Journal: EBioMedicine

Article Title: Tumor cells hijack enteric glia to activate colon cancer stem cells and stimulate tumorigenesis

doi: 10.1016/j.ebiom.2019.09.045

Figure Lengend Snippet: EGCs express IL-1R, and IL-1α/β are highly enriched in the tumor microenvironment. a/b. Agarose gel electrophoresis of PCR products corresponding to IL-1R cDNA amplicons in human primary EGC cultures ( a ) ( HOG-EGC , amplicon size 152 bp) and in rat non-transformed EGC line ( b ) ( JUG-EGC , amplicon size 63 bp). Lanes #1, #2 and #3 ( a ) correspond to 3 cultures derived from 3 different patients. Lanes P25 and P30 ( b ) correspond to 2 different passages. c/d. ELISA data showed that IL-1α ( c ) and IL-1β ( d ) were highly enriched in supernatants of human colon adenocarcinomas ( Tumor ) compared with supernatants of patient-matched healthy colonic mucosa ( Healthy ). n = 4; Wilcoxon rank-sum test, a: p < 0.05 vs. Healthy. e/f . Real-Time qPCR data demonstrated that IL-1α gene expression ( e ) was downregulated in CD44 High -CD24 High CSC s as compared to unsorted HT29 ( Total ) and CD44 Low -CD24 Low non-CSC s, and IL-1β mRNA ( f ) was enriched in non-CSCs. Data are expressed as fold change to unsorted HT29 cells ( Total ) (mean ± SEM). n = 4, ANOVA, a: p < 0.05 vs. Total, b: p < 0.05 vs. non-CSC.

Article Snippet: IL-1α/β concentrations were assessed using human IL-1α/IL-1F1 and IL-1β/IL-1F2 Quantikine ELISA Kits (R&D).

Techniques: Agarose Gel Electrophoresis, Amplification, Transformation Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: EBioMedicine

Article Title: Tumor cells hijack enteric glia to activate colon cancer stem cells and stimulate tumorigenesis

doi: 10.1016/j.ebiom.2019.09.045

Figure Lengend Snippet: Tumor epithelial cell-derived IL1α/β activates a pro-tumorigenic phenotype in EGCs. a. RT-qPCR data demonstrating that addition of IL-1α or IL-1β strongly induced mPGES-1 gene expression in EGCs. n = 3. b. RT-qPCR data showing that addition of an antagonist of IL-1R ( IL-1Ra , 10 µM) to supernatants of human primary colon adenocarcinomas ( Tumor ) abolished the tumor-induced up-regulation of mPGES-1 expression in EGCs. Healthy represents supernatants of patient-matched healthy colonic mucosa. n = 8; ANOVA, Holm-Sidak multiple comparison test, a: p < 0.05 vs. Control, b: p < 0.05 vs. IL-1Ra, c: p < 0.05 vs. Healthy, d: p < 0.05 vs. Healthy+IL-1Ra, e: p < 0.05 vs. Tumor+IL-1Ra. c. PGE2 EIA confirmed that addition of IL-1Ra blocked the increase in PGE2 release in EGCs activated by supernatants of human colon adenocarcinomas ( EGC/Tumor ). EGC, Healthy, Tumor and EGC/Healthy represent EGC-CM, supernatant of patient-matched healthy mucosa, supernatant of tumors, and CM of EGCs stimulated with supernatant of healthy mucosa, respectively. n ≥ 4; ANOVA, a: p < 0.05 vs. EGC, b: p < 0.05 vs. EGC+IL-1Ra, c: p < 0.05 vs. Healthy, d: p < 0.05 vs. Tumor, e: p < 0.05 vs. EGC/Healthy, f: p < 0.05 vs. EGC/Healthy+IL-1Ra, g: p < 0.05 vs. EGC/Tumor+IL-1RA. d. Representative photographs ( left panel ) and quantification ( right panel ) to show that double IL-1α and β knock-down tumor epithelial cells ( TEC ) had lost their abilities to activate a pro-tumorigenic phenotype in EGCs, while single IL-1α or β knock-down TECs had impact similar to that of Control TECs. n = 4, ANOVA. Scale Bar: 1 mm.

Article Snippet: IL-1α/β concentrations were assessed using human IL-1α/IL-1F1 and IL-1β/IL-1F2 Quantikine ELISA Kits (R&D).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing

Journal: American Journal of Translational Research

Article Title: Soluble Flt-1 improves the repair of ankle joint injury in rats

doi:

Figure Lengend Snippet: High PLGF and pro-inflammatory cytokines are detected in ankle joint from AAJI patients. We randomly chose 20 AAJI patients with ankle joint injury at one side for this study. The ankle joint fluid was taken from both ankle joints of the patients and analyzed for the levels of PLGF and pro-inflammatory cytokines, IL-6, TNFα and IFNɤ. The uninjured site was used a control (NT). (A-D) ELISA for the levels of PLGF (A), IL-6 (B), TNFα (C) and IFNɤ (D) in the joint fluid from the injured ankle joint of the AAJI patients, compared to NT. N=20. *P<0.05.

Article Snippet: ELISA ELISA was performed, using a human PLGF, human or rat tumor necrosis factor α (TNFα), human or rat interleukin 6 (IL-6), and human or rat interferon ɤ (IFNɤ) ELISA Kit (R&D System, Los Angeles, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: American Journal of Translational Research

Article Title: Soluble Flt-1 improves the repair of ankle joint injury in rats

doi:

Figure Lengend Snippet: Suppression of PLGF signaling reduce pro-inflammatory cytokines in the injured ankle joint by carrageenan in rats. (A) The histology of the injured ankle joints showed significant increases in local inflammation, compared to CTL, while sFLT-1 reduced the carrageenan-induced inflammatory. The Yellow arrows point to inflammatory cells, which appeared to be stained strongly for hematoxylin. CTL: no Carr injection. Carr: rats that received carrageenan. Carr+sFlt-1: rats that received both Carr and sFlt-1. (B) Phosphorylation of VEGFR1 was analyzed by Western blot, showing that VEGFR1 phosphorylated was induced in the injured joint by carrageenan, but was attenuated by sFLT-1 treatment. (-) negative control of Western blot, in which no protein was loaded. (C-E) The levels of IL-6 (C), TNFα (D) and IFNɤ (E) in the injured ankle joint were analyzed by ELISA. N=10. *P<0.05. Scale bars are 100 μm.

Article Snippet: ELISA ELISA was performed, using a human PLGF, human or rat tumor necrosis factor α (TNFα), human or rat interleukin 6 (IL-6), and human or rat interferon ɤ (IFNɤ) ELISA Kit (R&D System, Los Angeles, CA, USA).

Techniques: Staining, Injection, Western Blot, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa -Derived DnaJ Induces the Expression of IL−1β by Engaging the Interplay of p38 and ERK Signaling Pathways in Macrophages

doi: 10.3390/ijms242115957

Figure Lengend Snippet: P. aeruginosa -derived DnaJ induces the expression of IL−1β. ( A ) Cells were treated for 4 h with recombinant P. aeruginosa DnaJ (1 μg/mL), which was pre-treated with proteinase K (20 μg/mL) for 1 h. ( B – D ) Cells were treated with either DnaJ at the indicated concentration for 4 h ( B ) or 1 μg/mL of DnaJ for the indicated time ( C , D ). After treatment, the increase in IL−1β mRNA level was quantified by qPCR analysis, and the level of IL−1β protein released from cells was measured with supernatant by ELISA analysis. Data are expressed as the mean ± SD ( n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. DnaJ treatment alone ( A ) or CON. A control extract was used as a negative control ( A ). PBS was used as a control ( B – D ).

Article Snippet: The amount of IL−1β released into the supernatants was determined using the human IL−1β ELISA kit (R&D System, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa -Derived DnaJ Induces the Expression of IL−1β by Engaging the Interplay of p38 and ERK Signaling Pathways in Macrophages

doi: 10.3390/ijms242115957

Figure Lengend Snippet: Reciprocal effects of p38 and ERK MAPK signaling pathways for the expression of IL−1β. ( A , B ) Cells were pre-treated with either 10 μM of chemical inhibitors (BAY11-7082, SB203580, PD98059, SP600125; ( A )) or 20 μM of chemical inhibitors (SB203580, PD98059; ( B )) for 1 h, followed by treatment with 1 μg/mL of DnaJ for 4 h. ( C – E ) Cells were transfected with 50 nM of either ERK1 siRNA or ERK2 siRNA. Forty-eight hours post-transfection, the transfected cells were treated with 1 μg/mL of DnaJ for 4 h ( C ). The effect of siRNA was verified by qPCR analysis ( D , E ). ( F ) Cells were treated with 1 μg/mL of DnaJ for the indicated time. ( G – I ) Cells were pre-treated with 10 μM of chemical inhibitors (SB203580 and PD98059 for ( G , I ); BAY11-7082 for ( H ) for 1 h, followed by treatment with 1 μg/mL of DnaJ for the indicated time. After treatment, the increase in IL−1β mRNA level was quantified by qPCR analysis, and the levels of protein were measured by either ELISA or immunoblot analysis. Data in ( A – E ) are expressed as means ± SD ( n = 3). Data in ( F – I ) are representative of three separate experiments. ***, p < 0.001 vs. DnaJ treatment alone ( A – C ), or no transfection ( D – E ).

Article Snippet: The amount of IL−1β released into the supernatants was determined using the human IL−1β ELISA kit (R&D System, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa -Derived DnaJ Induces the Expression of IL−1β by Engaging the Interplay of p38 and ERK Signaling Pathways in Macrophages

doi: 10.3390/ijms242115957

Figure Lengend Snippet: DnaJ-induced IL−1β expression is under the control of CD91/CD40 signaling pathway. Cells were transfected with either 10 or 20 nM of either CD91 siRNA or CD40 siRNA. Forty-eight hours post-transfection, the transfected cells were treated with 1 μg/mL of DnaJ for 4 h ( A – D ) or for the indicated time ( G ). The effect of siRNA was verified by qPCR analysis ( E , F ). After treatment, the increase in mRNA levels was quantified by qPCR analysis, and the levels of protein were measured by either ELISA or immunoblot analysis. Data in ( A – F ) are expressed as means ± SD ( n = 3). Data in ( G ) are representative of three separate experiments. **, p < 0.01; ***, p < 0.001 vs. DnaJ treatment alone ( A – D ) or no transfection ( E – F ).

Article Snippet: The amount of IL−1β released into the supernatants was determined using the human IL−1β ELISA kit (R&D System, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa -Derived DnaJ Induces the Expression of IL−1β by Engaging the Interplay of p38 and ERK Signaling Pathways in Macrophages

doi: 10.3390/ijms242115957

Figure Lengend Snippet: DnaJ-induced IL−1β expression is partially under the control of TLR2/TLR4 signaling pathway. ( A , B ) Cells were pre-treated with either OxPAPC ( A ) or CLI-095 ( B ) at the indicated concentrations for 1 h, followed by treatment with 1 μg/mL of DnaJ for 4 h. ( C ) Cells were pre-treated with 3 μM of CLI-095 for 1 h, followed by treatment with 1 μg/mL of DnaJ for the indicated time. ( D ) Cells were transfected with TLR2 siRNA at the indicated concentration. At 24 h post-transfection, the transfected cells were treated with 1 μg/mL of DnaJ for 4 h ( left panel ). The effect of siRNA was verified by immunoblot of TLR2 protein ( right panel ). ( E ) Cells were transfected with 20 nM of TLR2 siRNA. Twenty-four hours post-transfection, the transfected cells were treated with 1 μg/mL of DnaJ for the indicated time. After treatment, the increase in IL−1β mRNA level was quantified by qPCR analysis, and the levels of protein were measured by immunoblot analysis. Data in ( A , B , D ) are expressed as means ± SD ( n = 3). Data in ( C , E ) are representative of three separate experiments. **, p < 0.01; ***, p < 0.001 vs. DnaJ treatment alone.

Article Snippet: The amount of IL−1β released into the supernatants was determined using the human IL−1β ELISA kit (R&D System, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Expressing, Transfection, Concentration Assay, Western Blot

Journal: Oncotarget

Article Title: The histone methyltransferase G9a as a therapeutic target to override gemcitabine resistance in pancreatic cancer

doi: 10.18632/oncotarget.11256

Figure Lengend Snippet: ( A ) The conditioned media of PANC-1 and G9a-overexpressing PANC-1 cells were collected and the secreted cytokines were determined by using human cytokine array. Up-regulated or down-regulated cytokines were labeled by red and green square mark. The cytokines tested in the array were shown in the right panel. ( B ) The IL-8 mRNA level of PANC-1 and PANC-1-R cells was compared by using RT-qPCR assay. Columns represented the mean of triplicate PCR assays and normalized to GAPDH. * P < 0.05. ( C ) The IL-8 mRNA level in cells and the secreted IL-8 protein in the conditioned media were studied by RT-qPCR assay (top panel) and by ELISA assay (bottom panel). The IL-8 level of PANC-1-R cells was defined as 1, and relative level of G9a-depleted PANC-1-R cells was shown. * P < 0.05. ( D ) The IL-8 mRNA level in cells and the secreted IL-8 protein in the conditioned media of PANC-1 and G9a-overexpressing PANC-1 cells were studied by RT-qPCR assay (top panel) and by ELISA assay (bottom panel). Results from three independent assays were expressed as Mean ± SE. * P < 0.05.

Article Snippet: IL-8 concentration was measured using Quantikines Human IL-8 Immunoassay kit (R&D, Minneapolis, MN, USA).

Techniques: Labeling, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: The histone methyltransferase G9a as a therapeutic target to override gemcitabine resistance in pancreatic cancer

doi: 10.18632/oncotarget.11256

Figure Lengend Snippet: ( A ) Expression of CXCR1 and CXCR2 mRNA in PANC-1 cells and pancreatic satellite cells (PSC) was determined by RT-PCR analysis. ( B ) The expression of CXCR1 and CXCR2 in PANC-1, PANC-1-G9a, PANC-1-R and PANC-1-R-shG9a cells was studied by RT-qPCR analysis. Results from three independent assays were expressed as Mean ± SE. ns: not significant. ( C ) PANC-1 or G9a-overexpressing PANC-1 cells were treated with IL-8 (200 ng/ml) or anti-IL-8 (0.5 mg/ml) for 48 h and cellular proliferation was studied by MTT assay. * P < 0.05. ( D ) G9a-overexpressing PANC-1 cells were treated with GEM in combination with nonimmune immunoglobulin (C), anti-IL-8, or anti-CXCR1+anti-CXCR2 antibodies. Cell viability was assessed after treatment for 48 h by MTT assay. * P < 0.05. ( E ) PANC-1-R cells were treated with GEM in combination with nonimmune immunoglobulin (C), anti-IL-8, or anti-CXCR1+anti-CXCR2 antibodies. Cell viability was assessed after treatment for 48 h by MTT assay. * P < 0.05. ( F ) PANC-1 cells were pre-treated without or with different concentrations of IL-8 for 24 h and then incubated with 100 or 300 ng/ml of GEM for another 48 h. Cell viability was studied by MTT assay. * P < 0.05. ( G ) PANC-1-R and PANC-1-R-shG9a cells were cultured in low attachment plates in the presence or absence of IL-8 (200 ng/ml) for 14 days. The number of the spheres was counted. * P < 0.05. ( H ) PANC-1, PANC-1-R and PANC-1-R-shG9a cells were pre-incubated with nonimmune immunoglobulin (Ig) or anti-IL-8 antibody and subjected to trans-endothelial migration assay as described in MATERIALS and METHODS.

Article Snippet: IL-8 concentration was measured using Quantikines Human IL-8 Immunoassay kit (R&D, Minneapolis, MN, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, MTT Assay, Incubation, Cell Culture, Migration

Journal: Oncotarget

Article Title: The histone methyltransferase G9a as a therapeutic target to override gemcitabine resistance in pancreatic cancer

doi: 10.18632/oncotarget.11256

Figure Lengend Snippet: ( A ) PSC cells were treated with indicated concentrations of recombinant IL-8 for 48 h and cellular proliferation was assessed. ( B ) PSC cells were treated with indicated concentrations of IL-8 for 48 h, and the protein level of fibronectin and α-SMA were studied by Western blot analysis. ( C ) PSC cells were treated with indicated concentration of IL-8 for 48 h and the expression and deposition of fibronectin in PSC cells was analyzed by immunofluorescence. ( D ) PANC-1-R and G9a-depleted PANC-1-R cells were seeded into upper inserts of transwell plates for 24 h and co-cultured with PSC cells for another 48 h. The expression and deposition of fibronectin in PSC cells was analyzed by immunofluorescence. ( E ) The conditioned medium collected from PANC-1 or G9a-overexpressing PANC-1 cells were pre-incubated with non-immune IgG (—) or IL-8 antibody for 4 h and were used to treat PSC cells. After 48 h, the protein level of fibronectin was investigated by Western blot analysis. ( F ) PANC-1-R cells were treated without or with UNC0638 (2.5 μM) for 24 h and co-cultured with PSC cells for another 48 h. The expression and deposition of fibronectin in PSC cells was analyzed by immunofluorescence. ( G ) PANC-1-R cells were treated with indicated concentration of UNC for 24 h and co-cultured with PSC for another 48 h. The expression of α-SMA in PSC cells were analyzed by Western blot. ( H ) PSC cells were treated with UNC0638 (2.5 μM) or IL-8 (200 ng/ml) for 48 h and the number of lipid droplets was stained by Oil Red dye. Scale bar, 5 micrometer.

Article Snippet: IL-8 concentration was measured using Quantikines Human IL-8 Immunoassay kit (R&D, Minneapolis, MN, USA).

Techniques: Recombinant, Western Blot, Concentration Assay, Expressing, Immunofluorescence, Cell Culture, Incubation, Staining

Journal: Oncotarget

Article Title: The histone methyltransferase G9a as a therapeutic target to override gemcitabine resistance in pancreatic cancer

doi: 10.18632/oncotarget.11256

Figure Lengend Snippet: ( A ) Tumor growth was examined by biofluorescence imaging. Fluorescent images of the whole body of tumor-bearing mice received different treatments as indicated in MATERIALS and METHODS at 3 and 6 weeks after orthotopic inoculation of PANC-1-R cancer cells into the pancreas. ( B ) The photon number fluxed from the tumors in the control or drug-treated animals were measured at week 3 and 6. The increase of photon number (6 weeks versus 3 weeks) was compared in different experimental groups. The statistical difference between different groups was assessed. Control group: n = 3 and other drug-treated groups: n = 4. * P < 0.05. ( C ) Organs of tumor-bearing mice were shown to demonstrate tumor metastasis. GEM treatment alone induced stronger local invasion and distant metastasis to liver. Combination of GEM and UNC significantly reduced tumor growth and metastasis. a:control group; b:GEM treatment group; c:UNC0638 treatment group and d:GEM+UNC group. ( D ) Fluorescent images to demonstrate the tumor growth induced by PANC-1-R cells or PANC-1-R cells overexpressing dominant-negative G9a (PANC-1-R-DN-G9a) in mice. ( E ) The photon number fluxed from the tumors in the animals injected with PANC-1-R or PANC-1-R-DN-G9a cells were measured at week 2 and 4. The increase of photon number (4 weeks versus 2 weeks) was compared in two experimental groups. The statistical difference between two groups was assessed. PANC-1-R group: n = 3; PANC-1-R-DN-G9a group: n = 3. * P < 0.05. ( F ) Representative images of immunohistochemical staining for G9a, α-SMA, IL-8 and H3K9 di-methylation (H3K9me2) in the tumors of different treatment groups. Scale bar, 20 micrometer. The staining of H3K9me2 is monitored to validate the inhibitory effect of UNC0638 on G9a enzymatic activity.

Article Snippet: IL-8 concentration was measured using Quantikines Human IL-8 Immunoassay kit (R&D, Minneapolis, MN, USA).

Techniques: Imaging, Dominant Negative Mutation, Injection, Immunohistochemical staining, Staining, Methylation, Activity Assay