2 2 dipyridyl dp

For treatment of mMSC (murine mesenchymal stem cells) with the hypoxia-mimicking iron chelators 2,2′-dipyridyl (DP; dissolved in EtOH) and desferrioxamine (DFX; dissolved in water) (both Sigma) cells were cultured in six-well plates for 24 h to a density of about 60%. After that, the culture medium was replaced and DP/DFX was added at a concentration of 100 and 250 μM. Respective controls were treated with solvent only. 24 h later cells were harvested and total RNA or protein was isolated. Before incubation of mMSC at hypoxic conditions (1% O₂) cells were cultured in six-well plates for 24 h to a density of about 60%. After that, the culture medium was replaced and the cells were transferred into a New Brunswick Galaxy 48 series incubator (Eppendorf AG, Hamburg, Germany) flushed with 1% O₂ and 8% CO₂. Excessive O₂ was replaced by nitrogen. Respective controls were incubated at normoxia (21% O₂). After 24 h cells were harvested and total RNA or protein was isolated. Both experiments were repeated at least five times.

The Caulobacter crescentus strains used were: NA1000 (wild-type) (Evinger and Agabian, 1977 (link)), SP0057 (fur mutant) (da Silva Neto et al., 2009 (link)), GM10 (recA mutant) (Galhardo et al., 2005 (link)), and NA1000 and GM10 harboring the pRKlacZ290 vector containing the imuA promoter (imuA/lacZ fusion) (Galhardo et al., 2005 (link)). Cultures were grown aerobically at 30°C in minimal medium (M2), which contains 10 μM FeSO₄ (Ely, 1991 (link)). Iron-limiting conditions were obtained by adding the iron chelator 2,2-dipyridyl (DP) (Sigma, 100 μM) to the M2 medium (DP-treated M2) or by using a modified M2 medium without iron sulfate (iron-limited M2). Excess iron conditions were achieved by supplementing M2 medium with 100 μM FeSO₄ instead of 10 μM FeSO₄. The growth curves in each condition are shown in Supplementary Figure S1.

All the strains and plasmids used in this work are described in Table S3. Escherichia coli strains were cultured in LB medium and C. violaceum strains were cultured in LB medium or M9 minimal medium supplemented with 0.1% casein hydrolysate. When necessary, cultures were supplemented with kanamycin (50 µg/mL), ampicillin (100 µg/mL), nalidixic acid (4 µg/mL), gentamicin (40 µg/mL for C. violaceum or 20 µg/mL for E. coli), or tetracycline (5 µg/mL in liquid medium for C. violaceum, 10 µg/mL in agar plates for C. violaceum, or 12 µg/mL for E. coli). Iron-deficient conditions were obtained by supplementation with 2,2′-dipyridyl (DP) (Sigma) as previously defined for C. violaceum (28 (link)). Saccharomyces cerevisiae AH109 strain was cultured in yeast peptone dextrose adenine (YPDA) medium (51 (link)).