Additional file 1 of Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination

Supplementary Material

Model of the DDT Pathway in Mammalian Cells

<p>Recovery from a stalled replication fork at sites of DNA damage can occur by one of two alternative pathways. Previous work has shown that PCNA mono-ubiquitination by the RAD6/RAD18 complex stimulates lesion bypass through recruitment of the error-prone TLS polymerases. Here we show that an alternative error-free pathway requires formation of K63-polyUb chains. Blockade of this error-free pathway results in increased use of the TLS polymerases after DNA damage and a corresponding increase in mutations. As the TLS polymerases POLη and POLι both bind directly and avidly to polyUb chains [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-b020" target="_blank">20</a>], it is hypothesized that the interaction with K63-polyUb causes a disengagement of the polymerase from the DNA, allowing other proteins to migrate to the site of damage to perform error-free repair. This model predicts that K63-polyubiquitination acts to suppress environmental carcinogenesis by preventing genomic instability that would otherwise be introduced by the TLS polymerases.</p

Modification of PCNA by Polyubiquitin in Human Cells after DNA Damage

<div><p>(A) A549, 293T, and Hela cells were irradiated with 0 or 30 J/m² UV and lysed 6 h posttreatment followed by immunoblotting for PCNA.</p><p>(B) 293T cells were transfected with 100 nM of either control siRNA, siRNA Ubc13, or siRNA RAD18. Seventy-two hours post-transfection, cells were treated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g001" target="_blank">Figure 1</a>A. A darker and lighter exposure of the PCNA immunoblot is shown.</p><p>(C) A549, 293T, and Hela cells were irradiated with 30 J/m² UV and lysed in boiling SDS, diluted in lysis buffer and subjected to immunoprecipitation with a PCNA antibody and detected with PCNA or Ub antibodies. The controls in the immunoprecipitations were “no 1”, in which lysates were incubated with beads but no PCNA antibody, and “1 B” in which PCNA antibody was incubated with beads alone.</p><p>(D) 293T cells were transfected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g005" target="_blank">Figure 5</a>B. Seventy-two hours post-transfection, cells were irradiated with 30 J/m² of UV and lysed 6 h later in boiling SDS, diluted in lysis buffer, and subjected to immunoprecipitation with a PCNA antibody and immunoblotted for PCNA (upper panel) and Ub (lower panel). A lighter exposure of the PCNA IP immunoblotted for Ub is also shown. A PCNA immunoblot with darker and lighter exposure performed on protein lysates from the same samples used in the immunoprecipitations is also shown. Asterisks denote immunoglobulin heavy and light chains as detected on the immunoprecipitations.</p></div

Cells Deficient in K63-Ub Chain Formation Are Sensitized to Cisplatin Treatment while UV Sensitivity Is Revealed only upon POLη Knockdown

<div><p>(A and B) Clonogenic survival assays were used to determine sensitivity to 1 h acute treatment with cisplatin in untransfected A549 cells or in A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub.</i> The mean values of three independent experiments are shown with standard error of the mean (error bars). Cells expressing <i>K33R-Ub</i> or cells that lost <i>K63R-Ub</i> expression revert to WT-Ub cisplatin sensitivity.</p><p>(C) Cells were treated for 24 h with 100 μM cisplatin followed by Hoechst staining to detect apoptosis. The mean values of three independent experiments are shown with standard deviation.</p><p>(D) Clonogenic survival assays were used to determine sensitivity to UV irradiation in untransfected A549 cells or in A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub.</i></p><p>(E) Clonogenic survival of A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub</i> with or without POLη RNAi following 10 J/m² UV treatment.</p></div

Cells Deficient in K63-Ub Chain Formation Are Mutagenic in Response to UV Treatment

<div><p>(A and B) Cells were treated with cisplatin for 1 h or UV irradiation and subcultured for 7 d. Cells were then plated and grown in 6-TG to select for <i>HPRT</i> mutants. The mean values of three independent experiments are shown with standard deviation.</p><p>(C) Normal fibroblasts stably expressing <i>WT-Ub</i> or <i>K63R-Ub</i> were UV-irradiated (10 J/m²) and cultured for 5 d. Cells were then plated and grown in 6-TG to select for <i>HPRT</i> mutants.</p><p>(D) The number of <i>HPRT</i> mutants was quantitated for A549 cells stably expressing <i>WT-Ub</i> or <i>K63R-Ub</i> with or without POLη RNAi. Cells were treated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g003" target="_blank">Figure 3</a>C.</p><p>(E) Cells were UV-irradiated and plated in the absence or presence of 0.4 mM caffeine. The mean values of three independent experiments are shown with standard error of the mean (error bars).</p></div

Disrupting K63-PolyUb Chain Formation Increases Reliance of Cells on the Error-Prone TLS Pathway

<div><p>(A) HeLa cells stably expressing <i>WT-Ub-puro</i> or <i>K63R-Ub-puro</i> were transiently transfected with a plasmid expressing a <i>POLη-GFP</i> fusion. Twenty-four hours post-transfection, cells were UV-irradiated (10 J/m²). POLη (green) and PCNA (red) were detected using antibodies. Shown are representative confocal photographs of cells 6 h post-UV treatment.</p><p>(B) Kinetics of POLη foci formation in <i>WT-Ub–</i> and <i>K63R-Ub–</i>expressing HeLa cell lines.</p><p>(C) <i>HPRT</i> mutation spectra. RNA was isolated from 6-TG resistant 10 J/m² UV-treated clones followed by RT-PCR and sequence analysis of the <i>HPRT</i> locus. The UV-induced mutations are shown in the upper table. Most of the point mutations were G→A or C→T transitions indicated as G/C→A/T. The lower table in (C) shows the same mutants in sequence context.</p><p>(D) Foci were quantitated 6 h post-UV treatment using a live-cell imaging fluorescent microscope.</p><p>(E) The number of <i>HPRT</i> mutants was quantitated for A549 cells stably expressing <i>K63R-Ub</i> with or without RAD18 RNAi. Cells were treated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020116#pgen-0020116-g004" target="_blank">Figure 4</a>B.</p></div

Body and liver weights of Wt-tp and DLL4^del-tp mice.

<p><b>(A)</b> Relative weight gain was calculated from the body weights of Wt-tp and DLL4^del-tp mice. (<b>B)</b> Relative liver/total body ratio was measured from the liver and body weights of Wt-tp and DLL4^del-tp mice. (<b>C)</b> Plasma ALT levels were measured of Wt-tp and DLL4^del-tp mice. All data are represented as mean +/- SEM. Data are significant at * p< 0.05, ** p< 0.01, *** p< 0.001. Significance is compared to the chow group of the respective genotype.</p

Lipid level measurements in Wt-tp and DLL4^del-tp mice.

<p><b>(A-D)</b> Cholesterol and triglyceride levels were measured in plasma and in the liver of Wt-tp and DLL4^del-tp mice. (<b>E)</b>. Gene expression of <i>Abca1</i>, <i>Abcg1</i>, <i>Cd36 and Lxrα</i> were measured in the liver of Wt-tp and DLL4^del-tp mice. All data are represented as mean +/- SEM. Data are significant at * p< 0.05, ** p< 0.01, *** p< 0.001. Significance is compared to the chow group of the respective genotype.</p

Hepatic gene and protein expression analysis of DLL4 and its downstream targets.

<p><b>(A)</b> Gene expression of <i>Dll4</i> in the livers of Wt-tp and DLL4^del-tp mice. (<b>B-C)</b> DLL4 protein expression in BMDM and livers of mice, respectively. (<b>D-E)</b> Gene expression analysis of <i>Dll4</i> and <i>Hes1</i> in isolated KCs from Wt-tp and DLL4^del-tp mice. (<b>F-G)</b> Gene expression of <i>Hey1</i> and <i>Hes1</i> in the livers of Wt-tp and DLL4^del-tp mice. All data are represented as mean +/- SEM. Data are significant at * p< 0.05, ** p< 0.01, *** p< 0.001. Significance is compared to the chow group of the respective genotype.</p

Inflammatory response of bone marrow-derived macrophages from Wt and DLL4^del mice.

<p><b>(A)</b> Gene expression of <i>Dll4</i> was measured in BMDM of Wt and DLL4^del mice. (<b>B-C)</b> Gene expression of <i>Tnfα</i> and <i>Itgam</i> were measured in BMDM of Wt and DLL4^del mice. (<b>D)</b> TNFα cytokine production was measured in BMDM of Wt and DLL4^del mice. (<b>E)</b> Wt BMDM stimulated with immobilized recombinant DLL4. All data are represented as mean +/- SEM. Data are significant at * p< 0.05, ** p< 0.01, *** p< 0.001. Significance is compared to Wt BMDM.</p