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: Mouse hippocampal neuronal cell line HT-22 (Sigma-Aldrich, SCC129) and the APOE3 (E3/E3) genotype human neuroblastoma cell line SH-SY5Y (ATCC, CRL-2266) were cultured in Dulbecco's Modified Eagle Medium (Gibco) and Minimum Essential Medium (Gibc), respectively, at 37°C in a 5% CO2 humidified atmosphere.

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: Mouse hippocampal neuronal cell line HT-22 (Sigma-Aldrich, SCC129) and the APOE3 (E3/E3) genotype human neuroblastoma cell line SH-SY5Y (ATCC, CRL-2266) were cultured in Dulbecco's Modified Eagle Medium (Gibco) and Minimum Essential Medium (Gibc), respectively, at 37°C in a 5% CO2 humidified atmosphere.

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

Journal: F1000Research

Article Title: Identification of high-performing antibodies for Apolipoprotein E for use in Western Blot and immunoprecipitation

doi: 10.12688/f1000research.133899.3

Figure Lengend Snippet: Summary of the Apolipoprotein E antibodies tested.

Article Snippet: Bio-Techne , MAB41441 , ZRQ0318021 , AB_2289763 , monoclonal , recombinant fragment , 395004 , rat , 5.0 , Wb.

Techniques: Concentration Assay, Recombinant

Journal: F1000Research

Article Title: Identification of high-performing antibodies for Apolipoprotein E for use in Western Blot and immunoprecipitation

doi: 10.12688/f1000research.133899.3

Figure Lengend Snippet: HAP1 WT and APOE KO were cultured in serum free media. Media were collected, concentrated, and 30 μg of protein were processed for Western Blot with the indicated Apolipoprotein E antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies ab1907*,13366**,18254-1-AP, MA5-15852* and 66830-1-Ig* which were titrated to the concentrations listed below, as the signals were too weak when following the supplier’s recommendations. Antibody dilutions used: GTX635889* at 1/200, GTX635891* at 1/200, ab52607** at 1/200, ab51015** at 1/1000, ab1907* at 1/200, 13366** at 1/500, ARP54283 at 1/1000, 701241** at 1/200, MA5-41148** at 1/200, MA5-15852* at 1/200, MAB41441* at 1/200, NB110-60531* at 1/200, 18254-1-AP at 1/200, 66830-1-Ig* at 1/200. Apolipoprotein E predicted band size: 36 kDa. *Monoclonal antibody, **Recombinant antibody.

Article Snippet: Bio-Techne , MAB41441 , ZRQ0318021 , AB_2289763 , monoclonal , recombinant fragment , 395004 , rat , 5.0 , Wb.

Techniques: Cell Culture, Western Blot, Staining, Recombinant

Journal: Journal of Lipid Research

Article Title: Hormonal modulators of glial ABCA1 and apoE levels [S]

doi: 10.1194/jlr.M042473

Figure Lengend Snippet: Cholesterol efflux is enhanced by lynestrenol in CCF-STTG1 cells. CCF-STTG1 cells were seeded in 24-well plates and incubated in 3H-cholesterol containing media with cotreatment of DMSO alone (Ctrl), 1 µM of GW3965 (GW), 10 µM of lynestrenol (Lyn), or 10 µM of progesterone (Prog) for 24 h. Cholesterol efflux over 8 h in the absence (NA) or presence of 5 µg/ml of exogenous apoA-I or apoE3 along with the drug treatment was evaluated. Graphs represent means and SD of four independent experiments, each conducted in duplicate. Two-way ANOVA with a Tukey's post-test (comparing rows) was used to determine the drug effect over respective baselines (#P < 0.05, ##P < 0.01, ###P < 0.0001). Another Tukey's post-test (comparing columns) was used to determine the acceptor effect (**P < 0.01, ***P < 0.0001).

Article Snippet: Estrone, 17α-estradiol, 17β-estradiol, estriol, RU486 (Mifepristone), and recombinant human apoE3 were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Incubation

Journal: iScience

Article Title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

doi: 10.1016/j.isci.2024.110006

Figure Lengend Snippet: Microglial APOE4 particles exhibit distinct biochemical features and severer neurotoxicity compared with microglial APOE3 particles (A and B) ApoE particles from conditioned medium of primary APOE3 -TR or APOE4 -TR microglia were analyzed by native gel electrophoresis. Particle sizes were defined as large (>690 kDa), medium (232 kDa–720 kDa), and small (<232 kDa). The percentages of apoE particles in different size categories were quantified. (C) Schematic diagram of microglia-neuron co-culture system. (D) Neurons co-cultured with APOE3 -TR or APOE4 -TR microglia were stained for MAP2. Neuronal culture without microglia in the top insert was used as the control group. Scale bar, 20 μm. (E and F) Neurite lengths (from initiation site) and numbers were quantified and normalized to that of the control. Data are presented as mean ± SEM ( n = 3–5). n represents the number of independent experiments. Statistical significance was determined with t test or one-way ANOVA. ∗, p < 0.05; ∗∗∗, p < 0.001.

Article Snippet: APOE3 and APOE4 targeted replacement mice, which express human apoE isoforms driven by the endogenous murine Apoe promoter, were purchased from Taconic.

Techniques: Nucleic Acid Electrophoresis, Co-Culture Assay, Cell Culture, Staining

Journal: iScience

Article Title: Microglial apolipoprotein E particles contribute to neuronal senescence and synaptotoxicity

doi: 10.1016/j.isci.2024.110006

Figure Lengend Snippet:

Article Snippet: APOE3 and APOE4 targeted replacement mice, which express human apoE isoforms driven by the endogenous murine Apoe promoter, were purchased from Taconic.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Microscopy, Functional Assay, Microarray

Journal: PLoS ONE

Article Title: Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2

doi: 10.1371/journal.pone.0128974

Figure Lengend Snippet: (A-B) Starved VSMCs were incubated with 10% FBS in a culture dish (A) or on fibronectin-coated low or high stiffness hydrogels (B) with or without apoE3 treatment for 9 hr. Rho activity was measured. n = 4 . (C) VSMCs were incubated with 10% FBS in the absence or presence of apoE3 for 24 hr. Scale bar = 50 μm. Fixed cells were incubated with FITC-conjugated phalloidin to stain f-actin. (D) Average phalloidin intensity of single cells was analyzed using ImageJ and normalized to control cells. n = 4 independent experiments with 5–10 cells analyzed per experiment. Data information: Graphs show mean + SEM. * p <0.05 or ** p <0.01.

Article Snippet: Human recombinant apoE3 was dialyzed overnight at 4°C in PBS using a 10,000 MWCO Slide-A-Lyzer Dialysis Cassette from Thermo-Scientific.

Techniques: Incubation, Activity Assay, Staining

Journal: PLoS ONE

Article Title: Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2

doi: 10.1371/journal.pone.0128974

Figure Lengend Snippet: (A) VSMCs infected with adenoviruses encoding LacZ or Rho V14 were incubated in 10% FBS with or without apoE3 for 24 hr. Cells were co-stained with phalloidin and anti-paxillin. Scale bar = 50 μm. The zooms show magnified views of paxillin staining in the boxed areas. The average phalloidin (B) and paxillin (C) intensity of single cells was analyzed using ImageJ and normalized to control cells. n = 3 independent experiments with at least 8 cells analyzed per experiment. Data information: Graphs show mean + SEM. * p <0.05 or ** p <0.01.

Article Snippet: Human recombinant apoE3 was dialyzed overnight at 4°C in PBS using a 10,000 MWCO Slide-A-Lyzer Dialysis Cassette from Thermo-Scientific.

Techniques: Infection, Incubation, Staining

Journal: PLoS ONE

Article Title: Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2

doi: 10.1371/journal.pone.0128974

Figure Lengend Snippet: (A) VSMCs in 10% FBS were treated with apoE3 or Y27632, or their respective vehicle controls (Ctrl), for 24 hr. Intracellular stiffness was determined by AFM. n = 4 . (B) VSMCs were plated on fibronectin-coated low or high stiffness hydrogels in 10% FBS with or without apoE3 for 24 hr, and intracellular stiffness was determined by AFM. n = 4 . (C) VSMCs infected with adenoviruses encoding LacZ or Rho V14 were incubated with 10% FBS in the absence or presence of apoE3 for 24 hr, and intracellular stiffness was measured by AFM. n = 4 . Data information: Graphs show mean + SEM. * p <0.05.

Article Snippet: Human recombinant apoE3 was dialyzed overnight at 4°C in PBS using a 10,000 MWCO Slide-A-Lyzer Dialysis Cassette from Thermo-Scientific.

Techniques: Infection, Incubation

Journal: PLoS ONE

Article Title: Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2

doi: 10.1371/journal.pone.0128974

Figure Lengend Snippet: (A-E) Human VSMCs in 10% FBS were treated with apoE3 (A) or Y27632 for the times shown (C). Total cell lysates were immunoblotted for Cox2 and GAPDH (A and C). The bar graphs show Cox2 levels normalized to control (B and D). n = 4 . In E, Cox2 mRNA was quantified by RT-qPCR and plotted relative to 18S rRNA. n = 4 . (F) VSMCs in 10% FBS were treated with vehicle (DMSO), latrunculin B (LatB), or jasplakinolide (Jasp) for 6 hr. Cox2 mRNA was determined by RT-qPCR; n = 4. Data information: Graphs show mean + SEM. * p <0.05 or *** p <0.001.

Article Snippet: Human recombinant apoE3 was dialyzed overnight at 4°C in PBS using a 10,000 MWCO Slide-A-Lyzer Dialysis Cassette from Thermo-Scientific.

Techniques: Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2

doi: 10.1371/journal.pone.0128974

Figure Lengend Snippet: A working model depicting how apoE3 regulates Rho and intracellular stiffness and how these effects regulate mechanosensitive Cox2 expression.

Article Snippet: Human recombinant apoE3 was dialyzed overnight at 4°C in PBS using a 10,000 MWCO Slide-A-Lyzer Dialysis Cassette from Thermo-Scientific.

Techniques: Expressing

Journal: Journal of Molecular Endocrinology

Article Title: Upregulation of hepatic LRP1 by rosiglitazone: a possible novel mechanism of the beneficial effect of thiazolidinediones on atherogenic dyslipidemia

doi: 10.1530/jme-12-0119

Figure Lengend Snippet: Figure 3 The effect of rosiglitazone on ApoE uptake in HepG2 cells. HepG2 cells were treated with indicated concentrations of rosiglitazone for 48 h. Human recombinant ApoE3 was added to culture media and cells were incubated for 1 h. ApoE3 was reconstituted with lipid using DMPC before the treatment on HepG2 cells. Three independent experiments were performed for the representative figures. (A) Western blot analysis of ApoE3 in HepG2 cells incubated with or without added ApoE3. (B) Western blot analysis of ApoE3 in HepG2 cells incubated with added ApoE3. HepG2 cells were transfected with siRNA targeting human LRP1 (siLRP1) and non-targeting negative siRNA (siCTRL) before the rosiglitazone treatment and adding ApoE3.

Article Snippet: After 48 h, cells were washed once with PBS and then incubated with 25 mg/ml human recombinant ApoE3 (R&D Systems) for 1 h. ApoE3 was reconstituted with lipid using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) before the treatment on HepG2 cells using the method of previous studies (Innerarity et al. 1979).

Techniques: Recombinant, Incubation, Western Blot, Transfection