Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Ascites in peritoneal carcinomatosis (PC) is biologically active (A) Kaplan-Meier curve showing survival of PC patients who underwent cytoreductive surgery (n = 121), stratified by the presence or absence of ascites. (B and C) Proliferation curves of (B) Colo-205 and (C) SNU-C1 cells treated with varying concentrations of ascites, measured by CellTitre-Glo assay. Data are represented as cell viability at day 5 relative to day 0. Dotted line represents cell viability of Colo-205 treated with 10% fetal bovine serum (FBS) at day 5 relative to day 0. Graph shows mean ± SD. (D and E) Migration of (D) Colo-205 and (E) SNU-C1 cells pre-treated with serum-free media (SFM), 10% FBS medium or 5% cell-free ascites (CFA) medium, assessed by transwell migration assay. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with SFM and 10% FBS or 5% CFA was denoted by ∗. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant difference detected via unpaired two-sided t test between migration of cells pre-treated with 10% FBS and SFM or 5% CFA was denoted by # . # p < 0.05, ## p < 0.01, ### p < 0.001. Graph shows mean ± SD. (F) Enriched signaling pathways in colorectal PC cell lines upon treatment with CFA, identified by comparing gene expressions of cells treated with 5% versus 0.1% CFA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G and H) Treatment of (G) Colo-205 and (H) SNU-C1 cells with 5% CFA activated STAT3 signaling pathway through phosphorylation at Tyr705.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Glo Assay, Migration, Transwell Migration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Identification and validation of prognostic putative activators of STAT3 signaling in ascites (A) Workflow to identify clinically significant putative secreted STAT3 activators. (B) Kaplan-Meier survival curve illustrating poorer survival in patients expressing three biomarkers as compared to patients expressing 0–2 biomarkers. (C) Characterization of cytokine profiles in colorectal PC ascites and benign ascites. Top 25% most highly abundant cytokines in colorectal PC ascites (by mean abundance) are shown. Heatmap displays Z score of normalized mean pixel density of duplicate cytokine spots on the array. (D) Downregulated signaling pathways upon PAI-1 inhibition in Colo-205 cells exposed to CFA with high PAI-1 levels (PC085), CFA with low PAI-1 levels (PC249) and no PAI-1 (FBS control), identified using RNA microarray. Only signaling pathways with differential downregulation are presented. IL6-JAK-STAT3 signaling pathway was significantly downregulated in high PAI-1 CFA-treated cells upon PAI-1 inhibition. Normalized enrichment scores <0 indicate pathway suppression and scores >0 indicate pathway activation. (D) is representative of two independent biological experiments. ∗p < 0.05.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Expressing, Inhibition, Microarray, Activation Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Correlating PAI-1 level in ascites and intracellular STAT3 activation in cancer cells revealed distinct subgroups associated with differing susceptibility to PAI-1 inhibition (A) PAI-1 prevalence in colorectal PC ascites (n = 54). (B) Correlation between colorectal PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.4691, p = 0.0003). (C) PAI-1 prevalence in ascites of various histological PC subtypes (n = 156). † indicates benign ascites. (D) Correlation between various histological PC ascitic PAI-1 concentrations and ascites-treated Colo-205 cells p-STAT3(Y705) was determined by Pearson correlation coefficient test ( r = 0.6476, p < 0.0001). (A–D) PAI-1 concentrations are plotted on a log 2 scale to transform skewed data to normal distribution. p-STAT3(Y705) level was shown as optical density reading at 450 nm (OD450). (E) Untransformed values of PAI-1 and p-STAT3(Y705) levels from (D) were used for stratification strategy to identify patient subpopulations who might benefit from PAI-1 inhibition. Using 20 ng/mL PAI-1 level and 0.2 OD450 p-STAT3(Y705) level as cut-off values, three distinct subgroups of samples were observed: (i) high PAI-1 and high p-STAT3 levels, termed PAI-1 paracrine addicted (PPA) group (yellow region), (ii) low PAI-1 and high p-STAT3 levels, termed co-activators predominant (CAP) group (pink region) and (iii) low PAI-1 and low p-STAT3 levels, termed alternative pathways activation (APA) group (blue region). Each dot represents one patient ascites. Colors in each panel represent the various histological PC subtypes. All histological subtypes with less than five samples are grouped into other histological subtypes.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Activation Assay, Inhibition

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet: Cells dependent on PAI-1 to activate STAT3 are most susceptible to PAI-1 inhibition (A) Effect of TM5441 (PAI-1 inhibitor) on CFA-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow), CAP group (pink), APA group (blue) and FBS (control, black) demonstrated a left shift, indicating responsiveness to PAI-1 inhibition. (B) Differential sensitivity to TM5441 corresponding to the three subgroups, with PPA (n = 18) being the most sensitive to PAI-1 inhibition, followed by CAP (n = 59) and APA (n = 17). Graph shows mean ± SD. (C–E) Effect of (C) Napabucasin (STAT3 inhibitor), (D) BEZ235 (dual PI3K/mTOR inhibitor) and (E) Mitomycin C (conventional chemotherapeutic agent – DNA crosslinker) on the three subgroups of ascites-treated Colo-205 cells. Representative inhibitor dose-response curves of PPA group (yellow; n = 3), CAP group (pink; n = 3), APA group (blue; n = 1) and FBS (control, black). Targeting PAI-1, a dominant paracrine factor in ascites, was more effective than targeting downstream signaling pathway activated by ascites, proliferation pathway or DNA synthesis. (F) Evaluation of STAT3 suppression in Colo-205 cells treated with PPA CFA (PC085 and PC383), CAP CFA (PC249), APA CFA (PC010) and various concentrations of TM5441 by ELISA. STAT3 activation was shown as p-STAT3(Y705) and total STAT3 ratio at the indicated concentration relative to DMSO vehicle. Cells exposed to PPA CFA relied on PAI-1 to activate STAT3 as they required lower concentrations of TM5441 to suppress STAT3 activation. Graph shows mean ± SEM. Data in (A-E) are representative of at least three independent biological experiments and (F) is representative of two independent biological experiments. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Inhibition, DNA Synthesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Concentration Assay

Journal: Cell Reports Medicine

Article Title: Ligand-mediated PAI-1 inhibition in a mouse model of peritoneal carcinomatosis

doi: 10.1016/j.xcrm.2022.100526

Figure Lengend Snippet:

Article Snippet: Total STAT3 ELISA , Cell Signalling Technology , 7305C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Sequencing, Microarray, Cell Culture, Software

Journal: Cell

Article Title: Loss of IFN- γ pathway genes in tumor cells as a mechanism of resistance to anti-CTLA-4 therapy

doi: 10.1016/j.cell.2016.08.069

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Microarray gene expression analysis Pre- and post-treatment samples were collected for total RNA isolation using the Qiagen RNeasy Mini Kit.

Techniques: Recombinant, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Proliferation Assay, Microarray, Expressing, shRNA, Sequencing, Software

Journal: Nature medicine

Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

doi: 10.1038/s41591-018-0336-8

Figure Lengend Snippet: ( a ) ChPs were stained for complement C3 (red), anaphylatoxin C3a (cyan), and Ig (green). Bar 100 μm. ( b ) Complement C5. ChPs were stained for complement C5 (red). Bar 10 μm. ( c ) Liver-targeted C5-siRNA reduces serum C5. Control (n = 9 mice), C5 (n=9). ( d-f ) C5-siRNA attenuates leukocyte infiltration ( d ), CD68 + macrophage/DC infiltration ( e ), and CD3 + T cell infiltration ( f ) in ApoE -/- ChPs. Bars 10 μm. Control (n = 6 mice), C5 (6). ( g ) Low C4 and C3 protein levels in lipid deposits of HFD ApoE4 ChPs. Serial sections of ChPs as shown in were stained for C4 (green) and C3 (red). ( h ) Super-resolution (STED) microscopy shows colocalization of C1q (green) and ApoE (red) in HFD ApoE4 ChPs. Bar 10 μm. ( i ) ChP complement mRNA expression. WT (n=4 mice); ApoE -/- (n=5); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). Data in a,b,g,h are representative images from at least 3 independent mouse samples. Data represent means± SEM; Two-tailed Student´s t-test was applied to c,d,e,f. *** P <0.0001; one-way ANOVA with Tukey’s test was applied to i. Gene names in .

Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

Techniques: Staining, Microscopy, Expressing, Two Tailed Test

Journal: Nature medicine

Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

doi: 10.1038/s41591-018-0336-8

Figure Lengend Snippet: ( a ) ChPs were stained for C1q (red) and C4 (green). Bar 100 µm. ( b ) C5 siRNA treatment blocks C5 protein deposition in ApoE-/- ChPs; ( c ) ChPs were stained for C3. Ig represents lipid; ( d ) Serum C3 and C5. Serum C3 and C5 protein levels were measured by ELISA. ApoE-/-(n = 6 mice), HFD ApoE4 (n=5). ( e ) High resolution confocal microscopy shows colocalization of ApoE4 (ApoE, red) and Ig (green, represents lipid) in HFD ApoE4-KI ChPs. ApoE-/- ChPs serve as negative controls for ApoE staining; ( f) Complement regulators are expressed in ChPs. WT (n = 5 mice); ApoE-/-(n=4); ND ApoE3 (n=6); HFD ApoE3 (n=6); ND ApoE4 (n=6); HFD ApoE4 (n=6). ( g ) ChP Factor H expressed between WT and ApoE-/- mice. WT (n=5); ApoE-/-(n=4); (h) ChP factor H protein in ChPs. White arrows indicate lipid positive areas. Data in a,b,c,e,h are representative images from at least 3 biologically independent mouse samples. Data in d,f,g represent means ± SEM; Two-tailed Student's t-test was applied to d,g; one-way ANOVA with Tukey posttest was applied to f; Gene names in .

Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Two Tailed Test

Journal: Nature medicine

Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

doi: 10.1038/s41591-018-0336-8

Figure Lengend Snippet: ( a ) Human ChP sections were stained with ORO/HE. Bar 100 μm. ChPs lipid was quantified as described in . Non-dementia cases (n=13) and demented cases (n=17). ( b ) Pearson correlation of ChP lipid and neurofibrillary tangle stage (Braak & Braak). n=30. ( c-e ) ChP lipid correlate with Aβ score (Thal phase), neuritic plaque score (CERAD), and ApoE4 genotype. n = 30 biologically independent samples, ( f ) ChP lipid correlates with dementia in ApoE3/ApoE3 carriers. ApoE3/ApoE3 Non-dementia cases (n=10) and demented cases (n=7). ( g ) Human ChP sections were stained for C1q (green) and C5 (red). Bar 100 μm. C5 percentage of lipid- ChP and lipid+ ChP from the same case was quantified according the Methods. Lipid- (n=7 biologically independent samples), lipid+ (n=7). ( h ) STED microscopy shows colocalization of C1q (green) and ApoE (red). Bar 5 μm. ( i ) Binding of C1q-ApoE in vivo by PLA. Anti-ApoE, anti-C1q, or no primary antibodies were used as controls. The number of C1q-ApoE complexes of lipid-negative ChP or lipid-positive areas were quantified as described in . Lipid- (n=4 independent samples), lipid+ (n=4). Bar 5 μm. ( j ) Human brain sections were stained for Aβ/ApoE, pTau/ApoE, C1q/ApoE, or C1q alone. Protein-protein binding in vivo was detected by PLA. Blue for nuclei. Bar 5 μm. ( k ) 16 weeks AD (APPPS1-21) brain cortex sections were examined by the PLA assay for the presence of C1q/ApoE complexes, methoxy X04 to outline plaques. X04-(5), X04+ (5). Bars represent 10 μm. ( l ) Liver-targeted C5 siRNA reduces serum C5 in APPPS1-21 mice. Ctr (n=4 mice), C5 (n=5). ( m ) Brain sections were stained with iba1 for microglial cells (red), To-Pro-3 for nuclei, and X04 for Aβ plaque. White dashed circle represents the area within a 30 μm radius. The number of iba1 + /To-Pro-3 + cells per areawere quantified (> 30 μm radius represents non-Aβ plaque area). Plaques were further grouped into small plaques (X04% < 10% of 30 μm radius area), moderate plaques (X04% between 10 - 30% 30 μm radius area), and large plaques (X04% > 30% of 30 μm radius area). Percentage of iba1 positivity within a 30 μm radius of Aβ plaques and non-Aβ plaque areas were compared. 420 individual Aβ plaques and 40 fields of non-Aβ plaques from 4 control mice, 536 individual Aβ plaques and 51 fields of non-Aβ plaques from 5 C5 treated mice. Data in h,j are representative images from at least 3 independent samples. Data represent means ± SEM. Two-tailed Student´s t-test was applied to a,c,d,f; paired two-tailed Student´s t-test was applied to g,I,k; One-way ANOVA was applied to e; Two-way ANOVA was applied to l,m.

Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

Techniques: Staining, Microscopy, Binding Assay, In Vivo, Protein Binding, Two Tailed Test

Journal: Nature medicine

Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

doi: 10.1038/s41591-018-0336-8

Figure Lengend Snippet: ( a )16 weeks APPPS1-21 mouse brain sections were stained with Aβ/ApoE complexes (red) by PLA, methoxy X04 for Aβ plaque (blue). High resolution confocal images show the spatial location of Aβ-ApoE complexes and Aβ plaque in 3D view (lower panel). Bars represent 10 µm. ( b ) Brain sections were stained with methoxy-X04, ApoE, and LAMP1; the size of areas covered by methoxy-X04, ApoE, and LAMP1 was determined. ApoE/X04 and LAMP1/X04 (X04 > 150 µm2) were quantified. n = 123 plaques from 4 control mice, 147 plaques from 5 treated mice. Bars 100 µm. ( c ) Aβ plaque was stained with methoxy X04 (X04). Number of plaques per section and number of plaque per area were quantified. control (n=4 mice), C5 (n=5). Bar 1000 µm; ( d ) Total plaque volume was determined in 3D, plaques were further grouped according to the plaque volume. n = 71 random fields from 4 control mice, 88 fields from 5 C5 treated mice. Bar 100 µm; ( e ) 8-week old C57BL6 brain cortex sections were examined for the presence of C1q-ApoE complexes with methoxy X04. ApoE, or C1q only antisera were used as negative controls. Bar represents 10 µm. Data in a,e are representative images from at least 3 biologically independent mouse samples. Data in b,c,d represent means ± SEM; two-tailed Student's t-test was applied to b,c,d; Two-way ANOVA was applied to c,d.

Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

Techniques: Staining, Two Tailed Test

Journal: Nature medicine

Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

doi: 10.1038/s41591-018-0336-8

Figure Lengend Snippet: ( a ) Aorta complement gene mRNA expression. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE -/- (n=3); 32 weeks ApoE -/- (n=3); ( b ) Liver-targeted C5 siRNA reduces serum C5 in young ApoE -/- mice. Ctr (n=11 mice), C5 (12). ( c ) En face staining for whole aorta. Bar 0.5 cm. Atherosclerotic plaques were quantified as described in . Control (n=11 mice), C5 siRNA (n=12). ( d , e ) Aortic root sections were stained for ORO/HE and CD68 + macrophages/DCs. Bars 100 μm. Plaque size ( d ) and CD68 + macrophages/DCs size ( e ) were quantified as described in . Control (n=4 mice), C5-siRNA (n=4). ( f ) Human carotid artery parallel sections were stained for CD68, C1q, ApoE, and C5 by DAB and hematoxylin. Representative images from g. ( g ) CD68, C1q, ApoE, and C5 signal was quantify as described in . Control (n=5 independent samples), early plaque (n=6), advanced plaque (n=9). ( h ) C1q-ApoE complexes in human atherosclerosis plaque was determined by PLA. Intima (n=3 independent samples), media (3). Bar 5 μm. ( i ) High resolution microscopy shows colocalization of lipid (green) and malondialdehyde epitopes (MDA2, red) in human atherosclerotic plaque. Bar 10 μm. Representative images from at least 3 independent samples. ( j ) Schematic representation of the C1q-ApoE complex. Locally produced and/or serum-recruited C1q is activated in situ by a variety of surface activators including oxidized lipid, oxidized LDL, amyloid fibrils, and immunoglobulins. C1q activators have been implicated in diseases as varied as atherosclerosis and AD. Following activation, C1q acquires an active conformation that allows initiation of the CCC with resultant generation of C3a and C3b and C5 cleavage to generate C5a and C5b. ApoE inhibits the CCC activity by binding of ApoE at high affinity to the active C1q and forms the C1q-ApoE complex (upper part of the panel). By contrast, inflammation is amplified in the absence of ApoE by overactivation of the CCC (lower panel). C1q: inactive (yellow); activated (light green); overactivated (red). Data represent means ± SEM. Two-tailed Student´s t-test was applied to b,c,h; One-way ANOVA was applied to a,g; Two-way ANOVA was applied to d,e.

Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

Techniques: Expressing, Staining, Microscopy, Produced, In Situ, Activation Assay, Activity Assay, Binding Assay, Amplification, Two Tailed Test

Journal: Nature medicine

Article Title: ApoE attenuates unresolvable inflammation by complex formation with activated C1q

doi: 10.1038/s41591-018-0336-8

Figure Lengend Snippet: ( a ) Expression microarray analyses of aortas. Heatmaps show GO terms leukocyte migration, complement activation, phagocytosis, and cellular response to lipid. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( b ) aorta alternative complement pathway genes (factor B, factor H, factor D) mRNA expression in 6 weeks and 32 weeks old WT and ApoE-/- mouse aortas. 6 weeks WT (n=3 mice); 32 weeks WT (n=3); 6 weeks ApoE-/- (n=3); 32 weeks ApoE-/- (n=3); ( c - d ) plasma cholesterol and body weight; ( e - f ) blood leukocytes and percentage. For c-f, control (11 mice); C5 siRNA (12 mice). ( g ) blood CD4+ T cells, CD8+ T cells, and B220+ B cells by flow cytometry. Control (6 mice); C5 siRNA (6 mice). ( h ) super-resolution microscopy shows colocalization of C1q (green) and ApoE (red) in human atherosclerotic plaque. Representative images from at least 3 biologically independent mouse samples. Bar 5 µm. Data in b,c,d,e,f,g represent means ± SEM; Two-tailed Student's t-test was applied to c.d.e.f.g; one-way ANOVA with Tukey posttest was applied to b; abbreviations: WBC, white blood cells; RBC, red blood cells; PLT, platelets; LYM, lymphocytes; MO, monocytes; GRA, granulocytes. Gene names and statistics in .

Article Snippet: Immunofluorescence staining was performed as previously described , using marker antibodies anti-mouse collagen IV (2150-1470; AbDSerotec), immunoglobulins (715-166-151; Dianova), immunoglobulin isotype that do not react with mouse Ig (017-160-006; Dianova), anti-human/mouse C3 (A213, ComplementTech), anti-human/mouse C3a (A218, ComplementTech), anti-human C5 (A220, ComplementTech), anti-mouse C5 (ab11898, Abcam), isotype for C5 (ab27478, Abcam), anti-mouse C1q (HM1096BT, Hycult Biotech), anti-human C1q (ab71089, Abcam), anti-mouse C4 (HM1046, Hycult Biotech), anti-human Factor H (A312, Quidel), anti-mouse Factor H (HM1119F, Hycult Biotech), anti-human ApoE (ab52607, Abcam), anti-human ApoE (178479; Calbiochem), anti-mouse ApoE (ab183597, Abcam), anti-mouse CD68 (FA11; Serotec), anti-human CD68 (EMB11, DAKO), anti-mouse CD31 (553370; BD PharMingen), anti-mouse iba-1 (019-19741; WAKO), anti-mouse CD45 (BZL 01145; Biozol), anti-human/mouse cytokeratin (Z0622; DAKO), anti-human collagen IV (CIV22, DAKO), anti-beta-amyloid (4G8, Biolegend), anti-phospho-Tau (AT8, Thermofisher), anti-iba1 (polyclonal, WAKO), anti-LAMP1 (1D4B, Abcam), TO-PRO™-3 Iodide (642/661) (T3605, Thermo Fisher Scientific) or DAPI for DNA.

Techniques: Expressing, Microarray, Migration, Activation Assay, Flow Cytometry, Microscopy, Two Tailed Test