Journal: The Lancet. Rheumatology

Article Title: Autoantibodies against interleukin-1 receptor antagonist in multisystem inflammatory syndrome in children: a multicentre, retrospective, cohort study

doi: 10.1016/S2665-9913(22)00064-9

Figure Lengend Snippet: Neutralising and functional effect of anti-IL-1Ra antibodies in MIS-C (A) Free IL-1Ra plasma concentrations as measured by ELISA in patients with MIS-C (n=21), Kawasaki disease (n=6) and systemic juvenile idiopathic arthritis (n=10). Horizontal lines represent the mean and SD. Data were analysed by Brown–Forsythe and Welch ANOVA with Dunnett's T3 multiple comparisons. (B) IL-1β-signalling reporter assay on selected MIS-C plasma compared with an adult critical COVID-19 plasma sample (both 1:20 dilution) as well as commercially available anti-IL-1Ra antibody or control (anti-SLP2) antibody. The absorbance of secreted embryonic alkaline phosphatase, as a marker for IL-1β pathway activation in HEK IL-1β reporter cells, was detected at 650 nm. Error bars show mean (SD). MIS-C=multisystem inflammatory syndrome in children. IL-1Ra=interleukin-1 receptor antagonist. KD=Kawasaki disease. sJIA=systemic juvenile idiopathic arthritis. TNF=tumour necrosis factor. IL-1β=interleukin-1β.

Article Snippet: Subsequently, these compounds were added with either 2 ng/mL IL-1β (Biozol) or 2 ng/mL TNF (Biozol) in 100 μl Dulbecco's Modified Eagle Medium to HEK-Blue IL-1β reporter cells (2 × 10 4 cells per well) and incubated overnight at 37°C.

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Reporter Assay, Marker, Activation Assay

Journal:

Article Title: Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

doi:

Figure Lengend Snippet: Effects of growth factors (A and B), forskolin, and PMA (C) on FGF-2 content in human astrocytes: Western analysis. Subconfluent astrocytic cultures were maintained in serum-free medium containing 0.25% BSA for 24 h before the incubation with growth factors, PMA, or forskolin. Total cell lysates were subjected to Western blot analysis as described in MATERIALS AND METHODS. (A and B) Growth factor concentrations used (PDGFAB, 1 × 10−10 M; FGF-2, 5 × 10−10 M; IL-1β, 2.8 × 10−10 M [this Figure]; and EGF, 5.0 × 10−9 M [Figure 2]) are similar to their respective Kd values (Binger et al., 1990 ; Sorkin et al., 1991 ; Ban et al., 1993 ; Stachowiak et al., 1997a ) and maximally or near maximally increased or FGF-2 immunoreactivity in rat (Araujo and Cotman, 1992 ) and human astrocytes and astrocyte proliferation (Joy et al., 1997 ) (our unpublished observations). Similar results were obtained in two or three independent experiments with different astrocytic cultures. (C) Western analysis of PMA- and forskolin-induced changes in FGF-2 content in human astrocytes. Subconfluent astrocytes were maintained in serum-free medium for 24 h, after which the cells were treated with forskolin (10 μM) or PMA (100 nm) for the indicated times. Control cells were incubated with 0.007% DMSO used as a vehicle for forskolin and PMA. Similar results were obtained in three independent experiments.

Article Snippet: Materials Culture media were from Life Technologies (Grand Island, NY), epidermal growth factor (EGF) from Upstate Biotechnology (Lake Placid, NY), platelet-derived growth factor (PDGF) from Genzyme (Cambridge, MA), FGF-2 from Boehringer Mannheim (Indianapolis, IN), and interleukin-1β (IL-1β) from Bachem (Torrance, CA).

Techniques: Western Blot, Incubation

Journal:

Article Title: Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

doi:

Figure Lengend Snippet: Effects of growth factors, forskolin, or PMA on FGF-2 mRNA levels. Human astrocytic cultures at 40–60% confluence were maintained in serum-free medium for 24 h before they were treated for 24 h with EGF, FGF-2, or IL-1β (A) and with forskolin, PMA, or both (B) at the concentrations given in Figures ​Figures1 1 and ​and2.2. Total RNA was isolated and analyzed for FGF-2 and histone H3.3 mRNA levels using RT-PCR. Aliquots of FGF-2 cDNA were taken from cycles 24, 26, 28, 30, 32, 34, and 36 and of H3.3 cDNA from cycles 16, 18, 20, 22, 24, 26, and 28. DNA was electrophoresed through 2% agarose gels and stained with ethidium bromide (dark-field photograph). The FGF-2 cDNA products were also transferred to a nylon membrane and hybridized to 32P-FGF-2 cDNA (light-field autoradiograms) as described in MATERIALS AND METHODS.

Article Snippet: Materials Culture media were from Life Technologies (Grand Island, NY), epidermal growth factor (EGF) from Upstate Biotechnology (Lake Placid, NY), platelet-derived growth factor (PDGF) from Genzyme (Cambridge, MA), FGF-2 from Boehringer Mannheim (Indianapolis, IN), and interleukin-1β (IL-1β) from Bachem (Torrance, CA).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Staining

Journal:

Article Title: Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

doi:

Figure Lengend Snippet: Activation of the FGF-2 gene promoter by growth factors, forskolin, and PMA in astrocyte cultures. Human astrocytes (QG) were transfected with a luciferase reporter plasmid containing a −1800/+314-bp fragment (relative to the transcription start site) of the FGF-2 promoter. Treatment with growth factors, forskolin, or PMA was for 24 h in serum-free medium at the concentrations given in Figures ​Figures11 and ​and22 as described in MATERIALS AND METHODS. Bar graphs represent mean ± SEM from 6–15 samples obtained in two experiments. Analysis of variance showed an overall significant effect of treatments on luciferase expression (p < 0.000002). Luciferase expression was significantly increased in astrocytes treated with PMA (p < 0.00001), FGF-2 (p < 0.0005), IL-1β (p < 0.05), or EGF (p < 0.005).

Article Snippet: Materials Culture media were from Life Technologies (Grand Island, NY), epidermal growth factor (EGF) from Upstate Biotechnology (Lake Placid, NY), platelet-derived growth factor (PDGF) from Genzyme (Cambridge, MA), FGF-2 from Boehringer Mannheim (Indianapolis, IN), and interleukin-1β (IL-1β) from Bachem (Torrance, CA).

Techniques: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing

Journal:

Article Title: Activation of mitogen-activating protein kinase by glucose is not required for insulin secretion

doi:

Figure Lengend Snippet: Effect of glucose on protein kinase activities in INS-1 and 293 cells. (A) Activation of ERK1 and ERK2 detected by immunoblotting of INS-1 cell lysates with active ERK antibody (Upper) and Y691 (Lower). Lane 1, INS-1 cells were maintained in growth medium containing 11 mM glucose. Cells pretreated in either 0 (lanes 6–10) or 3 (lanes 2–5) mM glucose for 1 h and then treated in KRBH for 30 min as follow: 0 mM glucose (lane 6), 3 mM glucose (lanes 2 and 7), 15 mM glucose (lanes 3 and 8), 15 mM glucose and 10 μM forskolin (lanes 4 and 9), and 10 μM forskolin (lanes 5 and 10). (B) Immune complex kinase assays of ERK1 and MEK1. Cells were preincubated in 1 mM glucose for 1 h and then incubated in KRBH without glucose (lane 1), with 15 mM glucose (lane 2), with 10 μM forskolin (lane 3), or with 15 mM glucose plus 10 μM forskolin (lane 4) for 30 min. The activities of ERK1 and MEK1 were assayed [X837, ERK-1 (Upper) or A2227, MEK1 (Lower)] using myelin basic protein or ERK2 K52R as substrates. (C) Immune complex kinase assays of JNK/SAPK. Cells were preincubated as in B and incubated in KRBH alone (lane 1), with 15 mM glucose plus 10 μM forskolin (lane 2) for 30 min, or with 10 ng/ml TNF-α and 10 ng/ml IL-1β (lane 3) for 20 min. JNK/SAPK was immunoprecipitated with O977 and assayed with GST-c-Jun(1–221). (D) Immune complex kinase assays of p38. Cells were pretreated as in B and incubated in KRBH alone (lane 1), with 15 mM glucose (lane 2) or with 15 mM glucose plus forskolin (lane 3) for 30 min; in KRBH alone (lane 4), with 10 ng/ml TNF-α (lane 5) or with 10 ng/ml IL-1β (lane 6). p38 was immunoprecipitated with P287 and assayed with GST-ATF2(1–254). In B–D, autoradiograms are shown. (E) 293 cells were serum-starved overnight and glucose was removed for the final 1 h. The cells were incubated in KRBH alone for 5 min (lane 1), KRBH with 10% fetal bovine serum for 5 min (lane 2), KRBH for 30 min (lane 3), KRBH with 15 mM glucose (lane 4), and with 15 mM glucose plus forskolin (lane 5) for 30 min. Immunoblots with active ERK antibody (Upper) and Y691 (Lower). Experiments in A and B were performed five times and in C–E two to three times.

Article Snippet: Tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were from Promega.

Techniques: Activation Assay, Western Blot, Immune Complex Kinase Assay, Incubation, Immunoprecipitation