Time evolutions of [GSH] and [casp3] predicted by Model III in the presence of N₂O₃ and FeL_nNO.

<p>N₂O₃ is present in the model ([O₂] is non-zero) as well as FeL_nNO ([FeL_n]₀ is non-zero). Each column is a counterpart of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002249#pone-0002249-g002" target="_blank">Figure 2A–C</a> with different initial concentrations of GSH. A–C) [GSH]₀ = 10⁴ µM; D–F) [GSH]₀ = 10³ µM. Solid curve shows the time evolution of [casp3] and dotted curve that of [GSH].</p

Equilibrium levels and initial concentrations used in Model II

<p>Equilibrium levels and initial concentrations used in Model II</p

Time evolutions of A) GSH, B) N₂O₃, C) FeL_nNO, and D) ONOO⁻ predicted by Model II.

<p>N₂O₃ and FeL_nNO increase to high concentrations by a switch-like mechanism induced by a decrease in GSH concentration due to conversion of GSH to GSNO and subsequently to GSSG. [ONOO⁻] does not follow a similar switch-like increase in its concentration. Solid curve is for [GSH]₀ = 10⁴ µM, dotted curve for [GSH]₀ = 10³ µM, and dashed curve with diamonds for [GSH]₀ = 10² µM. The response is thus sharper and earlier in the presence of lower initial concentrations of GSH.</p

Time evolution of [casp3] predicted by a bistable model in response to different strengths of apoptotic stimuli, A) in a cell subjected to a weak EC apoptotic signal (reflected by the low concentration [caps8]₀); B) in a cell that is subjected to a stronger EC pro-apoptotic signal.

<p>Caspase-3 is activated at 60 minutes; C) in a cell that is subjected to a stronger EC pro-apoptotic signal than one in panel B. Caspase-3 is activated at 30 minutes. Panels A and B illustrate two opposite effects induced by different initial concentrations of caspase-8. The threshold concentration of [caps8]₀ required for the switch from anti-apoptotic to pro-apoptotic response is calculated to be 8.35×10⁻⁵ µM. Panels B and C illustrate the shift in the onset time of apoptosis depending on [casp8]₀. D) Dependence of apoptotic response time on the initial caspase-8 concentration. The ordinate is the onset time of caspase-3 activation, and the abscissa is the initial concentration of caspase-8 in excess of the threshold concentration required for the initiation of apoptosis (evidenced by increase in [casp3], see panels B–C). The onset time of caspase-3 activation exhibits a logarithmic decrease with Δ[casp8]₀ ([casp8]₀–8.35×10⁻⁵ µM).</p

Analysis of different group amino acid replacement of Glycine 120 of human LC3B and LC3B truncates effect on their mobility in SDS-polyacrylamide gel.

<p><b><i>A,</i></b> plasmids expressing Flag-LC3B, Flag-LC3B^G120A, Flag-LC3B^G120 or Flag-LC3^A120 was expressed in HEK293 cells for 24 hours. Cells were treated with 50 µM of CQ for two hours and harvested and lysed. Total cell lysates (10 µg) was used for immunoblotting by Flag antibody. <b><i>B,</i></b> G120 point mutant plasmids were expressed in HEK293 cells for 24 hours. Immunoblotting was conducted to compare the band’s mobility with Flag-LC3B, Flag-LC3B^G120A or Flag-LC3B^G120A as indicated. <b><i>C,</i></b> Flag-LC3B^G120D and Flag-LC3B^G120E were expressed in HEK293 cells for 24 hours. Cells were then treated with 50 µM of CQ for 2 hours. Expression patters of Flag-LC3B^G120D and Flag-LC3B^G120E were compared with endogenous LC3B by immunoblotting with LC3B antibody (upper panels). Adenoviral vector expressing GFP-LC3B, GFP-LC3B^G120D or GFP-LC3B^G120E was used to infect A549 cells for 24 hours. Cells were then treated with 50 µM of CQ for 2 hours. Images were recorded using fluorescence microscopy. Scale bar: 25 micron. <b><i>D,</i></b> Truncates of LC3B (see Fig. 1) were expressed in HEK293 for 24hours. Cells were treated with 50 µM of CQ for two hours before harvesting. Total cell lysates were used for Wb assay to compare the mobility with Flag-LC3B-I and Flag-LC3B-II. Flag antibody was used to detect the recombinant proteins.</p

Reactions bridging between Models I to II (*)

<p>(^*) The parameters used in the present study are k_18NO = 1 µM⁻¹s⁻¹ (varying the value between 0.01 µM⁻¹s⁻¹ and 100 µM⁻¹s⁻¹ does not affect the results), k_19NO = 10 µM⁻¹s⁻¹<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002249#pone.0002249-Wink2" target="_blank">[<i>78</i>]</a>, k_20NO = k_21NO = k_22NO = 66 µM⁻¹s⁻¹ (the same value as k_11NO).</p

Reactions in Model II

a<p>Same as k_11NO</p

G120A mutant of human LC3B has similar mobility to LC3B-II in SDS-polyacrylamide gel.

<p><b><i>Aa</i></b>, plasmids expressing Myc-LC3B, myc-LC3B^G120A or Myc-LC3B^G120 was expressed in HEK293 cells for 24 hours. Cells were harvested and lysed by sonication, and 10 µg of total protein of each lysate were separated by 15% of SDS-polyacrylamide gel, followed by immunoblotting with anti-LC3B antibody. <b><i>Ab,</i></b> plasmids expressing Flag-LC3B or Flag-LC3B^G120A was expressed in HEK293 cells for 24 hours. Cells were lysed followed by immnuoblotting using Flag-antibody. <b><i>B,</i></b> plasmids expressing LC3B, LC3B^G120A or LC3B^A120 was expressed in HEK293 cells for 24 hours. Cells were treated with 50 µM of CQ for 2 hours before collection. Transient expressed proteins were analyzed by immunoblotting using LC3B antibody.<b><i>Ca,</i></b> plasmids expressing GFP-LC3B or GFP-LC3^G120A was expressed in HEK293 cells for 24hours. Cells were then treated with 50 µM of Chloroquine (CQ) for 2 hours. Wb assays were performed using anti-GFP antibody. <b><i>Cb,</i></b> A549 cells infected with Adenoviral vectors expressing GFP-LC3B or GFP-LC3B^G120A at 5 MOI for 24 hours. Cells were then treated with 50 µM of CQ for two hours. GFP-LC3B punctuation was recorded by fluorescence microscopy. CM, complete media, CQ, complete media plus CQ. Scale bar: 25 micron.</p

Pro-human LC3B migrates at similar rate as G120A mutant and lipidated form in SDS-polyacrylamide gel.

<p><b><i>A,</i></b> pTriEX.1.1 plasmids encoding Flag-LC3B, Flag-LC3B^G120A, or Flag-LC3B^A120 was expressed in HEK293 or <i>E.coli</i> cells (Tuner cell), respectively. Recombinant protein expression in <i>E.coli</i> cells were induced with 1 mM of IPTG for four hours before harvesting. 10 µg of HEK293 or <i>E.coli</i> lysates were loaded onto SDS-polyacrylamide gel. Immunoblotting was conducted using Flag-antibody. <b><i>B,</i></b> 10 µg of <i>E.coli</i> lysates with Flag-LC3B or Flag-LC3B^G120A recombinant proteins was digested with recombinant Atg4B at 37°C for indicated time and analyzed by immunoblotting with LC3B antibody and Atg4B antibody as indicated.</p

Last five amino acids determine human LC3B faster migration in SDS-PAGE.

<p><b><i>A,</i></b> Flag-LC3B and indicated mutants were expressed in <i>E.coli.</i> 10 µg of cell lysates were then separated in 15% of SDS-polyacrylamide gel. Transferred membrane was stained with Ponceau S before immunoblotting with LC3B antibody. <b><i>B,</i></b> human LC3B and indicated mutants were expressed in HEK293 cells for 24 hours. Cells were then treated with 50 µM of CQ for 2 hours. Expression patters were analyzed by Wb using LC3B antibody. <b><i>C,</i></b> Same sets of expression plasmids were then expressed in <i>E.coli</i> cells. 10 µg of cell lysates were then separated in 15% of SDS-polyacrylamide gel. Transferred membrane was stained with Ponceau S before immunoblotting with LC3B antibody. Arrows point to wild-type and mutant LC3B recombinant protein band expressed in <i>E. coli.</i></p