Increased senescence associated (SA)-β-galactosidase activity in IMR-90 cells infected with <i>pks</i>+ <i>E. coli.</i>

<p>3, 6 or 9 days after infection, IMR90 cells were fixed with formaldehyde 4% and then stained with the galactosidase chromogenic substrate X-gal at pH 6 for 24h. Cells were examined for perinuclear blue staining by light microscopy at x20 magnification (A), and these SA-β-Gal positive cells (100-200 cells per condition) were quantified by a blinded observer (B). Results represent the mean and SEM of three independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, **P<0.01 comparing infected and uninfected groups; #P<0.05, # #P<0.01, # # #P<0.001 comparing <i>pks</i>+ and <i>pks</i>- groups.</p

Senescent <i>pks+ E. coli</i> infected IMR-90 cells promote the growth of bystander tumour cells.

<p>IMR-90 cells were infected for 4h with live <i>pks</i>+ or <i>pks</i>- <i>E</i>. <i>coli</i> with an MOI of 60 or 180 or left uninfected. At the end of the infection, the cells were washed and grown with gentamicin for 3 days. Then the IMR-90 cells were layered with 0.5% agar and 5000 HCT116 p53^-/- cells embedded in 0.35% agar were added on top of the first layer and co-cultured for 10 days. (A) Representative photomicrographs of HCT116 p53^-/- cell colonies, scale bars = 0.7 mm. (B) Large (>225 µm) microcolonies per microscopy field were counted by a blinded observer. The upper HCT116 p53^-/- cell layer was harvested and stained with MTT for 24h. (C) Representative photomicrograph of experimental 6-well culture plates with viable cancer cell colonies coloured by MTT, scale bars = 3 mm. (D) The number of microcolonies was quantified using Image-J. (E) Total cell proliferation was assessed using MTT. Results represent the mean and SEM of two independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05 comparing infected and uninfected groups; #P<0.05 comparing <i>pks</i>+ and <i>pks</i>- groups.</p

Induction of a senescence-associated secretory phenotype (SASP) in IMR-90 cells following infection with <i>pks</i>+ <i>E. coli.</i>

<p>IMR-90 cells were infected as before and incubated for 1-14 days. Conditioned cell culture media were prepared by incubating each culture in serum-free medium for 24 h. MMP-3, IL-6, IL-8 and MCP-1 secretion was quantified by Bio-Plex in one experiment (A) and by ELISA in two independent experiments (B) ; Results represent the mean and SEM, one-way ANOVA with Bonferroni’s multiple comparison test; **P<0.01, ***P<0.001 comparing infected and uninfected groups; # # #P<0.001 comparing <i>pks</i>+ and <i>pks</i>- groups.</p

Permanent cell-cycle arrest and increased cell-cycle inhibitors expression in IMR-90 cells infected with <i>pks</i>+ <i>E. coli.</i>

<p>(A) Cell-cycle distribution was analyzed by flow cytometry 3 days after infection. (B) 3 days after the infection the cells were incubated with EdU for 2 hours and its incorporation in S-phase cells was analyzed by flow cytometry. (C) Western blot analysis of p21 and p16 protein content in IMR-90 cells 4h, 1 and 6 days after infection. Actin was probed as a protein loading control. Results represent the mean and SEM of two independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, ***P<0.001 comparing infected and uninfected groups; # #P<0.01, # # #P<0.001 comparing <i>pks</i>+ and <i>pks</i>- groups.</p

Increased intracellular and mitochondrial ROS production in IMR-90 cells infected with <i>pks</i>+ <i>E. coli.</i>

<p>(A) Cells were examined for intracellular ROS with the ROS sensor dihydro-dichloro-fluorescein diacetate (H2-DCFDA) (green). Pictures of uninfected and MOI 180-infected cells (at 3 days) are shown, scale bars = 10µm. (B) Mean H2-DCFDA fluorescence intensity was quantified by flow cytometry in 2x10⁴ cells, 3 and 6 days after infection. (C) Cells were examined for mitochondrial superoxide production with the mitochondrial superoxide probe MitoSOX (orange) and for DNA (blue). Pictures from uninfected and MOI 180-infected cells (at 3 days) are shown, scale bars = 10µm. (D) Mean MitoSOX fluorescence intensity was quantified by flow cytometry 3 or 6 days after infection. Results in panels B and D represent the mean and SEM of three independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; **P<0.01, ***P<0.001 comparing infected and uninfected groups; # #P<0.01, # # #P<0.001 comparing <i>pks</i>+ and <i>pks</i>- groups.</p

Persistent γH2AX foci in IMR-90 cells infected with <i>pks</i>+ <i>E. coli.</i>

<p>Non-transformed human IMR-90 cells were infected for 4h with live <i>pks</i>+ or <i>pks</i>- <i>E</i>. <i>coli</i> with a multiplicity of infection (MOI) of 20, 60 and 180 bacteria per cell or left uninfected. At the end of the infection, the cells were washed and further grown in medium supplemented with gentamicin. (A) Cells were examined for DNA (blue) and γH2AX (green) 3, 6 or 9 days after infection. Pictures of uninfected and MOI 180 -infected cells are shown, scale bars = 10µm. (B) The numbers of γH2AX foci per cell were quantified by a blinded observer in 30-100 nuclei for each condition in two independent experiments. Results represent the mean and standard error of the mean (SEM). Statistical significance was examined by one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, **P<0.01, ***P<0.001 comparing infected and uninfected groups; #P<0.05, # #P<0.01, # # #P<0.001 comparing <i>pks</i>+ and <i>pks</i>- groups.</p

Accumulation of heterochromatin foci in IMR-90 cells infected with <i>pks</i>+ <i>E. coli.</i>

<p>(A) Cells were stained with DAPI 6, 9 or 14 days after infection and examined for formation of heterochromatin foci (arrows) by confocal microscopy. Pictures of uninfected and MOI 180 –infected cells are shown, scale bars = 10µm. (B) Cells were stained with DAPI (blue) and with antibodies against H3K9me3 (green) 14 days after infection and examined by confocal microscopy, scale bar = 50µm. (C) Heterochromatin foci-positive cells (60-100 nuclei for each condition) were counted by a blinded observer. Results represent the mean and SEM of three independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, **P<0.01 comparing infected and uninfected groups; #P<0.05 comparing <i>pks</i>+ and <i>pks</i>- groups.</p

<i>Pks+ E. coli</i> infected IMR-90 cells induce bystander γH2AX foci formation and SA-β-Gal expression in uninfected IMR-90 cells.

<p>Naïve IMR-90 cells were treated for 1 day (γH2AX) or 3 days (SA-β-Gal) with conditioned media (CM) prepared 3-6-10 days after infection with <i>pks</i>+ or <i>pks</i>- <i>E</i>. <i>coli</i> with a MOI 180 (A, left panel). Cells were treated for 24 h with a 6 days-old CM and examined for DNA (Blue) and γH2AX (green), scale bars = 10µm (A, right panel). Cells were treated for 3 days with a 6 days old CM and examined for SA-β-Gal staining (Blue) (x20 magnification) (B) Numbers of γH2AX foci per cell (B) and percentages of SA-β-Gal positive cells (C) were quantified as before, 30-100 nuclei or 100-200 cells were evaluated for each condition. Results represent the mean and SEM of two independent experiments, one-way ANOVA with Bonferroni’s multiple comparison test; *P<0.05, ***P<0.001 comparing infected and uninfected groups; #P<0.05, # # #P<0.001 comparing <i>pks</i>+ and <i>pks</i>- groups. (D) IMR-90 grown on Transwells, were infected for 4h with live <i>pks</i>+ or <i>pks</i>- <i>E</i>. <i>coli</i>, washed and grown with gentamicin for 3 days. The Transwells were then transferred on top of naive IEC-6 cells and incubated for 24 hours with or without n-acetylcysteine (1mM). The IEC-6 cells were fixed and γH2AX was quantified by In-Cell Western. Results represent the mean and SEM of one experiments including triplicates for each conditions, two-way ANOVA with Bonferroni’s multiple comparison test; **P<0.01, ***P<0.001 comparing vehicle and n-acetylcysteine groups.</p

Growth parameters of Nissle 1917 and Csr mutants with glucose and gluconate as carbon source.

<p>μ, specific growth rate; <i>q_S</i>, carbon source uptake rate; <i>v_Ac</i>, acetate production rate; Y<i>_Ac</i>, acetate production yield ( = <i>v_Ac</i>/<i>q_S</i>); <i>v_Pyr</i>, pyruvate production rate; Y<i>_Pyr</i>, pyruvate production yield ( = <i>v_Pyr</i>/<i>q_S</i>). Values represent the mean ± standard deviation of three independent biological replicates.</p>a<p>n.d.: not detected.</p

Metabolic flux distributions upon growth on glucose (A) and gluconate (B) as carbon source.

<p>Values from top to bottom: wild-type strain, Δ<i>csrBC</i> and Δ<i>csrA</i>51. Flux values are normalized to the specific glucose uptake rate of each strain, which was arbitrarily given the value of 100. Values represent the mean ± standard deviation of three independent biological replicates for each strain. Arrow thicknesses represent the flux values in the wild-type strain. Box arrows represent fluxes towards biomass synthesis.</p