Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 2. APOE transcripts and protein are upregulated independent from cholesterol levels in SLC25A1 and SLC25A4 mutants. (A) MesoScale electrochemiluminescence solid phase ELISA determinations of human APOE in wild-type (column 1), SLC25A1Δ (columns 2 and 3), and SLC25A4Δ (column 4) HAP1 mutant cell lysates and conditioned media. Two independent SLC25A1Δ clones were tested (columns 2–3). Column 5 depicts complete media not exposed to cells. n=4. (B) APOE immunoblot of cellular extracts from wild-type, SLC25A1Δ, and SLC25A4Δ HAP1 mutant cells. HSP90 was used as a loading control. Lane 4 presents recombinant human APOE (rAPOE). In bold is the predicted molecular weight of rAPOE. (C) MesoScale ELISA measurements of human APOE in wild-type and SLC25A1Δ/Δ SH-SY5Y mutant cell lysates and conditioned media. (D–F) qRT-PCR quantification of APOE, sterol metabolism annotated genes, and housekeeping controls (VAMP2 and RPS20) in wild-type and diverse mutant cell lines. (D) and (E) show transcript levels in SLC25A1Δ and SLC25A4Δ HAP1 mutant cells, respectively. (F) depicts transcript levels in SLC25A1Δ/Δ SH-SY5Y mutant cells. All data are expressed as transcript ratio between mutant and wild-type. n=3 for D-F. (G–I) Volcano plots of positive mode untargeted lipidomics performed in SLC25A1Δ, SLC25A4Δ, and SLC25A1Δ/Δ mutant HAP1 and SH-SY5Y cells and their controls. Upper inserts present the distribution of cholesterol ester and triglyceride species marked by triangles. Depicted are log10 p values and log2 fold of change. n=4 per clone for the two SLC25A1Δ clones, n=4 for SLC25A4Δ, and n=4 for

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Electrochemiluminescence, Enzyme-linked Immunosorbent Assay, Mutagenesis, Clone Assay, Western Blot, Control, Recombinant, Molecular Weight, Quantitative RT-PCR

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 3. The integrity of respiratory chain complex I is required to control APOE expression. (A) Expression of respiratory complex subunits in wild-type and SLC25A1Δ HAP1 cells quantified by TMT mass spectrometry. Kendal Tau hierarchical clustering analysis. (B) Immunoblots with OxPhos antibody mix in mitochondrial fractions from wild-type and SLC25A1Δ cells. (C) Blue native electrophoresis of mitochondrial fractions from wild-type and SLC25A1Δ cells. Shown are Coomassie stained native gel and immunoblots probed with antibodies against complex, I, III, IV, and SLC25A1. (D) Blue native electrophoresis followed by SDS-PAGE then immunoblot with antibodies against complex, I, II, III, and V in mitochondrial fractions from wild-type and SLC25A1Δ cells. (E–G). Seahorse stress test, APOE qRT-PCR, and APOE MesoScale ELISA analysis respectively in wild-type and NDUFS3Δ HAP1 cells. In (F), APOE was measured with two primer sets. (H–J) Seahorse stress test, APOE qRT-PCR, and APOE MesoScale ELISA analysis respectively in wild-type and NDUFAF7Δ HAP1 cells. VAMP2 or RER1 transcripts were used as controls in F and J. All qRT-PCR data are expressed as ratio between mutant and wild-type. (E to M) average ± SEM. One-Way ANOVA followed by Šydák’s multiple correction (F), or unpaired t-test with Welch’s correction (G, I, and J). Arrows in E (n=4) and H (n=3) show sequential addition of oligomycin, FCCP, and rotenone-antimycin during the Seahorse stress test. (K) SLC25A1Δ cells are more sensitive to antimycin than wild-type HAP1 cells. Wild type and SLC25A1Δ cells were exposed to vehicle or increasing concentrations of antimycin. Basal cellular respiration was measured for 90 min after additions (arrow) using Seahorse. Data are presented normalized to basal respiration in the absence of drug. Average ± SEM, n=3, Gray square shows significant differences between wild-type and SLC25A1Δ drug- treated cells as determined by multiple unpaired t-tests followed by corrections with the Benjamini-Krieger-Yekuiteli method (FDR = 5%). (L–M) APOE qRT-PCR and APOE MesoScale ELISA in wild-type and SLC25A1Δ HAP1 cells, respectively, treated with vehicle or antimycin. Twenty nM antimycin was used in qPCR experiments. Twenty to 80 nM was used in MesoScale ELISA experiments. RER1 (columns 1–4) and PCBP1 transcripts (columns 5–8) were used as housekeeping controls. All qRT-PCR data are expressed as ratio between mutant and wild-type. Average ± SEM, One-way ANOVA followed by Benjamini-Krieger-Yekuiteli multiple comparison corrections (FDR = 5%). See available source data for (B and C).

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Control, Expressing, Mass Spectrometry, Western Blot, Electrophoresis, Staining, SDS Page, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Mutagenesis, Comparison

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 4. Robustness and redundancy of adenine nucleotide translocators regulating APOE expression. (A) Volcano plots of TMT proteomic data from wild-type HEK293 cells (n=4), SLC25A4Δ/Δ and triple knock-out SLC25A4,5,6Δ/Δ (B, n=4), depicted are log10 p values and log2 fold of change. (B). Venn diagram of protein hits in SLC25A4Δ/Δ and triple knock-out SLC25A4,5,6Δ/Δ mutants annotated in Mitocarta 3.0 or the Human Secretome (Uhlén et al., 2019). (C) Hierarchical clustering of all proteins annotated to the human secretome across genotypes. (D) MesoScale ELISA determinations of human APOE in wild-type and mutant HEK293 cells. Shown are APOE content in lysates and conditioned media. Mann-Whitney test. (E) Mitocarta 3.0 annotated hits in the triple knock-out SLC25A4,5,6Δ/Δ proteome, see panel B. Red font indicates increased levels in mutant. Hierarchical clustering of all proteins annotated to electron transport chain subunits and SLC25A transporters across genotypes. (F) Blue native electrophoresis followed by immunoblotting with antibodies against complex, I, II, III, and IV in mitochondrial fractions from wild-type and SLC25A4Δ/Δ and triple knock-out SLC25A4,5,6Δ/Δ cells. n-dodecyl-β-d-maltoside (DDM) was used to disrupt supercomplexes. Red arrow and font denote region blotted with SDHA antibodies. (G) Seahorse stress test wild-type and triple knock-out SLC25A4,5,6Δ/Δ cells as in Figure 3. N=3. See available source data for (F).

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Expressing, Knock-Out, Enzyme-linked Immunosorbent Assay, Mutagenesis, MANN-WHITNEY, Electrophoresis, Western Blot

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 5. Direct and indirect disruption of complex IV increases APOE expression. (A) Direct (COX17-20, HIGD1A) and indirect (SLC25A3, SLC31A1, ATP7A) mechanisms required for complex IV assembly. (B) MesoScale ELISA determinations of human APOE in wild-type and HEK293 cell clones null for the genes indicated in blue font. Shown are APOE content in lysates and conditioned media. (C) MesoScale ELISA determinations of human APOE in wild-type and two independent COX17Δ/Δ mutant SH-SY5Y cell clones. (D) MesoScale ELISA determinations of human APOE in wild-type and two independent SLC31A1Δ/Δ mutant SH-SY5Y cell clones were studied. (E) MesoScale ELISA determinations of human APOE in wild-type and two independent ATP7AΔ/Δ mutant SH-SY5Y cell clones transfected with the endocytosis-deficient ATP7A-LL construct. (F) MesoScale ELISA determinations of human APOE in wild-type and two independent ATP7AΔ/Δ mutant SH-SY5Y cell clones. (G) MesoScale ELISA determinations of human APOE in wild-type and SLC25A3Δ mutant HAP1 cells were studied. N=8 for B and n=4 for C-G. (H) NanoString mRNA quantification of human APOE and TBP transcripts in wild-type, and two independent mutant clones of either COX17Δ/Δ or SLC31A1Δ/Δ mutant SH-SY5Y cells. Wild type and SLC31A1Δ/Δ cells were treated with vehicle or 200 micromolar of the copper chelator bathocuproinedisulfonic acid (BCS). TBP was used as a housekeeping control transcript. n=3. (I) Seahorse basal cellular respiration across different genotypes normalized to the corresponding wild-type cell. (J) Correlation between APOE in conditioned media with either basal cellular respiration (OCR) or the extracellular acidification rate determined by Seahorse (ECAR, n=3–9). Simple linear regression fit and 95% confidence interval is shown. All data are presented as average ± SEM. For B to F, and H One-way ANOVA followed by Benjamini-Krieger-Yekuiteli multiple comparison corrections (FDR = 5%). (G) unpaired t-test with Welch’s correction.

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Disruption, Expressing, Enzyme-linked Immunosorbent Assay, Clone Assay, Mutagenesis, Transfection, Construct, Control, Comparison

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 6. APOE expression is increased in human astrocytes after complex III inhibition. (A) MesoScale ELISA determinations of human APOE in wild-type iPSC-derived human neurons and astrocytes treated with vehicle or 40–80 nM antimycin for 48 h. APOE determinations were performed in cell lysates and conditioned media and expressed normalized to a control value. n=4. p was obtained with two-sided estimation statistics. Untreated iPSC-derived astrocytes secrete ~70 x 104 pg/µg lysate. (B–G) Present analyses of changes in mRNA expression measured with NanoString Neuroinflammation panel. (B) Volcano plots of wild-type, SLC25A1Δ, and SLC25A4Δ HAP1 cells (n=3 per genotype) and iPSC-derived astrocytes treated with vehicle or 80 nM antimycin for 48 hr (n=3). Yellow symbols represent upregulated genes in mutant or drug treated cells. (C) mRNA expression of APOE and TBP was expressed as APOE /TADA2B or TBP/TADA2B ratios. TBP and TADA2B are both housekeeping control transcripts One-Way ANOVA followed by Benjamini-Krieger-Yekuiteli multiple comparison corrections (FDR = 5%). (D) Magnitude of compromise of significantly upregulated mRNAs annotated to secreted and mitochondrial proteomes in antimycin-treated astrocytes and compared to all genes present in the Neuroinflammation NanoString panel. p value was calculated with exact hypergeometric probability. (E) Circos plot of shared upregulated hits in SLC25A1Δ, SLC25A4Δ HAP1 cells, and iPSC-derived astrocytes treated with 80 nM antimycin. Outside arc represents the identity of each gene list. Inside arc represents a gene list, where each gene member of that list is assigned a spot on the arc. Dark orange color represents genes that are shared by multiple lists and light orange color represents genes that are unique to that gene list. Shared genes are presented by purple lines and different genes that belong to the same functional ontologies are connected by light blue lines. (F) Metascape ontology analysis and clustering of genes upregulated in SLC25A1Δ and SLC25A4Δ HAP1 cells and iPSC-derived astrocytes treated with 80 nM antimycin. Cumulative hypergeometric p-values.

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Mutagenesis, Comparison, Functional Assay

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 7. Correlative studies of APOE expression and electron transport chain subunits in an Alzheimer’s mouse model and aging humans. (A) Protein expression similarity matrix of APOE, complexes I to V (CI-CV) of the electron transport chain, and transporters of the SLC25A family in wild-type and 5xFAD mouse models. Data were obtained by TMT mass spectrometry from mouse cortices. Similarity was calculated with Spearman Rank correlation. (B) Kendall Tau Hierarchical clustering of complex I subunits and APOE across ages and genotypes. (C) Correlation of composite protein abundance for complexes I, II, and IV in wild-type and 5xFAD mouse cortices with APOE levels. (D) Quantification of human A beta 42 peptide, APP, APOE, three mitochondrial Alzheimer’s risk factors (NDUFS3, NDUFAF7, and COX7C), and the composite protein abundance for complex I to V, as well as members of the SLC25A family of mitochondrial transporters in wild-type and 5xFAD mice (grey and blue symbols, respectively). Two-way ANOVA followed by Šídák’s multiple comparisons tests. Factors are age (A), genotype (G), and their statistical interaction (I). Asterisks denote significant differences between genotypes at a defined age. Red asterisks denote the earliest age with differences between genotypes. See Supplementary file 1 for all Mitocarta hits in the 5xFAD mouse study. (E) The SLC25A1 RNAseq, proteome, and interactome correlate with the cognitive trajectory of human subjects. Cytoplasmic and mitochondrial ribosome subunits were used as controls. Graphs depict the correlation between the cognitive trajectory (Mini-Mental State Examination (MMSE)) in subjects belonging to the Banner collection that were longitudinally followed for an average of 14 years (n=106) (Beach et al., 2015; Wingo et al., 2019). The SLC25A1Δ RNAseq, up and downregulated hits, proteome hits, as well as the SLC25A1 interactome hits principal components were derived by estimating eigenvectors of the expression matrix of protein abundance data. Best-fit regression line drawn in blue and the 99.9% confidence interval for the regression line shaded in gray. No covariate was applied because sex, age at enrollment, and education have been regressed out of cognitive trajectory (Wingo et al., 2019). Blue circles represent males, pink circles represent females (48.1%).

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Expressing, Mass Spectrometry, Quantitative Proteomics, Derivative Assay

Journal: Brain Communications

Article Title: Apolipoprotein B-100-mediated motor neuron degeneration in sporadic amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac207

Figure Lengend Snippet: Apolipoprotein B-100 is upregulated in sALS CSF and downregulated post-filtration. ( A ) CSF protein intensities of apolipoprotein B-100 (ApoB), myelin oligodendrocyte glycoprotein (MOG), haptoglobin, apolipoprotein C-III (ApoC-III), apolipoprotein E (ApoE), and chitotriosidase1 (CHIT1) in patients with sALS ( n = 5) or primary progressive multiple sclerosis (PPMS) ( n = 5), and healthy individuals (HC) ( n = 5), as determined by global proteome profiling using HRM™ MS. ( B ) CSF protein levels in sALS CSF pre- and post-filtration with a 5 kDa MWCO tangential flow hollow-fibre filter. Data plotted as mean ± SEM. Each point represents an individual subject ( A and B ). ** P < 0.01, * P < 0.05.

Article Snippet: Proteins injected included the following: human ApoB purified from plasma (Millipore), human MOG protein 35–55 (Sigma-Aldrich), native human haptoglobin (abcam), human ApoC-III purified from plasma (Athens Research & Technology), recombinant human ApoE (Novus Biologicals) and recombinant human CHIT1 (ABclonal).

Techniques: Filtration

Journal: Brain Communications

Article Title: Apolipoprotein B-100-mediated motor neuron degeneration in sporadic amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac207

Figure Lengend Snippet: Apolipoprotein B-100 induces motor neuron disability and motor neuron death. ( A and B ) Motor deficit scores and normalized forelimb grip strength at 1 DPI of saline ( n = 16 mice) or 1.5 µg/µL and 0.75 µg/uL: ApoB ( n = 12 mice), MOG ( n = 3 mice), haptoglobin ( n = 4 mice), ApoC-III ( n = 7 mice), ApoE ( n = 6 mice), and CHIT1 ( n = 3 mice). ( C ) Representative images of ChAT immunostaining in cervical spinal cords at 1 DPI of saline or 0.75 µg/µL ApoB, MOG, haptoglobin, ApoC-III, ApoE, and CHIT1. Scale bar, 100 µm. ( D ) Quantification of the number of ChAT + motor neurons in cervical ventral horns at 1 DPI of saline ( n = 13 mice) or 1.5 µg/µL and 0.75 µg/µL: ApoB ( n = 11 mice), MOG ( n = 3 mice), haptoglobin ( n = 4 mice), ApoC-III ( n = 5 mice), ApoE ( n = 6 mice), and CHIT1 ( n = 3 mice). ( E ) ChAT immunostaining of human iPSC-derived motor neurons cultured in motor neuron maintenance medium for 8 days, then treated for 24 h with 0.05 µg/µL ApoB or haptoglobin. Scale bar, 100 µm. ( F ) Quantification of the area of ChAT + human motor neuron clusters 24 h following treatment with 0.05 µg/µL ApoB or haptoglobin. ( G ) ELISA measurements of ApoB levels in sALS CSF before and after ApoB immunodepletion. ( H and I ) Motor deficit scores and normalized forelimb grip strength at 1 DPI of saline ( n = 3 mice), sALS CSF ( n = 2; n = 9 mice), ApoB-depleted sALS CSF ( n = 2; n = 9 mice). ( J ) Representative images of ChAT immunostaining in cervical spinal cords at 1 DPI of sALS CSF or ApoB-depleted sALS CSF. Scale bar, 100 µm. ( K ) Quantification of the number of ChAT + motor neurons in cervical ventral horns at 1 DPI. Saline ( n = 3 mice), sALS CSF ( n = 9 mice), ApoB-depleted sALS CSF ( n = 9 mice). ( L ) Representative images of TDP-43 immunostaining in ChAT + motor neurons at 1 DPI of sALS CSF or ApoB-depleted sALS CSF. Scale bar, 25 µm. ( M ) Quantification of the number of ChAT + motor neurons displaying TDP-43 exclusively in the cytoplasm at 1 DPI of saline ( n = 3 mice), sALS CSF ( n = 9 mice) or ApoB-depleted sALS CSF ( n = 9 mice). Data plotted as mean ± SEM. Each point represents an individual mouse ( A , B , D , H , I , K , and M ), individual well ( F ), or CSF sample ( G ). One-way ANOVA with Bonferroni’s test or t -test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: Proteins injected included the following: human ApoB purified from plasma (Millipore), human MOG protein 35–55 (Sigma-Aldrich), native human haptoglobin (abcam), human ApoC-III purified from plasma (Athens Research & Technology), recombinant human ApoE (Novus Biologicals) and recombinant human CHIT1 (ABclonal).

Techniques: Saline, Immunostaining, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunodepletion

Journal: Journal of Cell Science

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1242/jcs.258894

Figure Lengend Snippet: Sortilin deficiency decreases FABP7 levels in primary neurons of apoE3 mice. (A) Immunodetection of FABP7 (red) and sortilin (green) in primary neuronal cultures from apoE3 mice either (WT) or genetically deficient for Sort1 (KO). FAPB7-stained cells are identified as neurons by expression of MAP2 (blue). Merged images show co-expression of FABP7 and sortilin. Images shown are representative of results from three independent experiments. Scale bar: 20 µm. (B) Detection of apoE3 and apoE4 in medium and lysate of neuronal cultures of the indicated genotypes. Detection of recombinant apoE4 served as control (ctr). (C) Western blot analysis of sortilin as well as FABP5 and FABP7 in primary neuronal cultures from apoE3 or apoE4 expressing mice, either wild-type (WT) or genetically deficient for Sort1 (KO). Detection of GAPDH served as loading control. (D) Levels of FABP7 are reduced in primary neurons from E3/KO and E4/WT mice as compared with neurons from E3/WT animals as determined by densitometric scanning of replicate blots (exemplified in panel C). Data are given as mean±s.e.m. with the E3/WT levels set to 100% ( n =8 for E3 groups, n =5 for E4/WT, n =4 for E4/KO). ** P <0.01; *** P <0.001 for expression levels between E3/WT and all other genotypes (unpaired, two-tailed Student's t -test; * P <0.05 for the interaction of APOE and sortilin genotypes (two-way ANOVA with Bonferroni post test). (E) Levels of sortilin are comparable in primary neurons from E3/WT and E4/WT animals as determined by densitometric scanning of replicate blots (exemplified in panel C). Data are given as mean±s.e.m. with the E3/WT levels set to 100% ( n =5–6 biological replicates per genotype). (F) Quantitative RT-PCR analysis of Gfap transcripts in primary neuronal cultures from E3/WT and E3/KO mice. Values are given as log 2 fold changes relative to E3/WT set to value 0 ( n =4 independent cultures per genotype). (G) Levels of Fabp7 transcript were determined in mouse primary neuronal cultures of the indicated genotypes by quantitative RT-PCR ( n =10 for E3/WT, n =7 for E3/KO, n =5 for E4/WT, n =6 for E4/KO). Values are given as log 2 fold change relative to transcript levels in the respective WT (set to value 0).

Article Snippet: The concentration of apoE in media batches from HEK293 or primary astrocytes was determined by western blot analysis comparing apoE signal intensities to those from serial dilutions of recombinant human apoE (MBL, JM-4699-500) included in the same blots.

Techniques: Immunodetection, Staining, Expressing, Recombinant, Control, Western Blot, Two Tailed Test, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Complement deposition on apolipoproteins was assayed using NHS and C1q-depleted human serum (ΔC1q). (A-D) C4b-, C3b-, C1q-, and C9 deposition from NHS, respectively using classical complement deposition conditions (DGVB++ buffer), on coated proteins h.a. IgG (2.5 μg/mL in A, B, D; 1 μg/mL in C), ApoE (10 μg/mL), and negative control A1AT (10 μg/mL). (E) C4b deposition from 2% NHS, 2% ΔC1q, and 2% C1q-reconstituted serum (ΔC1q + C1q, 3.2 μg/ml, based on 160 μg/ml in 100% serum), on coated proteins h.a. IgG (1.0 μg/mL), ApoE (10 μg/mL), and A1AT (10 μg/mL). (F) C1q and C1 binding to immobilized ApoE (10 μg/mL), h.a. IgG (0.5 μg/mL), and A1AT (10 μg/mL), detected using specific antibodies. (G) Fluorescently labeled ApoE (AF488) binding to immobilized C1q (10 μg/mL), and A1AT (10 μg/mL). (H) Fluorescently labeled ApoE (AF488) binding to full C1q protein, and isolated head-, and stalk domains of C1q (all 10 μg/mL). Mean ± SD (A-D, F, G) or mean + SD (E and H) of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA with Dunnett’s multiple-comparisons test (A-D, F), two-way ANOVA with Tukey’s multiple-comparisons test (E), two-way ANOVA with Sidak’s multiple-comparisons test (G), and one-way ANOVA with Dunnett’s multiple-comparisons test (H) were applied to determine statistically significant differences compared to control (A1AT), and per coated protein to compare C4b deposition from NHS, C1q-deficient serum, and C1q-replenished serum (E). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. NHS, normal human serum; h.a. IgG, heat-aggregated IgG; ApoE, recombinant human Apolipoprotein E (ε3 isoform from Novoprotein, AF488-labeled protein in G, 2 μg/mL in H); ApoB, Apolipoprotein B; ApoD, apolipoprotein D; A1AT, alpha-1-antitrypsin; RFU, relative fluorescence units.

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Negative Control, Binding Assay, Labeling, Isolation, Control, Recombinant, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Complement deposition on ApoE was assayed using NHS or Factor B (FB)-depleted serum (ΔFB). (A) C3b deposition using alternative complement deposition conditions (Mg2+-EGTA buffer). (B) C3b deposition from 3% sera: NHS, ΔFB, and FB-reconstituted serum (ΔFB + 6 μg/mL FB, based on 200 μg/mL in 100% serum). Mean ± SD (A) or mean + SD (B) of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA with Tukey’s (A) or Dunnett’s (B) multiple-comparisons test were applied to determine statistically significant differences. **p<0.01, ****p<0.0001. NHS, normal human serum; Zymosan (5 μg/mL); ApoE, recombinant human apolipoprotein E (ε3 isoform from Novoprotein, 10 and 3 μg/mL in A and B resp.); A1AT, alpha-1-antitrypsin (10 μg/mL).

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Complement deposition on ApoE was assayed using NHS. (A-D) C1q-, C4b-, C3b-, and C9 deposition using classical complement deposition conditions (DGVB++ buffer) on immobilized recombinant ApoE and plasma derived ApoE. (E, F) C1q and C4b deposition on different immobilized ApoE isoforms. Positive control h.a. IgG was coated at 1 μg/mL in A and E, and 2.5 μg/mL in B, C, D, and F; ApoE was coated at 10 μg/mL, and A1AT at 10 μg/mL. For each panel, mean ± SD of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA with Tukey’s multiple-comparisons test was applied to determine statistically significant differences between plasma-derived and recombinant ApoE or between ApoE isoforms. No statistically significant differences were found for all panels. NHS, normal human serum; IgG, heat-aggregated IgG; ApoE_R, recombinant human Apolipoprotein E of the ε3 isoform from Novoprotein; ApoE_P, plasma-derived Apolipoprotein E; A1AT, alpha-1-antitrypsin; Apo E2, recombinant human Apolipoprotein E of isoform ε2 from Athens Research; Apo E3, recombinant human Apolipoprotein E of isoform ε3 from Athens Research; Apo E4, recombinant human Apolipoprotein E of isoform ε4 from Athens Research.

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Recombinant, Clinical Proteomics, Derivative Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: We analyzed inhibition of C4b and sC5b-9 deposition on positive control (label ‘C’ on x-axis, coated heat-aggregated IgG, 2.5 μg/mL; A, B), and inhibition of sheep erythrocyte lysis by ApoE (panel C), using recombinant ApoE of different isoforms and plasma-derived ApoE. Mean + SD of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA (A, C), and one-way ANOVA (B) with Dunnett’s post-tests were applied to determine statistically significant differences compared to the uninhibited controls (label ‘C’ in panel A and B, and ‘0 μg/mL’ conditions per protein in panel C). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IgG, heat-aggregated IgG (2.5 μg/mL); ApoE, Apolipoprotein E (10 μg/mL in panel A); A1AT, alpha-1-antitrypsin (10 μg/mL); C, control on coated IgG, with amboceptor and without inhibitors; BSA, bovine serum albumin (10 μg/mL); C1-inh, C1-inhibitor (20 μg/mL); C4BP, C4b-binding protein; PRELP, Proline And Arginine Rich End Leucine Rich Repeat Protein (50 μg/mL).

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Inhibition, Positive Control, Lysis, Recombinant, Clinical Proteomics, Derivative Assay, Control, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging. In addition, ApoE with (F, H) and without (G, I) gold-label were allowed to adsorb to a dark stained surface, followed by adsorption of C1q. All experiments were performed once, and per condition 50 fields were evaluated. Quantification of mean + SD of at least 500 counted particles per field is depicted in panels E and J. Scale bar is 25 nm in panel I. Kruskal-Wallis test with Dunn’s post-test was applied to determine statistically significant differences between conditions, ****p<0.0001. N-terminal ApoE-Au, gold-labeled ApoE bound to the N-terminal stalk region of C1q; C-terminal ApoE-Au, gold-labeled ApoE bound to the C-terminal globular heads of C1q. N-terminal ApoE, non-labeled ApoE bound to the N-terminal stalk region of C1q; C-terminal ApoE, non-labeled ApoE bound to the C-terminal globular heads of C1q.

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Recombinant, Clinical Proteomics, Derivative Assay, Staining, Imaging, Adsorption, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein was incorporated into PC/PE liposomes which were then coupled to streptavidin Dynabeads and complement deposition from NHS was assessed using primary antibodies against C1q (panel A) and C4b (panel B), followed by AF488-coupled secondary antibodies and mean fluorescence intensity was measured with flow cytometry. In addition, we tested the inhibitory capacity of ApoE when incorporated into liposomes, using a hemolytic assay of sheep erythrocytes. Flow cytometry data are normalized to empty liposomes incubated with 5% NHS as a basal value which was set to 100%, and mean + SD of 8 individual experiments are shown. Flow cytometry samples per experiment were analyzed in duplicate and two-way ANOVA with Tukey’s multiple-comparisons test was applied to determine statistically significant differences between empty liposomes and ApoE-carrying liposomes. Hemolytic assay was performed three times, mean + SD are plotted as percentage of full lysis, and one-way ANOVA with Dunnetts’s multiple-comparisons test was applied to determine statistically significant differences compared with normal lysis. Concentrations provided on y-axis labels are in μg/mL. NHS, normal human serum.

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Recombinant, Liposomes, Fluorescence, Flow Cytometry, Hemolytic Assay, Incubation, Lysis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Oxidation, citrullination, and carbamylation were induced on ApoE-coated Maxisorp plates followed by incubation with either protein solutions of C1q (A, B), incubation with NHS to assay C4b deposition in GVB++ buffer (C, D), or incubation with protein solutions of AF488-labeled C4BP (E, F). C1q binding was assayed using primary antibody and HRP-labeled secondary antibody and colorimetric assay, and C4BP binding was assayed using Alexa Fluor 488-prelabeled C4BP and fluorescence measurements. C1q binding, C4b deposition on ApoE_ox and ApoE_cit, and C4BP were normalized to 1 μg/mL IgG (A, B), to 1 μg/mL IgG at 2% NHS (C) or to 10 μg/mL C4met (E, F). Mean + SD (A, B, E, F) or ± SD (C, D) of three individual experiments are shown. Samples per experiment were analyzed in duplicate and one-way ANOVA with Dunnett’s (A, B, E, F) or two-way ANOVA with Sidak’s (C, D) multiple-comparisons test were applied to determine statistically significant differences compared to untreated ApoE. *p<0.05, **p<0.01, ****p<0.0001. IgG, heat-aggregated IgG; ApoE, recombinant human ApoE of the ε3 isoform from Novoprotein; ApoE_ox, oxidized ApoE; ApoE_cit, citrullinated ApoE; ApoE_carb, carbamylated ApoE; A1AT, alpha-1-antitrypsin. C4met, methylamine-treated C4.

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Incubation, Labeling, Binding Assay, Colorimetric Assay, Fluorescence, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Oxidation, citrullination, and carbamylation were induced on ApoE-coated Maxisorp plates, followed by incubation with either NHS in Mg2+-EGTA buffer to assay C3b deposition (A, B), or with protein solutions of FH (C, D). Data per experiment were normalized to the positive controls (Zymosan for A and B; C3b for C and D), and are plotted as a percentage. FH binding was assayed using primary antibody and HRP-labeled secondary antibody and colorimetric assay. Mean + SD of three individual experiments are shown. Samples per experiment were analyzed in duplicate and one-way ANOVA with Dunnett’s multiple-comparisons test were applied to determine statistically significant differences compared to untreated ApoE. *p<0.05, **p<0.01, ****p<0.0001. ApoE, recombinant human ApoE of the ε3 isoform from Novoprotein (1 (A) and 10 μg/mL B-D)). ApoE_ox, oxidized ApoE; ApoE_cit, citrullinated ApoE; ApoE_carb, carbamylated ApoE; A1AT, alpha-1-antitrypsin (10 μg/mL). ApoE_ox, oxidized ApoE coated at 10 μg/mL; ApoE_cit, citrullinated ApoE coated at 10 μg/mL; ApoE_carb, carbamylated ApoE coated at 10 μg/mL; A1AT, alpha-1-antitrypsin (10 μg/mL), Zymosan (5 μg/mL), C3b (10 μg/mL).

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Incubation, Binding Assay, Labeling, Colorimetric Assay, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: A commercial ELISA was used for measuring ApoE (A), and an in-house developed ELISA (B) was used for measuring C4d in the SF of 30 RA patients. Assays were performed once, in single wells, to limit the amount of required patient material. ApoE and C4d concentrations were calculated using standard curves and plotted in ng/mL, with mean ± SD. Using the Spearman R test, no significant correlation was observed between C4d and ApoE in the SF of this cohort (C).

Article Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging.

Techniques: Enzyme-linked Immunosorbent Assay