Journal: Frontiers in Oncology

Article Title: Activin A Signaling Regulates IL13Rα2 Expression to Promote Breast Cancer Metastasis

doi: 10.3389/fonc.2019.00032

Figure Lengend Snippet: INHBA is correlated with IL13Rα2 expression in breast cancer cells. (A) MII cells were cultured in DMEMF/12 medium containing 0.2% horse serum (HS) overnight and then treated with 10 ng/ml Activin A for different time points. Western blotting was performed using whole cell lysates to assess phosphorylation of Smad2 (Ser465/467), Smad3 (Ser423/425), Akt (Ser 473), and Erk1/2 (Thr202/Tyr204). Detection of total Smad2, Smad3, Akt, and Erk1/2 was used as loading controls. Protein expression was quantified using ImageJ software. (B) MII cells were mock-treated (DMSO) or treated 1 μM EW-7197 or SB-505124, in the presence or absence of 10 ng/ml Activin A for 1 h. Western blotting was performed using whole cell lysates to assess the phosphorylation status of Smad2 and Smad3. Total Smad2, Smad3, and β-actin were detected as loading controls. (C) MIV cells were treated with 1 μM EW-7197 or 1μM SB-505124 for 24 h. Real time qPCR was used to measure the mRNA expression of IL13Rα2. Asterisk ( * ) indicates a statistically significant difference between Activin A-treated and mock-treated cells ( n = 6; p < 0.05). (D) MIV cells were stably transduced with lentiviral vectors expressing shSCR or shSmad2. Real time qPCR was used to measure the mRNA expression of IL13Rα2. Asterisk ( * ) indicates a statistically significant difference between MIV-shSCR and MIV-shSmad2 ( n = 3; p < 0.05). (E) Western blotting showing the protein expression of Smad2 and IL13Rα2 in MIV-shSCR and MIV-shSmad2 cells. Antibody against β-tubulin was used as a loading control. Protein expression was quantified using ImageJ software.

Article Snippet: To study the effects of small molecule inhibitors on Activin A-induced gene expression, MII cells were serum starved in 0.2% HS-containing DMEM/F12 overnight and then treated with 1 μM EW-7197 (TargetMol), 1 μM SB-505124 (TargetMol) or 10 ng/ml Activin A alone or in combination (as indicated in respective figures).

Techniques: Expressing, Cell Culture, Western Blot, Software, Stable Transfection, Transduction

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: APL Bioengineering

Article Title: Fibroblast senescence-associated extracellular matrix promotes heterogeneous lung niche

doi: 10.1063/5.0204393

Figure Lengend Snippet: Inhibition of TGFβR1 and MRTF-A induces divergent senescent lung phenotype. (a) 24 h prior to stress-induced premature senescence is induced by γ-irradiation (15 Gy), lung fibroblasts were treated with either selective TGβFR1 inhibitor (SB505124) or MRTF-A inhibitor (CCG-1423) and cultured over 14 days (Created with BioRender.com ). (b) Representative immunofluorescent images stained with Hoechst 33342 (blue), phalloidin rhodamine (red), anti-SMA (green) (obj. = 40×, N.A. = 1.3, scale bar = 50 μ m). (c) Inhibited senescent lung fibroblast nuclear area measured at day 14 post-irradiation (n ≥ 149, N = 3). (d) Inhibited senescent lung fibroblast cell area measured at day 14 post-irradiation (n ≥ 100, N = 3). (e) Percent of inhibited senescent fibroblasts with multinucleation (n ≥ 140, N = 3). (f) Percent of inhibited senescent fibroblasts positive for γH2A.X (n ≥ 100, N = 3). (g) Percent of inhibited senescent lung fibroblasts positive for αSMA stress-fibers (n ≥ 100, N = 3). Mean ± SEM; ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Senescent pulmonary fibroblasts were seeded into 96-well plates and treated with either culture media, DMSO, 1, 5, and 10 μ M SB505124 (Cayman Chemical, No. 11793) or CCG-1423 (Cayman Chemical, No. 10010350) every 48 h for 14 days.

Techniques: Inhibition, Irradiation, Cell Culture, Staining

Journal: APL Bioengineering

Article Title: Fibroblast senescence-associated extracellular matrix promotes heterogeneous lung niche

doi: 10.1063/5.0204393

Figure Lengend Snippet: TGFβR1 inhibition reduces SA-ECM remodeling heterogeneity. (a) Representative second-harmonic generation (SHG) images of collagen fiber organization of senescent lung fibroblast-derived matrices (FDMs) treated with DMSO, SB505124, and CCG-1423 at day 14 post-irradiation (obj. = 25×, N.A. = 1.05, scale bar = 100 μ m). (b) Inhibited senescent lung FDM SHG intensity normalized to cell number per image (n ≥ 20, N = 3). (c) Inhibited senescent lung FDM regional collagen signal (n ≥ 34, N = 3). (D) Global circular variance of inhibited senescent lung FDM individual collagen fibers (n ≥ 34, N = 3). (e) Anisotropic circular variance of inhibited senescent lung FDM individual collagen fibers (n ≥ 34, N = 3). ANOVA; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Senescent pulmonary fibroblasts were seeded into 96-well plates and treated with either culture media, DMSO, 1, 5, and 10 μ M SB505124 (Cayman Chemical, No. 11793) or CCG-1423 (Cayman Chemical, No. 10010350) every 48 h for 14 days.

Techniques: Inhibition, Derivative Assay, Irradiation

Journal: Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology

Article Title: Targeting cancer cells in the tumor microenvironment: opportunities and challenges in combinatorial nanomedicine

doi: 10.1002/wnan.1358

Figure Lengend Snippet: Nanocarriers for combination drug delivery. The anatomical drawing in the top half of the figure shows three targeting considerations used in nanocarrier design: tissue-, cell-, and macromolecule-level targeting. The bottom half of the figure depicts three novel nanocarriers designed to deliver multiple therapeutic agents to the tumor utilizing one or more of these targeting approaches. Nanocarrier (a) shows a biodegradable nanoscale liposomal polymeric gel engineered to release SB505124, a small molecule inhibitor of the TGFβ1 receptor, as well as IL-2 within the tumor.20 Nanocarrier (b) depicts a particle-within-a-particle encapsulation strategy, whereby dioleoyl phosphatidic acid-coated drug cores were created out of gemcitabine monophosphate (GMP) and cisplatin. GMP and cisplatin drug cores were subsequently loaded into PLGA nanoparticles to generate dual-loaded nanocarriers.21 Of the three nanocarriers depicted here, this nanocarrier is the only one that uses an active (cell-level) targeting approach: Anisamide was attached to the outside of these nanoparticles to target sigma receptor-overexpressing cancer cells. Nanocarrier (c) shows PLGA-PEG/G0-C14 nanoparticles with siRNA molecules contained in their core. A pro-drug form of cisplatin was embedded in the polymeric nanoparticle shell.22

Article Snippet: Nanocarrier (a) shows a biodegradable nanoscale liposomal polymeric gel engineered to release SB505124, a small molecule inhibitor of the TGF β 1 receptor, as well as IL-2 within the tumor.

Techniques: Encapsulation