Legends to Supplemental Material

Tabl. S1

The effect of hypoxia on the survival of E. coli and S. aureus in M

Mwere infected with either E. coli or S. aureus at a MOI of 10. One hour after infection extracellular bacteria were removed by washing with PBS followed by gentamicin treatment. 2 h after infection the cells were exposed to either normoxia (N) or hypoxia (H; 0.5 % oxygen). For reoxygenation (Re) the cells were first incubated for 8 h under hypoxic conditions and then kept in normoxia. Cell lysates were prepared and the bacterial load is given as CFU/ ml lysates prepared 2 h and 24 h after infection.

Fig. S1

Hypoxia does not promote the growth of E. coli and S. aureus

E. coli and S. aureus were grown under hypoxic and normoxic conditions in LB media as described in the Material & Methods section. At different time points the CFU/ ml of each sample was determined.

Fig. S2

Verification of Hif1a knock-down efficacy

(A) ns-siRNA (ns) or Hif1a-specific (Hif1a) siRNA was transferred into M or the cells were left untreated (untreated). 24 h later the cells were infected with S. aureus. After 2 h the infected cells were incubated in normoxic or hypoxic conditions for additional 24 h. qRT-PCR for Hif1a and Pgk1 was performed. Data are the mean + SEM of at least two independent experiments. (B) Mwere treated as described in (A). Cellular lysates were prepared and immunoblotting for HIF1A was performed. To demonstrate equal loading, the blots were also incubated with an ACTIN-specific antibody.

Fig. S3

Analysis of ROS- and NO-production of Cybb-/-/ Nos2-/- M upon infection with E. coli and S. aureus under normoxic and hypoxic conditions

M from Cybb-/-/ Nos2-/- mice or WT controls were infected with E. coli or S. aureus at a MOI of 10. One hour after infection extracellular bacteria were removed by washing with PBS followed by gentamicin treatment. 2 h after infection the cells were exposed to either normoxia (N) or hypoxia (H; 0.5 % oxygen). (A) After 24h the cells were labelled with CM-H2DCFDA, fixed with 3.5 % PFA and analyzed by flow cytometry. Grey filled line demonstrates the FL-1 fluorescence of labelled cells under normoxic conditions. Black dotted line shows FL-1 fluorescence of CM-H2DCFDA labelled cells under hypoxic conditions; black solid line demonstrates FL-1 fluorescence of CM-H2DCFDA labelled cells under normoxic conditions. A representative of two similar experiments is shown. (B) Cells were infected as described in (A). After 24 h supernatants were collected and nitrite was measured using the Griess reaction. Data are the mean + SEM of at least three independent experiments.

Fig. S4

Infection with E. coli and S. aureus does not induce a PHOX- or NOS2-dependent apoptosis or necrosis

Mfrom Cybb-/-/ Nos2-/- mice or WT controls were left untreated or infected with E. coli or S. aureus at MOI of 10 in a microcentrifuge tube. As additional control RAW 264.7 macrophage-like cells were left untreated for 24 h under normoxic conditions. The bone marrow-derived M were washed to remove non-internalized bacteria. To kill extracellular bacteria the cells were treated with gentamicin 100 µg/ ml for 1 h in the microcentrifuge tubes. Thereafter the cells were transferred to a low adherence plate and cultured in the presence of 25 µg/ ml gentamicin for the rest of the experiment under normoxic conditions. 24 h after infection apoptotic or necrotic effects were determined by FACS by performing an Annexin V-APC and Propidium Iodide (PI) staining. (A) shows a representative FACS analysis (B). % of total Annexin+ (apoptotic), PI+ (necrotic) or Annexin V-/ PI- (viable) cells are given.

Fig. S5

The viability of Cybb-/-/ Nos2-/- Mand rotenone treated Mis not impaired

Mfrom Cybb-/-/ Nos2-/- mice or WT controls were infected with E. coli or S. aureus at MOI of 10. After 2 h the infected cells were incubated in normoxic or hypoxic conditions for 24 h. The relative LDH release was determined 24 h after infection. Data are the mean + SEM of at least three independent experiments.

Fig. S6

Mitochondrial activity is restored within 5 min of reoxgenation.

Mwere infected with E.coli at MOI of 10 and subjected to normoxic and hypoxic conditions. Mitochondrial activity was assessed by determining the mitochondrial membrane potential (ΔΨM) as described in Material & Methods using the JC-1 dye. ΔΨM is given in arbitrary units (AU). 5 and 10 min after reoxygenation ΔΨM was determined. A representative of two similar experiments is shown.

Fig. S7

Effects of different rotenone concentrations on bactericidal activity of M

Mwere infected with E.coli. One hour after infection extracellular bacteria were removed by washing with PBS followed by gentamicin treatment. 2 h after infection the cells were exposed to normoxia, rotenone 10µM, rotenone 100µM or hypoxia (0.5 % oxygen). Cell lysates were prepared and the bacterial load is given as CFU/ ml lysates prepared 2 h and 24 h after infection. Data are the mean + SEM of two independent experiments

Fig. S8

Rotenone does not affect NO-production of infected mononuclear phagocytes

Mwere infected with E. coli or S. aureus. After 2 h the infected cells were subjected to normoxic or hypoxic conditions for 24 h and treated with rotenone where indicated. Nitrite was determined in the supernatant. Data are the mean + SEM of three independent experiments.

Fig. S9

Rotenone does not affect growth of E. coli or S. aureus in LB

E. coli and S. aureus were grown under hypoxic and normoxic conditions in LB media in the absence or presence of rotenone as described in the Material & Methods section. At different time points the CFU/ ml of each sample was determined.

Fig. S10

Influence of gas permeable dishes to the infection of M with S. aureus

Mwere infected with S. aureus in either conventional polystyrene dishes or gas permeable dishes. One hour after infection extracellular bacteria were removed by washing with PBS followed by gentamicin treatment. 2 h after infection the cells were exposed to either normoxia (N) or hypoxia (H; 0.5 % oxygen). Cell lysates were prepared and the bacterial load is given as CFU/ ml lysates prepared 2 h and 24 h after infection. Data are the mean + SEM of two independent experiments.

Fig. S11

Antimycin A does not affect growth of E. coli or S. aureus in LB

E. coli and S. aureus were grown under hypoxic and normoxic conditions in LB media in the absence or presence of antimycin A as described in the Material & Methods section. At different time points the CFU/ ml of each sample was determined.