Journal: Molecular Cancer Research

Article Title: Prostate Tumor Cell–Derived IL1β Induces an Inflammatory Phenotype in Bone Marrow Adipocytes and Reduces Sensitivity to Docetaxel via Lipolysis-Dependent Mechanisms

doi: 10.1158/1541-7786.mcr-19-0540

Figure Lengend Snippet: Figure 3. Adipocyte–tumor cell cross-talk: IL1b expression and secretion by prostate carcinoma cells augments COX-2 signaling in adipocytes. PC3 (A–C) and ARCaP(M) cells (D–F) were grown alone or in Transwell coculture with bone marrow adipocytes. TaqMan RT-PCR results show highly induced mRNA levels of IL1b in PC3 (A) and ARCaP(M) cells (D); graph representative of multiple experiments. Immunoblot analyses depicting increased levels of IL1b (1:1,000) protein in PC3 (B) and ARCaP(M) cells (E). Tubulin (1:2000) shown for equal loading control. C and F, ELISA assay results depicting levels of IL1b secreted by PC3 cells (C) or ARCaP(M) cells (F) grown alone or in Transwell with adipocytes. G, COX-2 protein levels in the absence or presence of recombinant IL1RA (200 ng/mL). H, Densitometry of COX-2 bands normalized to actin bands; data represent the mean of three experiments I, COX-2 gene expression in bone marrow adipocytes grown alone or in Transwell with ARCaP(M) cells in the absence or presence of IL1RA; J, siRNA-mediated IL1b knockdown in ARCaP(M) cells reduces COX-2 gene-expression levels in marrow adipocytes grown in Transwell cultures with ARCaP(M) cells as compared with cells transfected with scrambled control; K, mPGES mRNA levels in marrow adipocytes treated with IL1RA; L, mPGES gene expression in adipocytes upon siRNA-mediated knockdown of IL1b in tumor cells; mRNA levels of COX-2 (M) and mPEGS (N) in marrow adipocytes treated with recombinant IL1b. O, p65(NFkB) immunofluorescence in bone marrow adipocytes grown under control conditions or treated with recombinant IL1b. NFkB activation is demonstrated by the nuclear p65 staining in response to IL1b. P, Marrow adipocytes grown in Transwell with ARCaP(M) cells. Reduced nuclear p65 upon treatment with NFkB inhibitor BAY 11-0782. , P < 0.05; , P < 0.01; , P < 0.0001.

Article Snippet: Z-fixwas fromAnatech Ltd. StemXVivoAdipogenic Supplement, Cultrex, recombinant IL1b, and recombinant IL1RA were from R&D Systems. b-Tubulin (#E7-C) antibody was from Developmental Studies Hybridoma Bank. b-Actin antibody (#NB600-501) was from Novus Biologicals.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Recombinant, Gene Expression, Knockdown, Transfection, Activation Assay, Staining

Journal: Nature Communications

Article Title: TAK1 mediates microenvironment-triggered autocrine signals and promotes triple-negative breast cancer lung metastasis

doi: 10.1038/s41467-018-04460-w

Figure Lengend Snippet: Treatment with anakinra could reduce lung metastasis, but less so than TAK1 inhibition. a , c Changes in P38 phosphorylation levels in response to IL1α (10 ng/ml) in MDA-MB-231 cells ( a ) and 4T1 cells ( c ) overexpressing doxycycline-inducible dominant-negative TAK1 (TAK1-dn) with or without treatment of doxycycline and recombinant IL1RA (100 ng/ml). Representative examples of three experiments are shown. b , d Lung metastatic burden determined by measuring the bioluminescent signal in mice injected with MDA-MB-231 ( b ) and 4T1 ( d ) cells overexpressing doxycycline-inducible TAK1-dn and with or without treatment with doxycycline and/or anakinra. Bioluminescence was measured 4 and 3 weeks after tail vein injection of MDA-MB-231 and 4T1 cells, respectively. Scatter dot plots show means ± SD of signals obtained from eight ( b ) or seven ( d ) mice per group. e qPCR analysis of IL1α , IL1β , TGFβ , and TNFα expression in MDA-MB-231 cells cultured in the presence or absence of GFP-expressing fibroblasts for 5 days. RNA was extracted from cells separated by FACS based on GFP expression. Means ± SD of three independent experiments are shown. f Immunofluorescent staining of lungs dissected from mice injected with GFP and luciferase-tagged MDA-MB-231 cells. Representative examples of images taken from three immunostainings of two independent experiments are shown. GFP (yellow), myofibroblast marker α-SMA—also expressed in MDA-MB-231 cells (magenta), TNFα (white), DAPI (gray in single channel, blue in merge image), are shown. Scale bar = 20 μm. Statistical analyses: unpaired one-tailed Student’s t test ( b , d ), unpaired two-tailed Student’s t test ( e ). * p ≤ 0.05; ** p ≤ 0.01

Article Snippet: For in vitro experiments using recombinant human IL1RA (Peprotech), cells were treated with 100 ng/ml IL1RA for 30 min, followed by treatment with IL1α (10 ng/ml) for an additional 10 min. For experiments done in suspension conditions, cells were plated in poly-hema-coated dishes in RPMI media supplemented with B27 (Invitrogen), 20 ng/ml epidermal growth factor (Peprotech), and 20 ng/ml basic fibroblast growth factor (Peprotech).

Techniques: Inhibition, Phospho-proteomics, Dominant Negative Mutation, Recombinant, Injection, Expressing, Cell Culture, Staining, Luciferase, Marker, One-tailed Test, Two Tailed Test