TLR9 agonist ameliorates RIGS.

<p><b>A.</b> Representative (n = 3) histology from paraffin-embedded mid-jejunal sections of mice treated with TLR9 agonist+WBI versus WBI alone (20×). <b>Hematoxylin and Eosin staining</b> demonstrates larger crypt depth and elongated villi in the mid-jejunum of mice treated with TLR9 agonist and WBI, compared to mice treated with WBI. <b>BrdU immunohistochemistry</b> demonstrates increase in crypt cell proliferation after TLR9 agonist+WBI treatment, compared to WBI controls. Note an increase in BrdU-positive crypt cells in WBI-treated mice, which is further stimulated by TLR9 agonist treatment, 3.5 days after WBI. This is a physiological response of crypt survival after WBI. <b>TUNEL staining</b> demonstrates a decrease in TUNEL-positive, apoptotic cells in TLR9 agonist+WBI mice, when compared to WBI mice. <b>B–D. </b><b>Bar graph of crypt depth, proliferation and apoptosis of crypt epithelial cells in mice treated with TLR9 agonist+WBI versus WBI alone.</b> Effect of TLR9 agonist on intestinal crypt depth (<b>B</b>), crypt proliferation rate (<b>C</b>) and tunnel positive apoptotic cells (<b>D</b>) at 1day and 3.5day post WBI. Mice treated with TLR9 agonist showed a significant resistance to radiation-induced apoptosis (p<0.001) at 1day post WBI with the increase in proliferation rate (p<0.002) and crypt depth at 3.5day post WBI (p<0.001), compared to irradiated control.</p

TLR9 agonist-activated macrophage mitigates RIGS.

<p><b>A.</b> Macrophage cell line J774A.1 was incubated with TLR9 agonist in vitro for 24 hrs. BALB/c mice transplanted with syngeneic TLR9 agonist-activated J774A.1 macrophages, 1 hr after 9.4 Gy WBI, demonstrated significant improvement in survival, compared to WBI (p<0.002) or WBI+J774A.1 (p<0.005) cohorts. <b>B. Culture supernatant of TLR9 agonist-activated J774A.1 induces clonogenic survival of rat intestinal IEC6 following irradiation.</b> Incubation of IEC6 cells with culture supernatants from TLR9 agonist-activated J774A.1 cells followed by a clonogenic assay showed an increase in surviving fraction against incremental doses of radiation (2–6 Gy) compared to irradiated control.</p

FigureS1 from Auranofin Protects Intestine against Radiation Injury by Modulating p53/p21 Pathway and Radiosensitizes Human Colon Tumor

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FigureS4 from Auranofin Protects Intestine against Radiation Injury by Modulating p53/p21 Pathway and Radiosensitizes Human Colon Tumor

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FigureS5 from Auranofin Protects Intestine against Radiation Injury by Modulating p53/p21 Pathway and Radiosensitizes Human Colon Tumor

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TLR9 agonist treatment protected C57Bl/6 mice from radiation-induced mortality.

<p>Kaplan-Meier survival analysis of C57Bl/6 mice treated with TLR9 agonist prior to WBI (A) 8.4 Gy; (B) 9.4 Gy and (C) 10.4 Gy. A significant survival was observed in TLR9 agonist-treated group, exposed to WBI of 10.4 Gy (p<0.03), 9.4 Gy (p<0.008), 8.4 Gy (p<0.002) (log rank test) compared to WBI alone or WBI+TLR9 agonist+iMyd88 cohorts. Treatment with TLR9 agonist after radiation exposure (WBI+Post-TLR9 agonist) had mitigation effect only at 8.4 Gy WBI. Administration of inhibitory peptide against Myd88 (iMyd88) completely inhibited radioprotective effect of TLR9 agonist.</p

TLR9 agonist treatment increase the number of macrophages in pericryptal and lamina propria of intestine of mice treated with WBI.

<p>A. F480 Immunhistochemistry and confocal microscopic analysis and B. Quantification of number of intestinal macrophages. The number of F480+ve macrophages (indicated with arrow) increased at 1 day and 3.5 d post-WBI in the WBI+TLR9 agonist treatment group, compared to the WBI cohort. Nucleus was stained with DAPI (pseudo colored with red). Confocal microscopic images (40×) were magnified 2.3× (inset).</p

Figure S3 from Auranofin Protects Intestine against Radiation Injury by Modulating p53/p21 Pathway and Radiosensitizes Human Colon Tumor

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FigureS6 from Auranofin Protects Intestine against Radiation Injury by Modulating p53/p21 Pathway and Radiosensitizes Human Colon Tumor

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Supplement Table 1 from Auranofin Protects Intestine against Radiation Injury by Modulating p53/p21 Pathway and Radiosensitizes Human Colon Tumor

mRNA expression of UPR downstream proapoptotic molecule</p