Spectral characteristics of <i>Synechococcus</i> (A, C, E, G, I) and <i>Synechocystis</i> (B, D, F, H, J) over a 120 hour iron depletion time course.

<p>Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate time course experiments. (A, B) ln A₇₅₀ to track optical scattering, a proxy for culture cell suspension density. We fit measurements over the first 72 h with a linear regression to estimate the cell specific growth rates. Dotted lines show 95% confidence intervals on the slope of the regression; Data presented are mean +/− standard error, n = 5 or 6. (C, D) ln (A₆₈₀–A₇₅₀) to track chlorophyll content of the cultures. Dotted lines show 95% confidence intervals on the slope of the regression; Data presented are mean +/− standard error, n = 5 or 6. (E, F) (A₆₈₀–A₇₅₀)/(A₇₅₀) to track chlorophyll per cell. (G, H) The wavelength for the chlorophyll absorbance peak, an optical measure of the accumulation of chlorophyll bound to IsiA. (I, J) (A₆₃₀–A₇₅₀)/(A₆₈₀–A₇₅₀) to track phycobilisome absorbance normalized to chlorophyll absorbance. Data presented are mean +/− standard error, n = 5 or 6.</p

Chlorophyll allocations in <i>Synechococcus</i> (A) and <i>Synechocystis</i> (B) over a 120 hour iron depletion timecourse.

<p>Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate time course experiments. (A, B) Solid bars: IsiA subunit contents per μg of total cellular protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059861#pone-0059861-g001" target="_blank">Figure 1</a>) were multiplied by 12 chlorophyll bound per IsiA monomer; Horizontal cross-hatching: PsaC subunit contents per μg of total cellular protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059861#pone-0059861-g001" target="_blank">Figure 1</a>) were multiplied by 100 chlorophyll bound per PSI monomer; Diagonal cross-hatching: PsbA subunit contents per μg of total cellular protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059861#pone-0059861-g001" target="_blank">Figure 1</a>) were multiplied by 36 per PSII monomer. Data presented are mean +/− standard error, n = 5 or 6.</p

Photosystem II maximum quantum yield (F_v/F_m) (A, B) or Photosystem II quantum yield for electron transport (Φ_PSII) (C, D, E, F) in <i>Synechococcus</i> (A, C, E) or <i>Synechocystis</i> (B, D, F) over a 120 hour iron depletion timecourse.

<p>(A, B) F_v/F_m measured from cells under 0 μmol photons·m⁻²·s⁻¹. (C, D) Φ_PSII measured from cells under the growth light level of 65 μmol photons·m⁻²·s⁻¹. (E, F) Φ_PSII measured from cells under saturating light of 262 μmol photons·m⁻²·s⁻¹, 4X higher than the growth light level. Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate measurements from 6 or 5 separate cyanobacterial cultures. All yield data were captured using blue light excitation of fluorescence. Data presented are mean +/− standard error, n = 5 or 6.</p

Content of key protein subunits in <i>Synechococcus</i> (A, C, E, G, I, K) and <i>Synechocystis</i> (B, D, F, H, J, L) over a 120 hour iron depletion time course.

<p>Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate immunoblots of cyanobacterial protein extracts from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate time course experiments (A, B) IsiA, (C, D) PsaC, (E, F) PetC,(G, H) PsbA, (I, J) PsbD, (K, L) AtpB. Protein subunit contents are expressed in femtomoles of protein per μg of total cellular protein. Data presented are mean +/− standard error, n = 5 or 6. Curve fits are second order polynomials with 95% confidence intervals plotted as outer dotted lines, except for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059861#pone-0059861-g002" target="_blank">Figure 2A</a> which was fit with an logistic growth function, since IsiA reached a clear plateau.</p

Functional absorption cross section serving PSII chemistry (σ_PSII) in <i>Synechococcus</i> (A, C, E) and <i>Synechocystis</i> (B, D, F) over a 120 hour iron depletion timecourse.

<p>Closed symbols are σ_PSII for red light; open symbols are σ_PSII for blue light. (A, B) σ_PSII measured for all active PSII centers in cells, measured after 1 min dark acclimation. (C, D) σ_PSII' measured for PSII centers remaining open in cells exposed to actinic light of 66 μmol photons·m⁻²·s⁻¹ for 10 seconds before measurement. (E, F) σ_PSII'' measured for PSII centers remaining open in cells under actinic light of 262 μmol photons·m⁻²·s⁻¹for 10 seconds before measurement. Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate measurements from 6 or 5 separate cyanobacterial cultures. Data presented are mean +/− standard error, n = 5 or 6.</p

Light response curves for electron transport per PSII (A, B) or per total cellular protein (C, D) in <i>Synechococcus</i> (A, C) or <i>Synechocystis</i> (B, D) after 0 (closed symbols) or 120 hours (open symbols) of iron depletion.

<p>Data are expressed in electrons per PSII per second (A, B) or in pmol electrons per μg protein per s. Data were compiled from 6 (<i>Synechococcus</i>) or 5 (<i>Synechocystis</i>) replicate measurements of σ_PSII and q_P performed on 6 or 5 separate cyanobacterial cultures. For estimation of pmol electrons per μg protein per s we also approximated PSII content as fmol PsbA ug total protein⁻¹. Curve fits are photosynthesis/irradiance curves with 95% confidence intervals plotted as outer dotted lines. Iron depletion led significant changes in the curve fits.</p

TcdB, but not TcdA triggers CXCL8/IL-8 production and release from Caco-2 cells in a manner dependent on extracellular nucleotides and the P2Y₆ receptor.

<p>(A) CXCL8/IL-8 release from Caco-2 cells treated with purified TcdA or TcdB (16 hr). N = 5; * denotes p<0.05 compared to TcdA. (B) TcdB, but not TcdA, treatment of Caco-2 cells increases cell death as assessed by LDH release. N = 5; * denotes p<0.05 compared to TcdA. TcdB-induced CXCL8/IL-8 release is significantly reduced by (C) MRS2578 (10 μM) and (D) co-treatment with apyrase (20 u/mL). N = 5; * denotes p<0.05 compared to no treatment; # denotes p<0.05 compared to all groups. </p

<i>C. difficile</i> TcdA/B triggers the release of UDP from Caco-2 cells that express a functional P2Y₆ receptor.

<p>(A) Western blot analysis of lysates reveals the expression of the P2Y₆ receptor in differentiated Caco-2 cells and PMA-differentiated THP-1 macrophages (included as positive control). (B) Stimulation of the Caco-2 cells with the selective P2Y₆ receptor agonist 5-OMe-UDP (1 μM) increases intracellular calcium concentrations as assessed by fluorescence imaging. (B-i) Pseudocolour images of Caco-2 cells before and after 5-OMe-UDP treatment. (B-ii) Representative traces of individual cells challenged with 5-OMe-UDP. (B-iii) The mean of the 5-OMe-UDP-induced calcium responses (n=46; grey denotes the standard error of the mean). (C) P2Y₆ receptor agonist 5-OMe-UDP triggers CXCL8/IL-8 release from Caco-2 cells, an effect that blocked by the potent P2Y₆ receptor antagonist MRS2578. N = 6; * denotes p<0.05 compared to control; # denotes p<0.05 compared to vehicle; % denotes p<0.05 compared to vehicle and 1 μM MRS 2578. (D) TcdA/B triggers the release of UDP as assessed by HPLC. i – control treated culture supernatant; ii – UDP-spiked control culture supernatant (100 μM UDP); iii – TcdA/B-spiked control culture supernatant (10 μg/mL); iv – TcdA/B-treated cell culture supernatant (10 μg/mL; 16 hr). (E) Summary data from HPLC measurement of TcdA/B-induced UDP release. N=5; * denotes p<0.05.</p

TcdA/B-induced CXCL8/IL-8 production from Caco-2 IECs involves the NFκB activation, an effect that is inhibited by pharmacological blockade of the P2Y₆ receptor by MRS2578.

<p>(A) TcdA/B-induced CXCL8/IL-8 release is inhibited by pretreatment with the selective NFκB pathway inhibitor BAY 11-7085 (20 μM). N=6; ** denotes p<0.005 compared to vehicle-treated TcdA/B stimulated cells (10 μg/mL). (B) Representative western blot for phosphorylated p65 (P-p65) in lysates from TcdA/B (10 μg/mL) stimulated Caco-2 IECs over the course of 60 min in the presence of the P2Y₆ antagonist MRS2578 (10 μM) or vehicle control (DMSO). (C) The summarized western blot data for P-p65 expressed as a percentage of the total p65. N = 4, *, denotes p<0.05 compared to time 0 min; # denotes p<0.05 compared to respective vehicle control (DMSO). </p

Inhibition of the P2Y₆ receptor attenuates TcdA/B-induced intestinal epithelial barrier dysfunction in Caco-2 IECs.

<p>(A) TcdA/B-induced (10 μg/mL) FITC-flux is significantly reduced by the selective P2Y₆ receptor antagonist MRS 2578 (10 μM). N=4; * denotes p<0.05 compared to vehicle and MRS2578. # denotes p<0.05 compared to TcdA/B. (B) 5-OMe-UDP (100 μM) increases FITC-flux in Caco-2 monolayers, an effect that is significantly attenuated by MRS2578 (10 μM). N=4; * denotes p<0.05 compared to vehicle and MRS2578. # denotes p<0.05 compared to 5-OMe-UDP. (C) Apical administration of TcdA/B (10 μg/mL) or 5-OMe-UDP (100 μM) for 4 hr triggers a redistribution of ZO-1 in Caco-2 monolayers, an effect that is blocked by pretreatment with MRS 2578 (10 μM; N=4).</p