human il 6 receptor il6r  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: NB cells stimulated IL6 secretion from bone marrow mesenchymal stem cells. (A) Schematic diagram of iBMSC stimulated with NB cells CM or co-cultured with NB cells. That is, iBMSC were cultured with CM derived from 6 NB cells or co-cultured with NB cells for 48 h. (B) The content of IL6 was detected by ELISA in the supernatant of the corresponding culture system. NB cells were co-culture with iBMSC or stimulated by NB cells-priming iBMSC CM. (C) The protein expression levels of pSTAT3, STAT3, pERK1/2 and ERK1/2 in NB cells were detected by WB. (D) The phosphorylation levels and localization of pSTAT3 and pERK1/2 in NB cells were detected by immunofluorescence. Data represents three independent experiments and is presented as mean ± SD ( ***p < 0.001 vs. iBMSC-Monoculture).

Article Snippet: Human IL6, IL6R and Gal-3BP ELISA kit (Boster Biological Technology) were used to determine the level of secretion or expression of specific proteins in cell lysates, culture supernatants, or tumor tissue, according to manufacturer’s instructions.

Techniques: Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Expressing, Phospho-proteomics, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: LPC hinders the tumorigenic effect of iBMSC by inhibiting the secretion of IL6 in BMSC mediated by NB cells. NB cells pretreated with different doses of LPC were continued to be cultured in fresh medium for another 48, then the supernatant was extracted and as LPC pretreatment condition medium (LPC-CM). LPC-CM was used to prime the iBMSC, which were then co-cultured with non-LPC pretreated NB cells for a new round of culture. [ **p < 0.01 and ***p < 0.001 vs. NC; #p < 0.01, ##p < 0.01 and ###p < 0.001 vs. LPC (1 μM)] (A) Wound-healing assay. (B) Transwell migration assay. (C) Colony formation assay. (D,E) IL6 secretion in the supernatant of iBMSC cultured with LPC-CM was detected by ELISA. (F) WB method was performed to detect the phosphorylation levels of STAT3 and ERK1/2 in IMR32 cultured with CM from LPC-CM stimulated-iBMSC. The CM from iBMSC/Vector or iBMSC/IL6 cells primed by LPC-CM were used for culturing with NB cells. (G) EdU incorporation assay. (H) Wound-healing assay. (I) The supernatant of iBMSC stimulated by LPC-CM was used for NB cells stimulation. ( ***p < 0.001 vs. iBMSC/Vector) Transwell migration assay was used to detect the migration ability of IMR32 and SK-N-AS cultured with hrIL6 supplemented into LPC-CM stimulated iBMSC supernatant. ( **p < 0.01 and ***p < 0.001 vs. NC) Data represents three independent experiments.

Article Snippet: Human IL6, IL6R and Gal-3BP ELISA kit (Boster Biological Technology) were used to determine the level of secretion or expression of specific proteins in cell lysates, culture supernatants, or tumor tissue, according to manufacturer’s instructions.

Techniques: Cell Culture, Wound Healing Assay, Transwell Migration Assay, Colony Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Plasmid Preparation, Migration

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: LPC suppresses the carcinogenic effect of IMR32-priming iBMSC in vivo , which could be reversed by exogenous overexpression of IL6. (A) The schematic diagram shows the preparation procedure of cells to be transplanted and the construction process of subcutaneous xenograft tumor and lung metastasis model in nude mice. Briefly, IMR32 was injected into the flanks of nude mice alone or mixed with IMR32-derived CM-priming iBMSC, LPC-CM-priming iBMSC or LPC-CM-priming IL6-expressing iBMSC (iBMSC/IL6), respectively. Meanwhile, the cells prepared in the same manner were intravenously injected into nude mice to establish the lung metastasis model. During modeling, another intravenous injection of iBMSC were performed at the second month of vaccination. (B) Representative images of tumor obtained from mice at 40 days after subcutaneous injection. (C) Tumor growth curve. (D) Tumor weight. (E) The expression level of IL6 in tumor tissues was determined by ELISA. (F) Representative images of IHC staining of Ki67, pSTAT3 and pERK1/2 in tumor tissues. (G) Representative HE staining images obtained from mouse lungs 2 months after tail vein injection. (H–J) The number of tumors on the lung surface. (K) Lung weight. (L) IL6 expression in lung metastatic lesions. (M) Survival curve of mice. ( *p < 0.05, **p < 0.01 and ***p < 0.001 vs. IMR32; #p < 0.01, ##p < 0.01 and ###p < 0.001 vs. + iBMSC; & p < 0.05, && p < 0.01 and &&& p < 0.0001 vs. + iBMSC + LPC).

Article Snippet: Human IL6, IL6R and Gal-3BP ELISA kit (Boster Biological Technology) were used to determine the level of secretion or expression of specific proteins in cell lysates, culture supernatants, or tumor tissue, according to manufacturer’s instructions.

Techniques: In Vivo, Over Expression, Injection, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: LPC suppresses iBMSC-derived IL6 production through inhibiting Gal-3/Gal-3BP signal in NQO1-dependent manner. LPC-CM obtained from NB cells treated with indicated doses of LPC was used for iBMSC culture. (A) The relative mRNA level of IL6 in iBMSC was detected by qPCR. (B) Gal-3BP secretion from NB cells CM was detected by ELISA. [ *p < 0.05, **p < 0.01 and ***p < 0.001 vs. LPC (0 μM); #p < 0.01, ##p < 0.01 and ###p < 0.001 vs. LPC (0.5 μM); & p < 0.05, && p < 0.01 and &&& p < 0.0001 vs. LPC (1.0 μM)] (C) The relative protein expression levels of Gal-3BP in NB cells and Gal-3 in iBMSC were detected by western blot. (D) The expression and localization of Gal-3BP in NB cells were detected by IF. (E) iBMSC was cultured with LPC-CM supplemented with or without hrGal-3BP, and the IL6 secretion was detected by ELISA. NB cells were pretreated with or without dicoumarin before LPC stimulation, and their CM was used to stimulate iBMSC. (F) The protein expression levels of Gal-3BP in NB cells were detected by western blot. ( **p < 0.01 and ***p < 0.001 vs ctrl; ###p < 0.001 vs LPC) (G) NB cells transfected with Gal-3BP siRNA or control siRNA were treated as shown in , and IL6 secretion in the iBMSC CM was detected by ELISA. ( ***p < 0.001 vs. LPC; ###p < 0.001 vs. LPC + Dic) Data represents three independent experiments.

Article Snippet: Human IL6, IL6R and Gal-3BP ELISA kit (Boster Biological Technology) were used to determine the level of secretion or expression of specific proteins in cell lysates, culture supernatants, or tumor tissue, according to manufacturer’s instructions.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Cell Culture, Transfection, Control

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: NB cells stimulated IL6 secretion from bone marrow mesenchymal stem cells. (A) Schematic diagram of iBMSC stimulated with NB cells CM or co-cultured with NB cells. That is, iBMSC were cultured with CM derived from 6 NB cells or co-cultured with NB cells for 48 h. (B) The content of IL6 was detected by ELISA in the supernatant of the corresponding culture system. NB cells were co-culture with iBMSC or stimulated by NB cells-priming iBMSC CM. (C) The protein expression levels of pSTAT3, STAT3, pERK1/2 and ERK1/2 in NB cells were detected by WB. (D) The phosphorylation levels and localization of pSTAT3 and pERK1/2 in NB cells were detected by immunofluorescence. Data represents three independent experiments and is presented as mean ± SD ( ***p < 0.001 vs. iBMSC-Monoculture).

Article Snippet: Human IL6, IL6R ELISA kit were purchased from Boster Biological Technology (Nanjing, China).

Techniques: Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Expressing, Phospho-proteomics, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: LPC hinders the tumorigenic effect of iBMSC by inhibiting the secretion of IL6 in BMSC mediated by NB cells. NB cells pretreated with different doses of LPC were continued to be cultured in fresh medium for another 48, then the supernatant was extracted and as LPC pretreatment condition medium (LPC-CM). LPC-CM was used to prime the iBMSC, which were then co-cultured with non-LPC pretreated NB cells for a new round of culture. [ **p < 0.01 and ***p < 0.001 vs. NC; #p < 0.01, ##p < 0.01 and ###p < 0.001 vs. LPC (1 μM)] (A) Wound-healing assay. (B) Transwell migration assay. (C) Colony formation assay. (D,E) IL6 secretion in the supernatant of iBMSC cultured with LPC-CM was detected by ELISA. (F) WB method was performed to detect the phosphorylation levels of STAT3 and ERK1/2 in IMR32 cultured with CM from LPC-CM stimulated-iBMSC. The CM from iBMSC/Vector or iBMSC/IL6 cells primed by LPC-CM were used for culturing with NB cells. (G) EdU incorporation assay. (H) Wound-healing assay. (I) The supernatant of iBMSC stimulated by LPC-CM was used for NB cells stimulation. ( ***p < 0.001 vs. iBMSC/Vector) Transwell migration assay was used to detect the migration ability of IMR32 and SK-N-AS cultured with hrIL6 supplemented into LPC-CM stimulated iBMSC supernatant. ( **p < 0.01 and ***p < 0.001 vs. NC) Data represents three independent experiments.

Article Snippet: Human IL6, IL6R ELISA kit were purchased from Boster Biological Technology (Nanjing, China).

Techniques: Cell Culture, Wound Healing Assay, Transwell Migration Assay, Colony Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Plasmid Preparation, Migration

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: LPC suppresses the carcinogenic effect of IMR32-priming iBMSC in vivo , which could be reversed by exogenous overexpression of IL6. (A) The schematic diagram shows the preparation procedure of cells to be transplanted and the construction process of subcutaneous xenograft tumor and lung metastasis model in nude mice. Briefly, IMR32 was injected into the flanks of nude mice alone or mixed with IMR32-derived CM-priming iBMSC, LPC-CM-priming iBMSC or LPC-CM-priming IL6-expressing iBMSC (iBMSC/IL6), respectively. Meanwhile, the cells prepared in the same manner were intravenously injected into nude mice to establish the lung metastasis model. During modeling, another intravenous injection of iBMSC were performed at the second month of vaccination. (B) Representative images of tumor obtained from mice at 40 days after subcutaneous injection. (C) Tumor growth curve. (D) Tumor weight. (E) The expression level of IL6 in tumor tissues was determined by ELISA. (F) Representative images of IHC staining of Ki67, pSTAT3 and pERK1/2 in tumor tissues. (G) Representative HE staining images obtained from mouse lungs 2 months after tail vein injection. (H–J) The number of tumors on the lung surface. (K) Lung weight. (L) IL6 expression in lung metastatic lesions. (M) Survival curve of mice. ( *p < 0.05, **p < 0.01 and ***p < 0.001 vs. IMR32; #p < 0.01, ##p < 0.01 and ###p < 0.001 vs. + iBMSC; & p < 0.05, && p < 0.01 and &&& p < 0.0001 vs. + iBMSC + LPC).

Article Snippet: Human IL6, IL6R ELISA kit were purchased from Boster Biological Technology (Nanjing, China).

Techniques: In Vivo, Over Expression, Injection, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: Frontiers in Pharmacology

Article Title: Beta-Lapachone Attenuates BMSC-Mediated Neuroblastoma Malignant Transformation by Inhibiting Gal-3/Gal-3BP/IL6 Axis

doi: 10.3389/fphar.2021.766909

Figure Lengend Snippet: LPC suppresses iBMSC-derived IL6 production through inhibiting Gal-3/Gal-3BP signal in NQO1-dependent manner. LPC-CM obtained from NB cells treated with indicated doses of LPC was used for iBMSC culture. (A) The relative mRNA level of IL6 in iBMSC was detected by qPCR. (B) Gal-3BP secretion from NB cells CM was detected by ELISA. [ *p < 0.05, **p < 0.01 and ***p < 0.001 vs. LPC (0 μM); #p < 0.01, ##p < 0.01 and ###p < 0.001 vs. LPC (0.5 μM); & p < 0.05, && p < 0.01 and &&& p < 0.0001 vs. LPC (1.0 μM)] (C) The relative protein expression levels of Gal-3BP in NB cells and Gal-3 in iBMSC were detected by western blot. (D) The expression and localization of Gal-3BP in NB cells were detected by IF. (E) iBMSC was cultured with LPC-CM supplemented with or without hrGal-3BP, and the IL6 secretion was detected by ELISA. NB cells were pretreated with or without dicoumarin before LPC stimulation, and their CM was used to stimulate iBMSC. (F) The protein expression levels of Gal-3BP in NB cells were detected by western blot. ( **p < 0.01 and ***p < 0.001 vs ctrl; ###p < 0.001 vs LPC) (G) NB cells transfected with Gal-3BP siRNA or control siRNA were treated as shown in , and IL6 secretion in the iBMSC CM was detected by ELISA. ( ***p < 0.001 vs. LPC; ###p < 0.001 vs. LPC + Dic) Data represents three independent experiments.

Article Snippet: Human IL6, IL6R ELISA kit were purchased from Boster Biological Technology (Nanjing, China).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Cell Culture, Transfection, Control

Journal:

Article Title: The Phenotypic Plasticity of Myeloma Plasma Cells as Expressed by Dedifferentiation into an Immature, Resilient, and Apoptosis-Resistant Phenotype

doi: 10.1158/1078-0432.CCR-05-0523

Figure Lengend Snippet: A–B, blocking IL-6 activity did not reverse the immature phenotype of cocultured myeloma cells. CD45/38 flow cytometry of multiple myeloma plasma cells from two patients before culture (precultured), after 6 weeks in culture with osteoclasts (cocultured), and after 4 additional weeks in culture with osteoclasts, anti-IL6, and anti-IL6R. Note an up-regulation of CD45 expression by osteoclasts that was not reversed by inhibition of IL-6 activity.

Article Snippet: Neutralizing polyclonal antibodies against human interleukin-6 (IL-6) and human IL-6 receptor (IL6R) were obtained from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Blocking Assay, Activity Assay, Flow Cytometry, Clinical Proteomics, Expressing, Inhibition