Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Mutation of H231 and D232 residues in the HD-GYP domain of <i>Xoc</i> RpfG disrupts the PDE activity and regulatory function.

<p>(A) Purified RpfG_AA-GYP completely lost its PDE activity detected by colorimetric assays. (B–D) RpfG_AA-GYP lost the ability to restore EPS production (B), biofilm formation (C) and virulence to rice (D) of <i>ΔrpfG</i> to the wild-type level. WT: wild-type, EV: empty vector.</p

Effects of mutations of genes encoding several putative glycosyltransferases on biofilm formation and EPS production in <i>Xoc</i> wild-type and <i>ΔrpfG</i> mutant backgrounds.

<p>(A) Elevated biofilm formation in <i>Xoc ΔrpfG</i> was reduced to the wild-type level when <i>xagA</i>, <i>xagB</i> and <i>gumD</i>, but not <i>pgaA</i> or <i>pgaC</i> was deleted singly in the <i>ΔrpfG</i> genotype. The full length <i>xagA</i> and <i>gumD</i> genes restored the ability of biofilm formation in <i>ΔrpfG/xagA</i> and <i>ΔrpfG/gumD</i> mutants, respectively. (B) The ability of <i>ΔgumD</i> to form biofilm was greatly attenuated in the wild-type background, but was restored by complementation. Biofilm formation in the <i>ΔpgaA</i>, <i>ΔpgaC, ΔxagA</i> and <i>ΔxagB</i> single mutants was not altered compared to the wild-type strain. (C) EPS production was significantly reduced in the <i>ΔgumD</i> mutant, but not in <i>ΔpgaA</i>, <i>ΔpgaC, ΔxagA</i> and <i>ΔxagB</i> mutants compared to the wild-type. These experiments were repeated at least three times with similar results.</p

Virulence assays of the wild-type and <i>rpfG-, hgdA- and hgdC-</i>related single, double and triple mutant strains on rice cv. Jingang 30.

<p>(A) The length of disease lesions was measured at 14 days after pressure inoculation of the wild-type (WT), <i>ΔrpfG</i>, complemented <i>ΔrpfG (rpfG)</i>, <i>ΔhgdA</i>, <i>ΔhgdA/hgdC</i>, <i>ΔhgdA/rpfG</i>, <i>ΔrpfG/hgdC</i>, <i>ΔhgdA/rpfG/hgdC</i> strains, respectively. Ten to 15 leaves were scored for each strain; means ± standard error (SE) are shown. (B) <i>In planta</i> bacterial populations of <i>Xoc</i> RS105, <i>ΔrpfG</i> and <i>ΔrpfG(rpfG)</i> at the specific time points after inoculation. Data are presented as means ± SE.</p

The effect of <i>gumD</i> deletion on bacterial virulence in <i>Xoc.</i>

<p><i> Xoc</i> virulence to rice was greatly attenuated when <i>gumD</i> was deleted, but was unaltered in the <i>ΔpgaA</i>, <i>ΔpgaC, ΔxagA</i> and <i>ΔxagB</i> mutant strains. Virulence of the <i>ΔgumD</i> mutant was restored by the introduction of full-length <i>gumD</i> into the <i>ΔgumD(gumD)</i> strain. The length of disease lesions was measured at 20 days after pressure inoculation. Ten to 15 leaves were scored for each strain; means ± SE are shown. The experiments were repeated three times with similar results.</p

The effect of RpfG on the expression of <i>hrp</i>-related genes in <i>Xoc</i>.

<p>(A) Gene expression of <i>hrpG</i>, <i>hrpX</i> and <i>hrpA</i> in wild-type Xoc (RS105), <i>ΔrpfG</i> and complemented strain <i>ΔrpfG(rpfG)</i> was detected by qRT-PCR. 16S RNA was used as an internal control for data analyses. (B) Gene expression of <i>hrpG</i>, <i>hrpX</i> and <i>hrpA</i> in RS105, <i>ΔrpfG</i> and <i>ΔrpfG(rpfG)</i> strains was examined by GUS activities of appropriate promoter-GUS fusions. WT: wild-type.</p

Effect of pharmacologic inhibition of CCR2 on mouse mammary carcinoma development.

<p>(<b>A</b> and <b>B</b>) Overall survival and tumor-free survival of <i>neu</i>^<i>+</i> mice treated with the CCR2 antagonist CCX872 (n = 26) or vehicle (n = 25). Median survivals were compared using Log-rank (Mantel-Cox) test. Mice were sacrificed when the diameter of any single tumor reached 2 cm, if a tumor became necrotic, or if mice were unable to reach food or water (<b>C</b> and <b>D</b>) Tumor growth in mice treated with CCX872 or vehicle measured as total tumor volume in a mouse <b>(C)</b> or as the volume of the single largest tumor mass in a mouse <b>(D)</b>. Tumors were measured twice weekly and averaged. Comparisons were made by two-way ANOVA.</p

CCL2 production associated with MMTV-neu-driven tumors.

<p>(<b>A</b>) CCL2 concentrations were measured by ELISA in sera from 4 wild type and 6 <i>neu</i>^<i>+</i> mice at the indicated ages. The difference between wild type and tumor bearing mice was significant by two-way ANOVA (p < 0.05). (<b>B</b>) Cell lines were developed from <i>neu</i>^<i>+</i>, <i>neu</i>^<i>+</i>/<i>CCL2</i>^<i>-/-</i>, and <i>neu</i>^<i>+</i>/<i>CCR2</i>^<i>-/-</i> mice. CCL2 released into the culture medium by 10⁶ cells over a 72 hr period was measured by ELISA.</p

Measurement of tumor-associated leukocytes and endothelial cells.

<p>Tissue microarrays were prepared from MMTV-<i>neu</i>-driven tumors in wild type, <i>Ccl2</i>^<i>-/-</i>, and <i>Ccr2</i>^<i>-/-</i> mice at 60, 80, 100, 120 and 140 days of age. Four or five mice were included per data point. Tissue microarray slides treated with fluorescent antibodies against (<b>A</b>) Ki-67, (<b>B</b>) vWF, (<b>C</b>) CD31, and (<b>D</b>) Mac2 and imaged by fluorescence or immunohistochemistry. Quantified stains were compared using one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Gene sets enrichment analysis of genes enriched in wild type compared to <i>Ccr2</i>^<i>-/-</i> monocytes.

<p>(A) Enrichment plots of wound healing, inflammation and locomotory behavior pathways. (B) Common genes enriched in similar pathways.</p