Journal: Cells

Article Title: Human ApoE2 Endows Stronger Contractility in Rat Cardiomyocytes Enhancing Heart Function

doi: 10.3390/cells12030347

Figure Lengend Snippet: ApoE2 expressing cardiomyocytes exhibited elevated energy production through mitochondrial respiration/fatty acid β-oxidation. Western blotting detected the protein expression levels of rat ApoE (anti-rApoE) and human ApoE (anti-hApoE) in heart lysate of adult rats ( A ), H9C2 cells transfected with different human ApoE isoforms ( B ), and rat neonatal cardiomyocytes transfected with different human ApoE isoforms ( C ). Seahorse XF assays, a method to detect the mitochondrial function, were applied to measure the oxygen consumption rate (OCR) using isolated cardiomyocytes of adult rats at age 7–8 months ( D ), transfected H9C2 cells ( E ), and transfected rat neonatal cardiomyocytes ( F ). Representative Seahorse XF assay traces, basal OCR, ATP production rate, maximal OCR, and proton leak were compared between groups of cells. Seahorse data were normalized with total cellular protein content using BCA assays. Data were summary of 17–45 reads from 4 independent assays for each group. ( G – I ) The expression levels of fatty acid transporting proteins (anti-FAT/CD36) on cell membrane and carnitine palmitoyltransferase 1 (anti-CPT1) on the outer membrane of mitochondria, detected with Western blotting in heart lysate of adult rats and compared between rat groups. One-way ANOVA followed by a Tukey post hoc test. a vs. b, A vs. B, p < 0.05, NS, no significant difference. Four individual rats/experiments were performed for each Seahorse assays. Four to six rats per group were used for Western blotting analysis.

Article Snippet: Human ApoE2, 3, or 4 proteins were expressed in H9C2 cells using the pCMV4-ApoE2, 3, or 4 plasmids (Plasmid # 87085, 87086, 87087, Addgene, Watertown, MA, USA) and Lipofectamine™ 2000 Transfection Reagent (Cat. # 11668019, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Transfection, Isolation, XF Assay

Journal: Cells

Article Title: Human ApoE2 Endows Stronger Contractility in Rat Cardiomyocytes Enhancing Heart Function

doi: 10.3390/cells12030347

Figure Lengend Snippet: The possible mechanism of how ApoE2 is responsible for enhanced heart function. hApoE2 rat shows enhanced left ventricular function in comparison to hApoE3, E4, WT, and ApoE KO rats via echocardiography. Isolated adult hApoE2 cardiomyocytes showed stronger cellular contractility and higher intracellular Ca 2+ concentration after electric stimulation. Two mechanisms might contribute to the potent contractility of hApoE2 cardiomyocytes: sufficient energy supply and potent Ca 2+ -induced Ca 2+ release from the sarcoplasmic reticulum (SR). The former was a result of robust mitochondrial fatty acid β-oxidation and enhanced fatty acid uptake by fatty acid transporting protein CD36. The latter was a consequence of the considerable Ca 2+ release from the sarcoplasmic reticulum through increased ryanodine receptors (RyR) accompanied by the slightly decreased expression of Ca 2+ reuptake protein sarcoplasmic reticulum Ca 2+ ATPase (SERCA). The red-colored objects denote those augmented signals in hApoE2 cardiomyocytes. The green-colored objects denote the suppressed signals. (Created with BioRender.com ).

Article Snippet: Human ApoE2, 3, or 4 proteins were expressed in H9C2 cells using the pCMV4-ApoE2, 3, or 4 plasmids (Plasmid # 87085, 87086, 87087, Addgene, Watertown, MA, USA) and Lipofectamine™ 2000 Transfection Reagent (Cat. # 11668019, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Techniques: Isolation, Concentration Assay, Expressing

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure1. ThelevelofAoligomersinthebrainofAPOE4/4ADpatientswassignificantlyhighercomparedwithAPOE3/3ADpatients.A,Immunoblottingof50gofTBS-solublefractions from4control,5APOE3/3AD,and5APOE4/4ADprefrontalbrains.Ananti-AmAb82E1revealedAmonomers(arrow)anddimers(arrowhead).B,QuantificationofTBS-solubleAfrom 8control(whitesquares),6APOE2/xAD(darksquares),10APOE3/3AD(darktriangles),and10APOE4/4ADbrains(blackcircles).ThelevelofAinAPOE4/4ADbrainswassignificantly highercomparedwithcontrolbrains,APOE2/xADbrains,andAPOE3/3ADbrains.*p0.05,**p0.01,one-wayANOVAtest(Tukey’sposthoctest).C,Amyloidburden(%)intheprefrontal cortex of 8 control (white squares), 6APOE2/x AD (dark squares), 10APOE3/3 AD (dark triangles), and 10APOE 4/4 AD brains (black circles) analyzed in this study. There is no significant difference amongAPOE2/x AD, betweenAPOE3/3 AD andAPOE4/4 AD brains, one-way ANOVA test (Kruskal–Wallis test).D, Correlation analysis between the level TBS-soluble A and the level of A amyloid burden in 6 APOE 2/x AD (dark squares), 10 APOE 3/3 AD (dark triangles), and 10 APOE 4/4 AD brains (black circles). There is no significant difference. E, QuantificationofapoEconcentrationintheTBS-solublefractionof8control(whitesquares),6APOE2/xAD(darksquares),10APOE3/3AD(darktriangles),and10APOE4/4ADbrains(black circles).F,ImmunoblottingofSEC-separatedfractionsfromAPOE4/4ADbrain.Anti-AmAb82E1and6E10revealedA(arrow)andsAPP(arrowhead).Aelutedfrom94kDato217kDaas HMWAandelutedfrom8.6to16kDaasLMWA.Estimatedmolecularweight(kDa)wasindicatedabove(arrowheads).G,Representativedataoftheseparationof200mgofTBS-solublefractions of APOE 3/3 AD (triangles) and APOE 4/4 AD (squares) brains by double Superdex 75 SEC columns. The concentration of A40 is measured by A specific ELISA (BNT77-BA27) (Wako). Estimated molecular weight (kDa) was indicated above (arrowheads). A in TBS-soluble fraction formed dimer, trimer and HMW oligomers.

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Molecular Weight

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure3. PurifiedapoE-containingHDLparticlesenhancedoligomerformationofsyntheticA1–42invitro.A,Immunoblot- tingforAafterincubationof0.1mg/mlsyntheticA1–42withPBS,10gofpurifiedapoE2,10gofapoE3,or10gofapoE4 fortheindicatedtimes(hours).Anti-AmAb6E10revealedAmonomer,dimer,trimer,andtetramer(arrows).B,Bandintensity of remaining A in SDS-polyacrylamide gels after incubation of synthetic A1–42 oligomers with PBS (no), 5 g/ml purified lipidated apoE2, apoE3, or apoE4 for 12 h using an anti-A mAb 6E10. Lipidated apoE4 significantly increased the level of A trimer and tetramer compared with no lipidated apoE samples. N 6, average SD, *p 0.05, one-way ANOVA test (Bonfer- roni’stest).C,Luminescencefromconditionedmediacontainingsplit-luciferase-taggedAoligomersincubatedwith0,0.1,0.3, 0.6, 1.25, 2.5, 5, or 10 g of purified apoE2 (lipid apoE2, squares), purified apoE3 (lipid apoE3, triangles), or purified apoE4 (lipid apoE4, circles) for 24 h. N 6, average SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). D, Incubation of LMW A isolated from TBS-soluble fractions of the AD brains with (apoE) or without (PBS) 5 g of purified lipid apoE3 and separated the samplesbydoubleSuperdex75SECcolumns.TheconcentrationofA40wasmeasuredbyA-specificELISA(BNT77-BA27,WAKO Chemicals) and obtained the ratio of HMW A measured (in fraction 7 and 8). N 4, average SD, *p 0.05, student’ t test.

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Western Blot, Incubation, Purification, Luciferase, Isolation

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure 4. ApoE enhanced the level of A oligomers in an isoform-dependent manner. A, TransienttransfectionofGFP(control),apoA-II,apoE2,apoE3,orapoE4intodouble-expressing HEK293cells.Luminescenceofconditionedmediawasmeasured.apoE3significantlyincreased the luminescence compared with apoE2 and apoE4 significantly increased the luminescence compared with apoE3. N 6, average SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). B, Immunoblotting of conditioned media from GFP (control), apoE2, apoE3, or apoE4 transiently transfected double-expressing HEK293 cells by an anti-apoE mAb 3H1. C, Transient transfectionofapoA-II,apoE2,apoE3,orapoE4intodouble-expressingHEK293cells.Lumines- cenceofcelllysateswasmeasured.Thereisnosignificantdifferenceoftheluminescenceamong apoE2-, apoE3-, or apoE4-expressing cells. N 6, average SD, one-way ANOVA test (Bon- ferroni’s test). D, Transient transfection of apoA-II, apoE3, apoE4, or apoE4 R61T mutant into double-expressing HEK293 cells. Luminescence of conditioned media was measured. apoE4 significantly increased the luminescence compared with apoE4 R61T mutant. N 6, aver- age SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). E, Transient transfection of apoE2, apoE3, or apoE4 into double-expressing HEK293 cells and separation of conditioned media by a SEC column Superdex 200. Representative data of the luminescence profile of the elutants from conditioned media of apoE2 (circles)-, apoE3 (triangles)-, or apoE4 (squares)- transfected cells. Two peaks, HMW oligomers and dimers (arrows) were observed. F, Average ratiobetweenHMWoligomersanddimers.N 3,averageSD,*p0.01,one-wayANOVA test (Bonferroni’s test).

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Control, Expressing, Western Blot, Transfection, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure 6. Lipid-binding domain of apoE was necessary for the enhancement of A oligomers. A, Schematic structure of apoE and apoE fragments. The epitopes of mAb 6C5 and mAb 3H1 is illustrated. B, Transient transfection of GFP, apoE3, apoE2 NTF, apoE3 NTF, apoE4 NTF, apoE CTF, or both apoE3 and apoE3 NTF into double-expressing HEK293 cells. Lumines- cence of conditioned media was measured. apoE3 and apoE CTF significantly increased the luminescence, on the other hand, apoE2 NTF, apoE3 NTF, or apoE4 NTF did not increase the luminescence. N 6, average SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). C, Immunoblotting of conditioned media by the anti-apoE mAb 6C5 (top) and 3H1 (bottom panel). MAb 6C5 revealed 36 kDa band (full-length apoE, arrowhead) and 26 kDa band (apoE NTF, arrow). MAb 3H1 revealed also 36 kDa band (full-length apoE, arrowhead) and 10 kDa doublet band (apoE CTF, arrow). D, Transient transfection of GFP, apoA-II, apoE3, apoE3 NTF, apoE CTF, apoE 231–299, apoE 243–299, apoE 192–272, and apoE 192–242 into double-expressing HEK293 cells. Luminescence of conditioned media was measured. apoE3 significantly increasedtheluminescencecomparedwithapoA-II,apoECTFfragments.apoECTFsignificantlyincreasedtheluminescence compared with apoE 192–272 and apoE 192–272 significantly increased the luminescence compared with apoE 192–242. N 6,averageSD,*p0.05,one-wayANOVAtest(Bonferroni’stest).E,TransienttransfectionofGFP,apoA-II,apoE3, apoE3 NTF, apoE CTF, apoE3 243–272, apoE3 273–299, and apoE3 243–299 into double-expressing HEK293 cells. Luminescence of conditioned media was measured. apoE3 and apoE CTF significantly increased the luminescence com- pared with apoA-II. ApoE3 also significantly increased the luminescence compared with three apoE3 deletion mutants. ApoE3 273–299 significantly increased the luminescence compared with apoE3 243–272 or apoE3 243–299. N 6, average SD, *p 0.01, **p 0.05, one-way ANOVA test (Bonferroni’s test).

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Binding Assay, Transfection, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles *

doi: 10.1074/jbc.M115.669226

Figure Lengend Snippet: Factor H fragment FH5–7 interacted with serum apoE. A, in a pulldown assay, 0.2 mg of purified histidine-tagged FH5–7 protein (input) was first coupled on a HisTrap HP 1-ml column. After washing, the column was eluted with a salt gradient. An assay without FH5–7 coupling was used to control nonspecific binding of serum proteins to the column (Control). The collected fractions were run into SDS-PAGE gels under reducing conditions. An ∼34-kDa band in fractions eluted from the FH5–7 coupled column is seen in the sample but not in the control assay (0.8–1.8 m NaCl). This protein was identified as apoE by mass spectrometry analysis. B, for microscale thermophoresis analysis FH5–7 was incubated with labeled apoE2. The concentration of labeled ligand was 0.0013 μm, and the starting concentration for the unlabeled FH5–7 was 5 μm. The average of two experiments suggested a Kd value of 0.202 μm.

Article Snippet: Microscale Thermophoresis ApoE2 protein was labeled with an N -hydroxysuccinimide-reactive Red dye (catalog no. L001) following the manufacturer's instructions (NanoTemper).

Techniques: Purification, Control, Binding Assay, SDS Page, Mass Spectrometry, Microscale Thermophoresis, Incubation, Labeling, Concentration Assay