human cortisol elisa kit  (Elabscience Biotechnology)

Journal: Theranostics

Article Title: Why 11β-HSD1 inhibitors show variable efficacy in Alzheimer's therapy: an APOE4-dependent HSD11B1 mechanism

doi: 10.7150/thno.126244

Figure Lengend Snippet: APOE4 protein enhances HSD11B1 expression and increases cortisol levels in neuronal cells. A. Cortisol levels were measured in HT-22 (left) and SH-SY5Y (right) cells treated with recombinant APOE4 (E4) or APOE3 (E3) recombinant proteins. Cells were co-treated with VLDL (25 μg/mL) or HDL (25 μg/mL) plus either APOE4 or APOE3 (10 μg/mL) for 3 days, followed by cortisone (0.4 μg/mL) treatment for an additional 24 hours. Cortisol in the culture supernatants was quantified using an ELISA kit. B. Schematic representation of local cortisol regulation by HSD11B enzymes. C. qRT-PCR analysis of HSD11B1 mRNA expression in HT-22 cells. Cells were treated under the same conditions as in (A), and HSD11B1 mRNA levels were quantified using GAPDH as the internal control. D. APOE4/HDL induces HSD11B1 expression and cortisol activation in primary EC neurons. Primary neurons derived from the EC were treated with recombinant APOE3 or APOE4 proteins in combination with HDL for 3 days, followed by cortisone for an additional 24 hours. Left: HSD11B1 mRNA levels were quantified by qRT-PCR and normalized to GAPDH. Right: Cortisol levels in culture supernatants were measured by ELISA. E. Predicted docking models of cortisone (left) and carbenoxolone (Cbxl; right) with human HSD11B1 using SwissDock. Cortisone, the natural substrate of HSD11B1, binds within a defined pocket on the enzyme surface. Cbxl, a known HSD11B1 inhibitor, occupies a similar docking site, suggesting competitive inhibition by blocking cortisone access. Binding sites are indicated by red dashed circles. F. Dose-dependent reduction in cortisol levels following HSD11B1 inhibition. HT-22 cells were co-treated with APOE4 and increasing concentrations of the HSD11B1 inhibitor carbenoxolone (Cbxl; 5, 10, 15 μM) for 24 hours in the presence of cortisone (0.4 μg/mL). Cortisol levels were measured by ELISA. G. HSD11B1 knockdown attenuates APOE4-induced cortisol activation in HT-22 cells. HT-22 cells were transfected with siRNA ( siHSD11B1 ) targeting Hsd11b1 or a non-targeting control siRNA ( siCtrl ), followed by treatment with recombinant APOE4 in the presence of HDL for 3 days and cortisone for an additional 24 hours. Cortisol levels in culture supernatants were quantified by ELISA.

Article Snippet: The culture medium was then collected, and cortisol levels were measured using the Cortisol ELISA Kit (Elabscience, Cat# E-EL-0157) according to the manufacturer's instructions.

Techniques: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Activation Assay, Derivative Assay, Inhibition, Blocking Assay, Binding Assay, Knockdown, Transfection

Journal: Theranostics

Article Title: Why 11β-HSD1 inhibitors show variable efficacy in Alzheimer's therapy: an APOE4-dependent HSD11B1 mechanism

doi: 10.7150/thno.126244

Figure Lengend Snippet: C/EBPβ mediates APOE4-induced HSD11B1 expression in neuronal cells. A. Identification of potential transcriptional regulators of HSD11B1. The top 50 genes co-expressed with HSD11B1 were identified using the ARCHS4 RNA-seq database. Enrichment analysis was performed with the Enrichr database and UCSC Genome Browser PWMs to pinpoint transcription factors potentially involved in HSD11B1 regulation. B. Schematic representation of two putative C/EBPβ ( CEBPB ) binding motifs within the HSD11B1 promoter, as identified through chromvatin immunoprecipitation (ChIP) analysis. Data were obtained from the ReMap ChIP-seq database via the UCSC Genome Browser. C. Proposed model illustrating how APOE4 activates C/EBPβ transcriptional activity, thereby promoting HSD11B1 transcription. D. ChIP assay demonstrating C/EBPβ binding to the HSD11B1 promoter in SH-SY5Y cells. Cells were co-treated with HDL and recombinant APOE3 or APOE4 for 3 days. ChIP-qPCR analysis quantified the enrichment of HSD11B1 promoter fragments immunoprecipitated with anti-C/EBPβ compared to IgG controls. The promoter sequence is shown with the transcription start site (+1) marked in dark red, putative C/EBPβ binding sites underlined and highlighted in brown, and the ChIP primer sites indicated in blue. E. qRT-PCR analysis of HSD11B1 mRNA levels following CEBPB (C/EBPβ) knockdown in SH-SY5Y cells. Cells transfected with siCEBPB or siCtrl were co-treated with HDL and APOE4 proteins, and mRNA levels for HSD11B1 and C/EBPβ were quantified. F. Western blot analysis showing the effect of C/EBPβ knockdown on HSD11B1 and C/EBPβ protein levels in SH-SY5Y cells. Cells transfected with siC/EBPβ or siControl were co-treated with HDL and either APOE4 or APOE3 proteins. Total cell lysates were probed with antibodies against HSD11B1, phosphorylated C/EBPβ (Thr235), total C/EBPβ, and GAPDH. G. ELISA quantification of cortisol levels in SH-SY5Y cells after C/EBPβ knockdown. Following transfection with siCEBPB or siCtrl , cells were co-treated with HDL and APOE4, then treated with cortisone for 24 hours. Cortisol levels in the culture supernatants were measured using a Cortisol ELISA Kit.

Article Snippet: The culture medium was then collected, and cortisol levels were measured using the Cortisol ELISA Kit (Elabscience, Cat# E-EL-0157) according to the manufacturer's instructions.

Techniques: Expressing, RNA Sequencing, Binding Assay, Immunoprecipitation, ChIP-sequencing, Activity Assay, Recombinant, ChIP-qPCR, Sequencing, Quantitative RT-PCR, Knockdown, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay