are shown. b An overview of the optimized human pluripotent stem cell ( hPSC ) culture and directed RPE and limbal epithelial stem cell ( LESC ) differentiation protocols, as well as key media and matrix components used (not to scale). Blebb. blebbistatin, col IV collagen type IV, E8 Essential 8™ Flex Medium, FGF fibroblast growth factor, LN-521 recombinant laminin-521, SB SB-505124 hydrochloride hydrate, TGFβ transforming growth factor beta, XF-Ko-SR Knock-out™ serum replacement XenoFree CTS™

Article Snippet: For LESC differentiation, EBs were subjected to surface ectodermal induction: 1 day in XF-Ko-SR medium supplemented with 10 μM SB-505124 and 50 ng/ml human basic fibroblast growth factor (bFGF; PeproTech Inc., Rocky Hill, NJ, USA), followed by 2 days in XF-Ko-SR medium supplemented with 25 ng/ml bone morphogenetic protein (BMP)-4 (PeproTech Inc.).

Techniques: In Vivo, In Vitro, Recombinant, Knock-Out

Journal: Stem Cell Research & Therapy

Article Title: Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method

doi: 10.1186/s13287-017-0738-4

Figure Lengend Snippet: Directed differentiation of feeder-free hPSCs led to rapid hPSC-LESC production. a Schematic illustration of the differentiation strategy. b Phase-contrast images showing cell morphology after 22 days of differentiation for hESC1-LESCs; scale bars = 100 μm. c Expression of LESC markers after 22 days of differentiation. Nuclei counterstained with DAPI; scale bars = 100 μm. d Percentage of p40- and p63α-positive cells quantified from cytospin samples after 24 days of differentiation; n = 10 images, 1986 cells. bFGF basic fibroblast growth factor, Blebb. blebbistatin, BMP bone morphogenetic protein, IF immunofluorescence, SB SB-505124 hydrochloride hydrate

Article Snippet: For LESC differentiation, EBs were subjected to surface ectodermal induction: 1 day in XF-Ko-SR medium supplemented with 10 μM SB-505124 and 50 ng/ml human basic fibroblast growth factor (bFGF; PeproTech Inc., Rocky Hill, NJ, USA), followed by 2 days in XF-Ko-SR medium supplemented with 25 ng/ml bone morphogenetic protein (BMP)-4 (PeproTech Inc.).

Techniques: Expressing, Immunofluorescence