Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: siRNA Library Screening Identifies a Druggable Immune-Signature Driving Esophageal Adenocarcinoma Cell Growth

doi: 10.1016/j.jcmgh.2018.01.012

Figure Lengend Snippet: LIF hierarchically regulates STAT3 phosphorylation, IL6 expression, and cell proliferation in EAC cells. SiRNA-mediated silencing of LIF expression results in reduced LIF mRNA ( A ) expression, ( B ) secretion, and ( C ) a recombinant-recoverable reduction in cell proliferation in CP-D, SKGT4, and OE-33 EAC cell lines. Treatment of LIF silenced cells with recombinant LIF protein (rLIF) results in ( D ) recovery of LIF mRNA in CP-D, SKGT4, and OE33 EAC cells and ( E ) protein expression levels in SKGT4 EAC cells. ( F ) STAT3-Y705 phosphorylation levels in LIF-silenced SKGT4 EAC cells are rescued by treatment with recombinant LIF protein as determined by Western blot. ( G ) Silencing LIF expression in SKGT4 EAC cells results in decreased IL6 mRNA expression that is recovered upon exposure to rLIF protein. ( H ) Neither silencing IL6 expression by siRNA-mediated silencing nor treatment with anti-IL6 antibody affects LIF mRNA levels or SKGT4 EAC cell growth. ( I ) Silencing LIF expression results in decreased expression of C1QA mRNA but not TREM2 in SKGT4 EAC cells. ∗ P < .05, ** P < .01, *** P < .001, and **** P < .0001 in either Student t test (proliferation) or Mann–Whitney testing (mRNA expression). siNT, nontargeting siRNA pool; siLIF, LIF-targeted siRNA pool; rLIF, recombinant LIF protein; rIL6, recombinant IL6 protein.

Article Snippet: Treatments with recombinant leukemia inhibitory factor (LIF) (30 ng/mL, ab57665; Abcam), IL6 (30 ng/mL; R&D Systems, Minneapolis, MN), native C1q (75 μg/mL; Sigma, Oakville, Ontario), and IL6 blocking antibody (AB206NA) experiments were performed in either 6- or 96-well cell culture plates for protein and viability experiments, respectively.

Techniques: Phospho-proteomics, Expressing, Recombinant, Western Blot, MANN-WHITNEY

Journal: Biology of reproduction

Article Title: Impairment of the interleukin system in equine endometrium during the course of endometrosis.

doi: 10.1095/biolreprod.113.109447

Figure Lengend Snippet: FIG. 1. Representative pictures of immunohistochemical staining for IL1a (IL-1a; A–C), IL1b (IL-1b; D–F), IL6 (IL-6; G–I), IL1RI (IL-1RI; J–L), ILRII (IL-RII; M–O), and IL6Ra (IL-6a; P, R, S) in endometrial tissue in category I (first column; no degenerative changes) and category III (second and third column; severe endometrosis) endometrium during the estrous cycle. The negative controls are shown in T, U, and V. GE, glandular epithelium; GN, glandular nests; LE, luminal epithelium; S, stromal cells. Bar ¼ 100 lm (original magnification 320 for first and second column) and 50 lm (original magnification 340 for third column).

Article Snippet: Briefly, the sections were incubated with primary goat polyclonal antibodies against IL1a (anti-mouse; 15 lg/ml; #AF-400NA; R&D Systems), IL1b/IL1F2 (anti-equine; R&D System; #AF3340; 10 lg/ml), IL6 (anti-equine; 10 lg/ml; #AF1886; R&D Systems), IL6Ra (anti-mouse; 15 lg/ml; #AF1830; R&D Systems); IL1RI (anti-mouse; 5 lg/ml; #AF771; R&D Systems); IL1RII (antimouse; 15 lg/ml; #AF563; R&D Systems).

Techniques: Immunohistochemical staining, Staining

Journal: Biology of reproduction

Article Title: Impairment of the interleukin system in equine endometrium during the course of endometrosis.

doi: 10.1095/biolreprod.113.109447

Figure Lengend Snippet: FIG. 6. IL6 (IL-6) mRNA transcription (A) and the density of the immunohistochemical reaction (B; relative units; image analysis; Micro- Image) during the estrous cycle in Kenney category I (no degenerative changes), II, and III in equine endometrium (n ¼ 48). a,bStatistical differences between phases of the estrous cycle in Kenney category I. n,o,pStatistical differences between phases of the estrous cycle in Kenney category II. x,y,zStatistical differences between phases of the estrous cycle in Kenney category III. Asterisks indicate statistical differences between IL6 mRNA transcription and the density of the immunohistochemical reaction during the course of endometrosis within each phase of the estrous cycle (*P , 0.05; **P , 0.01; ***P , 0.001).

Article Snippet: Briefly, the sections were incubated with primary goat polyclonal antibodies against IL1a (anti-mouse; 15 lg/ml; #AF-400NA; R&D Systems), IL1b/IL1F2 (anti-equine; R&D System; #AF3340; 10 lg/ml), IL6 (anti-equine; 10 lg/ml; #AF1886; R&D Systems), IL6Ra (anti-mouse; 15 lg/ml; #AF1830; R&D Systems); IL1RI (anti-mouse; 5 lg/ml; #AF771; R&D Systems); IL1RII (antimouse; 15 lg/ml; #AF563; R&D Systems).

Techniques: Immunohistochemical staining

Journal: Biology of reproduction

Article Title: Impairment of the interleukin system in equine endometrium during the course of endometrosis.

doi: 10.1095/biolreprod.113.109447

Figure Lengend Snippet: FIG. 8. The influence of IL1a (IL-1a; 10 ng/ml), IL1b (IL-1b; 10 ng/ml), and IL6 (IL-6; 10 ng/ml) on PGE2 in vitro secretion from midluteal phase endometrial explants. The endometrial explants were assigned to category I (A), II (B), and III (C) according to Kenney. All values are expressed as n- fold change from the control. Asterisks indicate statistical differences between the groups (*P , 0.05; ***P , 0.001).

Article Snippet: Briefly, the sections were incubated with primary goat polyclonal antibodies against IL1a (anti-mouse; 15 lg/ml; #AF-400NA; R&D Systems), IL1b/IL1F2 (anti-equine; R&D System; #AF3340; 10 lg/ml), IL6 (anti-equine; 10 lg/ml; #AF1886; R&D Systems), IL6Ra (anti-mouse; 15 lg/ml; #AF1830; R&D Systems); IL1RI (anti-mouse; 5 lg/ml; #AF771; R&D Systems); IL1RII (antimouse; 15 lg/ml; #AF563; R&D Systems).

Techniques: In Vitro, Control

Journal: Biology of reproduction

Article Title: Impairment of the interleukin system in equine endometrium during the course of endometrosis.

doi: 10.1095/biolreprod.113.109447

Figure Lengend Snippet: FIG. 9. The influence of IL1a (IL-1a; 10 ng/ml), IL1b (IL-1b; 10 ng/ml), and IL6 (IL-6; 10 ng/ml) on in vitro PGF2a secretion from midluteal phase endometrial explants. The endometrial explants were assigned to category I (A), II (B), and III (C) according to Kenney. All values are expressed as n- fold change from the control. Asterisks indicate statistical differences between the groups (*P , 0.05; **P , 0.01).

Article Snippet: Briefly, the sections were incubated with primary goat polyclonal antibodies against IL1a (anti-mouse; 15 lg/ml; #AF-400NA; R&D Systems), IL1b/IL1F2 (anti-equine; R&D System; #AF3340; 10 lg/ml), IL6 (anti-equine; 10 lg/ml; #AF1886; R&D Systems), IL6Ra (anti-mouse; 15 lg/ml; #AF1830; R&D Systems); IL1RI (anti-mouse; 5 lg/ml; #AF771; R&D Systems); IL1RII (antimouse; 15 lg/ml; #AF563; R&D Systems).

Techniques: In Vitro, Control

Journal: Biology of reproduction

Article Title: Impairment of the interleukin system in equine endometrium during the course of endometrosis.

doi: 10.1095/biolreprod.113.109447

Figure Lengend Snippet: FIG. 10. The influence of IL1a (IL-1a; 10 ng/ml), IL1b (IL-1b; 10 ng/ml), and IL6 (IL-6; 10 ng/ml) on PTGS-2 mRNA transcription in midluteal phase endometrial explants incubated in vitro. The endometrial explants were assigned to category I (A), II (B), and III (C) according to Kenney. Asterisks indicate statistical differences between the groups (*P , 0.05; **P , 0.01; ***P , 0.001).

Article Snippet: Briefly, the sections were incubated with primary goat polyclonal antibodies against IL1a (anti-mouse; 15 lg/ml; #AF-400NA; R&D Systems), IL1b/IL1F2 (anti-equine; R&D System; #AF3340; 10 lg/ml), IL6 (anti-equine; 10 lg/ml; #AF1886; R&D Systems), IL6Ra (anti-mouse; 15 lg/ml; #AF1830; R&D Systems); IL1RI (anti-mouse; 5 lg/ml; #AF771; R&D Systems); IL1RII (antimouse; 15 lg/ml; #AF563; R&D Systems).

Techniques: Incubation, In Vitro

Journal: Biology of reproduction

Article Title: Impairment of the interleukin system in equine endometrium during the course of endometrosis.

doi: 10.1095/biolreprod.113.109447

Figure Lengend Snippet: FIG. 11. The influence of IL1a (IL-1a; 10 ng/ml), IL1b (IL-1b; 10 ng/ml), and IL6 (IL-6; 10 ng/ml) on PGES mRNA transcription in midluteal phase endometrial explants incubated in vitro. The endometrial explants were assigned to category I (A), II (B), and III (C) according to Kenney. Asterisks indicate statistical differences between the groups (*P , 0.05; **P , 0.01; ***P , 0.001).

Article Snippet: Briefly, the sections were incubated with primary goat polyclonal antibodies against IL1a (anti-mouse; 15 lg/ml; #AF-400NA; R&D Systems), IL1b/IL1F2 (anti-equine; R&D System; #AF3340; 10 lg/ml), IL6 (anti-equine; 10 lg/ml; #AF1886; R&D Systems), IL6Ra (anti-mouse; 15 lg/ml; #AF1830; R&D Systems); IL1RI (anti-mouse; 5 lg/ml; #AF771; R&D Systems); IL1RII (antimouse; 15 lg/ml; #AF563; R&D Systems).

Techniques: Incubation, In Vitro

Journal: Biology of reproduction

Article Title: Impairment of the interleukin system in equine endometrium during the course of endometrosis.

doi: 10.1095/biolreprod.113.109447

Figure Lengend Snippet: FIG. 12. The influence of IL1a (IL-1a; 10 ng/ml), IL1b (IL-1b; 10 ng/ml), and IL6 (IL-6; 10 ng/ml) on PGFS mRNA transcription in midluteal phase endometrial explants incubated in vitro. The endometrial explants were assigned to category I (A), II (B), and III (C) according to Kenney. Asterisks indicate statistical differences between the groups (*P , 0.05; **P , 0.01; ***P , 0.001).

Article Snippet: Briefly, the sections were incubated with primary goat polyclonal antibodies against IL1a (anti-mouse; 15 lg/ml; #AF-400NA; R&D Systems), IL1b/IL1F2 (anti-equine; R&D System; #AF3340; 10 lg/ml), IL6 (anti-equine; 10 lg/ml; #AF1886; R&D Systems), IL6Ra (anti-mouse; 15 lg/ml; #AF1830; R&D Systems); IL1RI (anti-mouse; 5 lg/ml; #AF771; R&D Systems); IL1RII (antimouse; 15 lg/ml; #AF563; R&D Systems).

Techniques: Incubation, In Vitro

Journal: Journal of leukocyte biology

Article Title: The PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by α-PD-L1 or α-IL6 antibodies.

doi: 10.1002/JLB.MA1217-495R

Figure Lengend Snippet: FIGURE 5 Cytokine expression from cocultures of PBMCs with IL27-pre-stimulated HCC cells in presence or absence of a blocking anti-PD- L1 antibody. (A) Scheme of the experimental layout. (B to D) Cytokine levels were analyzed by cytokine arrays. IL2 (B), MCP1, IL1𝛽, TNF-𝛼(C) and IL6 (D) levels are expressed as the mean ± standard deviation of 3 biological replicates. One-way ANOVA with Bonferroni posttest was performed using Graphpad InSTAT version 3.10 (www.graphpad.com). For statistical analysis error probabilities <0.05 were considered to be significant, ***: P < 0.001, **: P < 0.01, *: P < 0.05

Article Snippet: The blocking antibodies used to antagonize IL6-type cytokine signaling were anti-human IL6 (R & D Systems Cat# MAB206, RRID: AB_2127617), anti-human IL6Rα (R&DSystemsCat#MAB227, RRID: AB_2127908) and anti-human OSM (R & D Systems Cat# MAB295, RRID: AB_2298931).

Techniques: Expressing, Blocking Assay, Standard Deviation

Journal: Journal of leukocyte biology

Article Title: The PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by α-PD-L1 or α-IL6 antibodies.

doi: 10.1002/JLB.MA1217-495R

Figure Lengend Snippet: FIGURE 6 Blocking antibodies against IL6- type cytokine or receptors prevent down- regulation of IL27 signaling. (A) Expression of PD-L1 on the HCC cell lines HepG2, Huh7, and Hep3B was studied by flow-cytometry. Cells were left untreated (gray fill) or were stimulated with IL27 alone (red fill) or treated for 4 h with hy-IL6 or OSM prior to the IL27 treatment for 24 h (solid line). The dotted line indicates the sit- uation in which blocking antibodies against IL6, IL6R, or OSM were added together with the IL6 or OSM pre-treatment. (B) HepG2, Hep3B, Huh7, and PH5CH8 cells were stimulated as described in (A) but using only hy-IL6 and anti-IL6. Western blots of the lysates were immuno-detected for the STAT1 target genes IDO-1, TAP2, STAT1. Vinculin and tubulin detections were used as loading controls. Phosphorylation of STAT3 was investigated on a separate blot using STAT3, vin- culin and tubulin as controls. (C) HepG2, Hep3B, Huh7, and PH5CH8 cells were stimulated as described in (B). The cells were lysed after 6 h of treatment. SOCS3 mRNA expression was investigated by qPCR. Results show the mean of 3 biological replicates as a percentage of the highest SOCS3 induction with standard deviation

Article Snippet: The blocking antibodies used to antagonize IL6-type cytokine signaling were anti-human IL6 (R & D Systems Cat# MAB206, RRID: AB_2127617), anti-human IL6Rα (R&DSystemsCat#MAB227, RRID: AB_2127908) and anti-human OSM (R & D Systems Cat# MAB295, RRID: AB_2298931).

Techniques: Blocking Assay, Expressing, Flow Cytometry, Western Blot, Phospho-proteomics, Standard Deviation

Journal: Journal of leukocyte biology

Article Title: The PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by α-PD-L1 or α-IL6 antibodies.

doi: 10.1002/JLB.MA1217-495R

Figure Lengend Snippet: FIGURE 7 Major IL27-mediated signaling pathways in cancer cells and possible treatment options to improve immune clearance of cancer cells. Pathways and treatments that promote antitumor actions are indicated by blue arrows. Pathways that promote tumor immune escape are indicated using red arrows. Black boxes indicate those blocking antibodies that have been used in this study. Already FDA-approved blocking anti- bodies inhibiting the same targets mentioned above are shown in dark green boxes. An IDO-1 inhibitor that entered clinical trials but is not yet FDA-approved is indicated in a grey box. In tumors with high IL6-type cytokine levels, anti-cytokine-receptor- or anti-cytokine-antibodies could be used in addition to IL27, to relieve the SOCS3-mediated cross-inhibition of IL6-type cytokines on IL27 signaling. PD-L1 is expressed in all investi- gated cancer cell types in response to IL27 and thus anti-PD-L1 antibodies could be envisaged as standard co-treatment with IL27 (or Interferon-) therapies to prevent immune cell deactivation (e.g., a CD8+T-cell as shown in the scheme) by cancer cell-expressed PD-L1. IDO-1 is not induced by IL27 in all cells and thus IDO-1 inhibitor treatment could only be applicable to a subset of tumors

Article Snippet: The blocking antibodies used to antagonize IL6-type cytokine signaling were anti-human IL6 (R & D Systems Cat# MAB206, RRID: AB_2127617), anti-human IL6Rα (R&DSystemsCat#MAB227, RRID: AB_2127908) and anti-human OSM (R & D Systems Cat# MAB295, RRID: AB_2298931).

Techniques: Protein-Protein interactions, Blocking Assay, Clinical Proteomics, Inhibition