pcmv4 apoe2  (Addgene inc)

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: APOE4 decreases Kir4.1 and mitochondrial expression in rMC‐1. (a) Schematic showing the generation of rMC‐1 expressing human APOE isoforms. rMC‐1 was transiently transfected with human APOE2 / APOE3 / APOE4 , and EV was used as a control. (b) mRNA expression of Kcnj10 gene for Kir4.1 normalized to a housekeeping gene β‐actin. (c) Representative western blots of Kir4.1 expression and (d) quantification of integrated optical density (IOD) ratio of Kir4.1 and α‐tubulin showing decreased protein expression of Kir4.1 in APOE4 ‐transfected rMC‐1. (e) Representative images of rMC‐1 transfected with human APOE2 / APOE3 / APOE4 /EV showing decreased TOMM20 staining pattern in APOE4 ‐transfected rMC‐1, scale: 20 μm ( n : 3 independent experiments). (f) Quantification of TOMM20 staining intensity per cell area ( n : 15–24 cells/condition). (g) mRNA expression of Mfn1 , Mfn2 , and Dnm1 , showing that APOE4 ‐transfected rMC‐1 reduced Mfn1 , Mfn2 , and Dnm1 gene expression as compared to EV/ APOE2 / APOE3 ‐transfected rMC‐1 ( n : 4 independent experiments). Values are expressed as mean ± SEM. One‐way ANOVA followed by Tukey's multiple comparison test was used for statistical analysis. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The cells were cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

Techniques: Expressing, Transfection, Control, Western Blot, Staining, Gene Expression, Comparison

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: APOE4 impairs mitochondrial respiration and reduces metabolic flexibility in rMC‐1. (a) OCR traces in rMC‐1 expressing EV/ APOE2 / APOE3 / APOE4 in response to sequential addition of oligomycin (oligo), FCCP, and rotenone/antimycin A (Rot/AA). APOE4 expressing rMC‐1 showed consistently lower OCR across conditions. (b) Quantification of basal respiration, maximal respiration, and non‐mitochondrial respiration, with APOE4 expressing rMC‐1 showing significantly reduced maximal and non‐mitochondrial respiration. (c) Quantification of spare respiratory capacity, ATP‐linked respiration, and proton leak. APOE4 ‐expressing rMC‐1 exhibited a marked reduction in spare respiratory capacity, while ATP‐linked respiration showed a downward trend. (d) ECAR profile in rMC‐1 expressing EV/ APOE2 / APOE3 / APOE4 in response to oligomycin (oligo), FCCP, and rotenone/antimycin A (Rot/AA) shows comparable basal rates across groups. (e) Quantification of glycolytic reserve, basal, and maximal ECAR. APOE4 rMC‐1 displayed a significantly reduced glycolytic reserve compared to EV, APOE2 , and APOE3 ‐transfected rMC‐1. (f) Quantification of glycolytic capacity and non‐glycolytic ECAR showing no significant changes across groups. (g) PPR traces in rMC‐1 expressing EV/ APOE2 / APOE3 / APOE4 in response to oligomycin (oligo), FCCP, and rotenone/antimycin A (Rot/AA) show overall comparable levels across groups. (h) Quantification of basal and maximal PPR confirms no significant APOE isoform differences. (i) Quantification of glycolytic PPR and non‐glycolytic PPR also showing no significant differences across groups ( n : 3 independent experiments, with 3–4 technical replicates per condition). Values are expressed as mean ± SEM. One‐way ANOVA with Tukey's test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

Techniques: Expressing, Transfection

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: MitoQ restores Kir4.1 gene and protein expression in rMC‐1 transfected with APOE4 . (a) mRNA expression of Kcnj10 gene for Kir4.1 normalized to housekeeping gene for β‐actin after treating rMC‐1 with 1 μM MitoQ and vehicle. mRNA expression of Kir4.1 was significantly increased in APOE4 ‐transfected rMC‐1 upon treatment with 1 μM MitoQ compared to the vehicle. (b) Representative western blots of Kir4.1 expression and quantification of IOD ratio of Kir4.1 and α‐tubulin showing comparable protein expression of Kir4.1 in APOE4 ‐transfected rMC‐1 as compared to EV/ APOE2 /APOE3‐transfected rMC‐1 after treating with 1 μM MitoQ. Values are expressed as mean ± SEM. Two‐way ANOVA followed by Tukey's multiple comparison test was used for statistical analysis. * p < 0.05, ** p < 0.01. ( n : 3–4 independent experiments).

Article Snippet: The cells were cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

Techniques: Expressing, Transfection, Western Blot, Comparison

Journal: Glia

Article Title: Müller Glial Kir4.1 Channel Dysfunction in APOE4 ‐ KI Model of Alzheimer's Disease

doi: 10.1002/glia.70119

Figure Lengend Snippet: MitoQ decreases mitochondrial ROS in APOE4 ‐transfected rMC‐1. Representative images of unstained rMC‐1 and rMC‐1 transfected with EV/ APOE2 / APOE3 / APOE4 and treated with (a) vehicle or (b) MitoQ (1 μM). Cells were analyzed on a flow cytometer with 610/20 nm bandpass emission filter. (c) Bar graph showing quantification of % of MitoSox Red positive cells. Mitochondrial reactive oxygen species (ROS) was decreased upon treating APOE4 ‐transfected rMC‐1 with 1 μM MitoQ. Values are expressed as mean ± SEM ( n : 3 independent experiments). One‐way ANOVA followed by Tukey's multiple comparison test was used for statistical analysis. * p < 0.05, ** p < 0.01.

Article Snippet: The cells were cultured in low glucose, no phenol red, DMEM (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS, 1% L‐glutamine (Corning, VA, USA), and 1% antibiotic‐antimycotic (Thermo Fisher Scientific, MA, USA). rMC‐1 was grown in DMEM overnight and transfected with 1 μg of plasmids encoding human APOE isoforms: pCMV4‐ APOE2 (Cat. #87085, addgene, MA, USA), pCMV4‐ APOE3 (Cat. #87086, addgene), and pCMV4‐ APOE4 (Cat. #87087, addgene).

Techniques: Transfection, Flow Cytometry, Comparison

Journal: International journal of molecular sciences

Article Title: Neuronal ApoE Regulates the Cell-to-Cell Transmission of α-Synuclein.

doi: 10.3390/ijms23158311

Figure Lengend Snippet: Figure 1. ApoE deficiency inhibits α-syn uptake into neurons. (A) ApoE expression in SH-SY5Y and A53T-α-syn OE SH-SY5Y cells was analyzed by RT-PCR and Western blot. Values were derived from three independent experiments (n = 3). (B) After treatment with 1 µM α-syn monomers and fibrils for 24 h, the cells were analyzed by RT-PCR and Western blot. Values were derived from three independent experiments (n = 3). (C, D) NT, ApoE KD #1 and #2 SH-SY5Y cells (C) and primary neurons (D) were co-cultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 24 h using a dual chamber. The cells were then fixed and immunostained with anti-EGFP or anti-Tuj-1 antibodies. Values were derived from three independent experiments (n = 3). (E) SH-SY5Y cells were transiently transfected with plasmids of ApoE isoforms (ApoE2, ApoE3, or ApoE4). The cells were co-cultured with differentiated A53T-α-syn-EGFP OE SH-SY5Y cells using a dual chamber. The cells were then fixed and immunostained with anti-EGFP and anti-ApoE antibodies. Values were derived from three independent experiments (n = 3). (F) NT, ApoE KD #1, and #2 SH-SY5Y cells were incubated with 50 nM BOIPY® FL C5-Lactosylceramide and 2.5 µg/mL rhodamine-conjugated transferrin for 60 min. The intensity was analyzed with ImageJ and the LASX program. Values were derived from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 against control; one-way ANOVA or unpaired t-tests. Blue indicates DAPI staining. Scale bars indicate 20 µm.

Article Snippet: Plasmids for ApoE2, ApoE3, and ApoE4 were provided by Bradley Hyman (Addgene plasmids #87085, #87086, and #87087, respectively) [83].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Cell Culture, Transfection, Incubation, Control, Staining

Journal: International journal of molecular sciences

Article Title: Neuronal ApoE Regulates the Cell-to-Cell Transmission of α-Synuclein.

doi: 10.3390/ijms23158311

Figure Lengend Snippet: Figure 5. ApoE deficiency inhibits α-syn release in SH-SY5Y cells. (A) Differentiated NT and ApoE KD #1, #2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured in serum-free media for 1 day, and culture media were collected. The cells were lysed with RIPA buffer and culture media were 50-fold concentrated. The samples were then analyzed by Western blot. Values were derived from three independent experiments (n = 3). (B) The α-syn in culture media was analyzed by ELISA (n = 3). (C) Culture media were analyzed by α-syn RT-QUIC. Values were derived from triplicate and a representative of three independent experiments (n = 3). (D) A53T α-syn-EGFP OE SH-SY5Y cells were transfected with plasmids for ApoE isoforms (ApoE2, ApoE3, and ApoE4) and differentiated by 50 µM RA for 5 days. The cells were then cultured in serum-free media for 1 day and culture media were collected. Cells were lysed with RIPA buffer and culture media were 50-fold concentrated. The samples were then analyzed by Western blot. Values were derived from three independent experiments (n = 3). (E) Culture media were analyzed by α-syn RT-QUIC. Values were derived from triplicate and a representative of three independent experiments (n = 3). (F) SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, ApoE KD #1, or #2/A53T α-syn-EGFP OE SH-SY5Y cells for 24 h. The cells in the lower chamber were immunostained with an anti-EGFP antibody. Values were derived from three independent experiments (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 against the control; one-way ANOVA. Blue indicates DAPI staining. Scale bar indicates 20 µm.

Article Snippet: Plasmids for ApoE2, ApoE3, and ApoE4 were provided by Bradley Hyman (Addgene plasmids #87085, #87086, and #87087, respectively) [83].

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