All cell lines were grown in RPMI 1640 (PAN-Biotech P04-16500) supplemented with 10% heat-inactivated fetal calf serum (FCS; PAN-Biotech), penicillin/streptomycin (PAN-Biotech) (thereafter designated as RPMI-based medium) and cultivated at 37°C under 5% CO2 in a humidified incubator. K562 cells (a kind gift from Daniela Männel, University of Regensburg, Germany; [62] (link)) were maintained in RPMI-based medium. The non-tumorigenic immortalized interleukin-3 (IL-3)-dependent mouse pro-B cell line Ba/F3 (a kind gift from Jacqueline Marvel, IFR 128 BioSciences Gerland-Lyon Sud, France; [63] (link)) was grown in RPMI-based medium supplemented with 2 ng/mL rmIL-3 (ImmunoTools). The Ba/F3-1*6 cell line stably expressing the constitutively active mouse STAT5A-1*6 mutant [64] (link) was generated according to German GenTSV (genetic engineering safety regulations; authorization AZ.55.1-8791.7.52) by electroporating Ba/F3 cells with a pcDNA3-based expression vector allowing expression of a mSTAT5A-1*6-FLAG fusion protein. Stably transfected cells were selected in IL-3-free medium in the presence of 500 µg/mL G418 (PAA). Individual IL-3-independent clones were isolated and characterized to verify mSTAT5A-1*6 transgene expression and constitutive activity, as originally described [64] (link). Clone Ba/F3-1*6 F7 was used for this study. Cells were maintained in RPMI-based medium supplemented with 500 µg/mL G418.
For cytokine stimulation of Ba/F3 cells, cells were washed twice in RPMI 1640 and rested in RPMI-based medium for 12 hours before addition of 5 ng/mL IL-3. Duration of IL-3 stimulation was contingent upon the downstream assay, as indicated in the figure legends. For chalcone and other inhibitor treatments, Ba/F3 cells were incubated with compounds or DMSO (vehicle) for 30 minutes prior to cytokine stimulation. Ba/F3-1*6 and K562 cells were treated for 60–90 minutes with the various compounds or DMSO (vehicle), as indicated. With the exception of cell viability assays (see below), all experiments were performed in the presence of equal amounts of DMSO.
For cytokine stimulation of Ba/F3 cells, cells were washed twice in RPMI 1640 and rested in RPMI-based medium for 12 hours before addition of 5 ng/mL IL-3. Duration of IL-3 stimulation was contingent upon the downstream assay, as indicated in the figure legends. For chalcone and other inhibitor treatments, Ba/F3 cells were incubated with compounds or DMSO (vehicle) for 30 minutes prior to cytokine stimulation. Ba/F3-1*6 and K562 cells were treated for 60–90 minutes with the various compounds or DMSO (vehicle), as indicated. With the exception of cell viability assays (see below), all experiments were performed in the presence of equal amounts of DMSO.
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