Fibroblast-derived ECM alter Lung Cancer Cell Line Morphology.

<p>(A) Phase contrast microscopy photos of A549, H358, and HPL1D cells on FN, WI38 ECM, IMR90, and HDF ECM. (B) Immunofluorescent confocal microscopy of A549 cells on WI38-derived ECM stained with Phallodin and DAPI. (C) The circularity of A549 on FN and 3 different fibroblast-derived ECM was calculated. n = 10. *, p ≤ 0.05.</p

Fibroblast-derived ECM alters mRNA profile of A549 and H358 Cells.

<p>Heat map significantly changed genes from microarray (fold-change >1.5 and p-value ≤ 0.05). Columns represent individual gene probes; rows are the different samples and each row represents one of the biological triplicates ran on the microarray. (B) Validation of microarray data. Representative genes were chosen for quantitative real-time qRT-PCR analysis. New biological triplicates were prepared, RNA extracted and converted to cDNA, and real-time qRT-PCR was performed. All samples tested were validated and all genes were changed in the same direction as the microarray, however some amplitudes of change were slightly different.</p

Fibroblast-derived ECM Protects Lung Cancer Cell Lines from Serum Deprivation.

<p>(A) A549, H358, and HPL1D cells were grown on fibronectin (FN) or on WI38 ECM in serum-free media for 48 hours and then photographed. (B) Relative cell numbers were quantified for A549 cells (B), H358 cells (C) and HPL1D cells (D) using Alamar Blue every 24 hours after cells were put into serum free media. By the fourth day in serum free media, basically all cells were dead.</p

Fibroblast-derived ECM alters Protein Levels of A549 Cells.

<p>(A) A549 cells were cultured on the indicated substrate for 48 hours and then cells were harvested and western blots were performed with the indicated antibodies. (B) Cells were treated as in panel A and westerns were performed with indicated antibodies. (C) A549 cells were seeded on either fibronectin (FN) or WI38-derived ECM and then plates were incubated under hypoxic or normoxic conditions. Cells were harvested and westerns were performed with the indicated antibodies.</p

Characterization of fibroblasts and their ECM.

<p>A. Microscopic analysis of fibroblasts and de-cellularized matrix. Left colum- 5X phase contrast imaging of confluent fibroblasts just prior to de-cellularazation. Middle colum- 40X phase contrast microscopy of ECM following decellularization. Right column- Immunofluorescent confocal microscopy of fibroblast-derived ECM using an NHS-ester probe conjugated to Alexa Fluor 488. (B) Protein content of ECM was quantitated after scraping of the ECM from decellularized plates. Protein amount is indicated a μg/cm², N = 3. (C) SDS-PAGE and colloidal blue stain of fibroblast-derived ECM. n = 3.</p

Fluorovinylsulfones and -Sulfonates as Potent Covalent Reversible Inhibitors of the Trypanosomal Cysteine Protease Rhodesain: Structure–Activity Relationship, Inhibition Mechanism, Metabolism, and In Vivo Studies

Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo­[b]­[1,4]­dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 μM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations

Fibroblast-derived ECM decreases Lung Cancer Cell Growth.

<p>(A) A549, H358 and HPL1D cells were grown on fibroblast derived matrices and every day one well of cells was trypsinized and manually counted in triplicate using trypan blue. (B) A549, H358 and HPL1D cells were grown on fibroblast derived matrices and every day Alamar Blue was added to wells of cells in triplicate and relative conversion of Alamar Blue was determined. n = 3. *, p-value ≤ 0.05.</p

Fibroblast-derived ECM alter Cell Migration of Lung Cancer Cell Lines in a Trans-well Chamber.

<p>(A) The under side of a boyden chamber was coated with fibronectin and seeded with the indicated fibroblast cells. After eight days the ECM was prepared as described in the methods. A549 cells were then seeded inside the boyden chamber and 48 hours later the cells that migrated through the pores to the under-side containing the ECM were stained with H&Eand photographed. (B) Quantification of the cell migration. Stained trans-well membranes showing migrated cells were counted and the average migratory cells per field (4 fields) averaged over two experiments was calculated. *, p-value ≤ 0.05.</p

Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties - Table 1

<p>Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties - Table 1 </p