Journal: bioRxiv

Article Title: Targeting C5aR1 Increases the Therapeutic Window of Radiotherapy

doi: 10.1101/2020.10.27.358036

Figure Lengend Snippet: (A) Graph shows the % of CD45+ and IL-10+ cells found in small intestines of WT or C5aR1 −/− mice. * = p<0.05, 2-tailed t-test. Individual points represent individual mice per group. (B) Representative FACS plots showing the % IL-10 positivity in F4/80+ and CX3CR1+ cells found in small intestines one WT or C5aR1 −/− mouse respectively. (C) Graph shows the % IL-10 positivity in F4/80+ and CX3CR1+ cells found in small intestines of WT or C5aR1 −/− mice. ** = p<0.01, 2-tailed t-test. Individual points represent individual mice per group. (D) HCT 116 cells were treated with either vehicle or PMX205 in phenol and serum-free media. Western blotting was carried on condition media from these cells with the antibodies indicated. A non-specific band (denoted n.s) was used as an indication of loading. 80 ng of human recombinant IL-10 (rhIL-10) was used as a positive control. n=3. (E) HT29 cells were treated with either vehicle or PMX205 and either IgG or IL-10 blocking antibody from 1 hour before irradiation (IR) with either 0 or 9 Gy. Cells were harvested 48 hours post-IR. Western blotting was carried out with the antibodies indicated. Tubulin was used as the loading control. (F) The graph represents the number of apoptotic/non-apoptotic cells expressed as a % of the whole population for HT29 cells treated with either vehicle or PMX205 and either IgG or IL-10 blocking antibody from 1 hour before irradiation (IR) with either 0 or 9 Gy. Cells were harvested 48 hours post-IR. Independent fields of view are show, n=3. **** = p<0.0001 by 2-way ANOVA with Tukey-Kramer comparison test. (G) The graph represents the number of apoptotic/non-apoptotic cells expressed as a % of the whole population for HCT 116 cells treated with either vehicle or PMX205 and either IgG or IL-10 blocking antibody from 1 hour before irradiation (IR) with either 0 or 9 Gy. Cells were harvested 48 hours post-IR. n=3. **** = p<0.0001 by 2-way ANOVA with Dunnett’s comparison test. (H) The graph represents the number of apoptotic/non-apoptotic cells expressed as a % of the whole population for HCT 116 cells transfected with either Scr or RelA siRNA and treated with either vehicle or PMX205 from 1 hour before irradiation (IR) with either 0 or 9 Gy. Cells were harvested 48 hours post-IR. Independent fields of view from a representative experiment are show, n=3. ** = p<0.01, * = p<0.05 by 2-way ANOVA with Dunnett’s comparison test. (I) The graph represents the number of TUNEL positive (+) cells found in MC38 subcutaneous tumors treated with either vehicle or PMX205 and either IgG or IL-10 blocking antibody 1 day before irradiation (10 Gy), on the day of and 1 day post-irradiation. Individual points represent independent fields of view from up to 3 different tumors per group. **** = p<0.0001, n.s = not significant, 2-way ANOVA with Dunnett’s comparison test. (J) Relative tumor growth curves are shown for MC38 colorectal cancer cells treated with 10 Gy single dose irradiation, PMX205 treatment and either IgG or IL-10 blocking antibody for 3 doses flanking the irradiation dose (on day 0, 1 and 2). * = p<0.05, ** = p<0.01 **** = p<0.0001 by 2-way ANOVA. Individual points represent individual mice per group. (K) Relative tumor growth curves are shown for MC38 colorectal cancer cells treated with 10 Gy single dose irradiation, vehicle treatment and either IgG or IL-10 blocking antibody for 3 doses flanking the irradiation dose (on day 0, 1 and 2). Individual points represent individual mice per group. (L) Working hypothesis model: targeting C5aR1 increases both IL-10 expression in the small intestine and IL-10 secretion by tumor cells. IL-10 attenuates RelA phosphorylation and mediates increased tumor cell apoptosis following irradiation.

Article Snippet: Antibodies used were: β-actin (Sigma # A5441, concentration: 1:5000). hFAB™ Rhodamine Anti-Tubulin (Bio-Rad #12004166), RelA/p65 (Cell Signaling #3034, concentration 1:1000), pRelA/p65-S536 (Cell Signaling #3033T, concentration 1:1000), STAT3 (Cell Signaling #30835, concentration 1:1000), pSTAT3-Ty705 (Cell Signaling #9131, concentration 1:1000), p53-S15 (Cell Signaling, #9284 concentration 1:1000), total AKT (Upstate #14276, concentration 1:1000), AKT-S473 (Cell Signaling #9271S, concentration 1:1000) and anti-human IL-10 (R&D #MAB217-100, concentration 1:1000).

Techniques: Western Blot, Recombinant, Positive Control, Blocking Assay, Irradiation, Transfection, TUNEL Assay, Expressing

Journal:

Article Title: Dose-dependent synergy of Th1-stimulating cytokines on bacille Calmette-Gu?rin-induced interferon-? production by human mononuclear cells

doi: 10.1111/j.1365-2249.2007.03413.x

Figure Lengend Snippet: (a) Bacille Calmette–Guérin (BCG) dose-dependent interferon (IFN)-γ production by human peripheral blood mononuclear cells (PBMCs). PBMCs were incubated with medium or five different doses of BCG ranging from 2·5 × 103 to 2·5 × 106 colony-forming units (CFU)/ml for 3 days and then assayed for IFN-γ production in the conditioned media. (b) The roles of endogenous T helper 1 (Th1) and Th2 cytokines in BCG-induced IFN-γ production by human PBMCs. PBMCs were incubated with 2·5 × 105 CFU/ml of BCG in the presence or absence of indicated cytokine-neutralizing antibodies (3 µg/ml) for 3 days and then assayed for IFN-γ production in the conditioned media. Species and isotype-matched control antibodies had no effect on IFN-γ production (data not shown). *Significantly decreased.

Article Snippet: Human cytokine-neutralizing antibodies were obtained from Schering for IFN-α (sheep polyclone) and from R&D Systems (Minneapolis, MN, USA) for IL-2 (clone 5334·21, mouse IgG1), IL-10 (clone 23738·111, mouse IgG2b) and IL-12 (goat polyclone).

Techniques: Incubation