Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Overexpression of ANGPTL3 inhibits IL-1β-induced NF-κB activation. ( A and B ) Overexpression of ANGPTL3 inhibits IL-1β-induced NF-κB activation in a dose-dependent manner as determined by the reporter assay in HEK293T (upper panel in A ) and Hep3B (upper panel in B ) cells. The expression levels of ANGPTL3-Flag in the indicated cell lysate were detected by western blotting (lower panels). ( C – E ) The effects of overexpressed ANGPTL3 on IL-1β-induced transcription of TNFA, CXCL2 , and IL8 were determined by qPCR in HEK293T ( C ), Hep3B ( D ), and primary hepatocytes ( E ). AN3, ANGPTL3. ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Over Expression, Activation Assay, Reporter Assay, Expressing, Western Blot

Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Knockdown of ANGPTL3 potentiates IL-1β-induced NF-κB activation. ( A ) ANGPTL3 shRNA plasmids significantly reduced the protein levels of both transfected Flag-tagged ANGPTL3 (upper panel, in HEK293T cells) and endogenous ANGPTL3 (lower panel, in Hep3B cells). ( B and C ) The effects of ANGPTL3 knockdown on IL-1β-induced NF-κB activation were determined by the reporter assay in HEK293T ( B ) and Hep3B ( C ) cells. ( D – F ) The effects of ANGPTL3 knockdown on IL-1β-induced transcription of TNFA, CXCL2 , and IL8 were determined by qPCR in Hep3B ( D ), HepG2 ( E ), and HEK293T ( F ) cells. ( G and H ) The effects of ANGPTL3 knockdown on IL-1β- ( G ) or LPS-induced ( H ) transcription of TNFA, CXCL2 , and IL8 were determined by qPCR in THP1 cells. AN3, ANGPTL3; Exo., exogenous; Endo., endogenous. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Knockdown, Activation Assay, shRNA, Transfection, Reporter Assay

Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Knockout of ANGPTL3 potentiates IL-1β-induced NF-κB activation. ( A ) ANGPTL3 sgRNA plasmids significantly reduced the protein levels of both transfected Flag-tagged ANGPTL3 (upper panel, in HEK293T cells) and endogenous ANGPTL3 (lower panel, in Hep3B cells). ( B and C ) The effects of ANGPTL3 deficiency on IL-1β-induced transcription of TNFA, CXCL2 , and IL8 were determined by qPCR in Hep3B ( B ) and HepG2 ( C ) cells. ( D and E ) The effects of ANGPTL3 deficiency on IL-1β- ( D ) and LPS-induced ( E ) transcription of TNFA, CXCL2 , and IL8 were determined by qPCR in THP1 cells. AN3, ANGPTL3; KO, knockout; Exo., exogenous; Endo., endogenous. * P < 0.05; *** P < 0.001; ns, not significant.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Knock-Out, Activation Assay, Transfection

Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: ANGPTL3 targets the IL1R1-associated complex. ( A ) The effects of ANGPTL3 knockdown on IL1R1–IL1RAP- or MyD88-mediated NF-κB activation were determined by the reporter assay in HEK293T cells. ** P < 0.01; ns, not significant. ( B ) The interaction of overexpressed ANGPTL3 with IL1R1, IL1RAP, MyD88, and IKKβ was determined by Co-IP in HEK293T cells. ( C ) The interaction of endogenous ANGPTL3 with IL1R1 and IL1RAP was determined by Co-IP in HepG2 cells. ( D ) The co-localization of ANGPTL3-Cherry with IL1R1-GFP and IL1RAP-GFP in HEK293T cells. Scale bar, 10 μm. ( E ) A schematic presentation of human IL1R1, IL1RAP, and their truncation mutants. ( F and G ) The interaction of ANGPTL3 with IL1R1, IL1RAP, and their truncation mutants was determined by Co-IP in HEK293T cells. AN3, ANGPTL3; FL, full-length; Ig, immunoglobulin domain; TM, transmembrane domain; TIR: Toll/IL-1R homology domain.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Knockdown, Activation Assay, Reporter Assay, Co-Immunoprecipitation Assay

Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: The intracellular FLD domain of ANGPTL3 is involved in the negative regulation of IL-1β-induced signaling. ( A ) The effects of intracellular and secreted ANGPTL3 on IL-1β-induced NF-κB activation were determined by the reporter assay in HEK293T cells. ( B ) The effects of secreted full-length or truncated ANGPTL3 on IL-1β-induced NF-κB activation were determined by the reporter assay and western blotting in HEK293T cells. ( C ) The effects of wild-type or secretion-defective mutants of ANGPTL3 on the regulation of IL-1β-induced NF-κB activation were determined by the reporter assay and western blotting in HEK293T cells. ( D ) A schematic presentation of human ANGPTL3 and its truncation mutants. ( E ) The effects of full-length and various mutants of ANGPTL3 on IL-1β-induced NF-κB activation were determined by the reporter assay in HEK293T cells. ( F ) The interaction of IL1R1 with full-length and truncation mutants of ANGPTL3 was determined by Co-IP in HEK293T cells. AN3, ANGPTL3; Sup., supernatant; FL, full-length; WT, wild-type; SP, signal peptide; CCD, coiled-coil domain; FLD, fibrinogen-like domain. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Activation Assay, Reporter Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: ANGPTL3 inhibits the formation of IL1R1-associated complex. ( A and B ) The effects of ANGPTL3 overexpression on IL1R1 or IL1RAP homo- or hetero-interactions were determined by Co-IP ( A ) and quantitative analysis ( B ) in HEK293T cells. ( C – F ) The effects of ANGPTL3 overexpression on the IL1R1–MyD88 and IL1R1–IL-1β interactions were determined by Co-IP ( C and E ) and quantitative analysis ( D and F ) in HEK293T cells. ( G and H ) The effects of ANGPTL3 deficiency on the IL1R1–IL1RAP and IL1RAP–IL1RAP interactions were determined by Co-IP ( G ) and quantitative analysis ( H ) in HepG2 cells. ( I and J ) The effects of ANGPTL3 deficiency on the IL1R1–MyD88 and IL1R1–IL-1β interactions were determined by Co-IP ( I ) and quantitative analysis ( J ) in HepG2 cells. Quantification results were based on at least three experimental repeats. AN3, ANGPTL3; KO, knockout. ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Over Expression, Co-Immunoprecipitation Assay, Knock-Out

Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Intracellular ANGPTL3 inhibits the IL1R1–IL1RAP interaction to attenuate IL-1β-induced signaling. ( A and B ) Full-length ANGPTL3 and its truncation mutants were overexpressed in HEK293T cells. The effects of the full-length ANGPTL3 and its truncation mutants on the IL1R1–IL1RAP interaction were determined by Co-IP ( A ) and quantitative analysis ( B ). ( C – F ) Wild-type ANGPTL3 and its loss-of-function mutants were overexpressed in HEK293T cells. ( C ) The effects of wild-type and mutant ANGPTL3 on the regulation of IL-1β-induced NF-κB activation were determined by the reporter assay (left panel). The expression levels of Flag-tagged wild-type and mutant ANGPTL3 in cell lysate and supernatant were detected by western blotting (right panel). ( D ) The interaction of overexpressed IL1R1 with wild-type and mutant ANGPTL3 was determined by Co-IP. ( E and F ) The effects of wild-type and mutant ANGPTL3 on the IL1R1–IL1RAP interaction was determined by Co-IP ( E ) and quantitative analysis ( F ). Quantification results were based on at least three experimental repeats. AN3, ANGPTL3; FL, full-length; Sup., supernatant; WT, wild-type. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, Activation Assay, Reporter Assay, Expressing, Western Blot

Journal: Journal of Molecular Cell Biology

Article Title: ANGPTL3 negatively regulates IL-1 β -induced NF- κ B activation by inhibiting the IL1R1-associated signaling complex assembly

doi: 10.1093/jmcb/mjad053

Figure Lengend Snippet: Primers used in this study.

Article Snippet: Antibodies against ANGPTL3 (R&D Systems, AF3829), MyD88 (Cell Signaling Technology, 4283), Flag tag (Sigma, F1804), HA tag (Sigma, H3663), and Myc tag (Cell Signaling Technology, 2276) were purchased.

Techniques:

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 1. ANGPTL4/8-mediated plasmin generation blocks ANGPTL3/8-mediated LPL inhibition. A: LPL stable expres- sion cells were incubated with lipase substrate after being pre- viously incubated with vehicle alone, ANGPTL4/8, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. LPL ac- tivity was calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 10 from 4 independent experi- ments) (*P < 0.001 vs. vehicle). B: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL3/8 after being previously incubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. Afterward, LPL activity was assessed following the addition of fluorescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 8 from three independent experiments) (*P < 0.001 vs. ANGPTL3/8 alone). C: Samples from Figure 1B that contained ANGPTL3/8 were assessed using ANGPTL3 antibody Western blotting. Re- sults are representative of three independent experiments, with two replicates for each experiment (total n = 6 from three in- dependent experiments). ANGPTL, angiopoietin-like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Article Snippet: Human plasminogen (1939-SE), anti-ApoC2 rabbit antibody (MAF4497), anti-ANGPTL3 rabbit polyclonal antibody (AF3829), and anti-LPL antibody (AF7197) were purchased from R&D systems.

Techniques: Inhibition, Incubation, Control, Expressing, Activity Assay, Western Blot

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 2. ANGPTL4/8-mediated plasmin generation blocks LPL inhibition by ANGPTL4 and ANGPTL3. A: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL4 after being previously preincubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. Afterward, LPL activity was assessed following the addition of fluorescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 6 from two independent experiments) (*P < 0.001 vs. ANGPTL4 alone). B: LPL stable expression cells were incubated with vehicle alone throughout the experiment or in the absence or presence of ANGPTL3 after being previously preincubated with vehicle alone, ANGPTL4/8 alone, tPA + plasminogen, or ANGPTL4/8 + tPA + plasminogen. After- ward, LPL activity was assessed following the addition of fluo- rescent lipase substrate and calculated as a percent of vehicle control. Results are shown as the mean ± SD (n = 4 from two independent experiments) (*P < 0.001 vs. ANGPTL3 alone). C: Samples from Figure 2B that contained ANGPTL3 were

Article Snippet: Human plasminogen (1939-SE), anti-ApoC2 rabbit antibody (MAF4497), anti-ANGPTL3 rabbit polyclonal antibody (AF3829), and anti-LPL antibody (AF7197) were purchased from R&D systems.

Techniques: Inhibition, Expressing, Incubation, Activity Assay, Control

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 5. Characterization of ANGPTL4/8-mediated plasmin generation and its effects on LPL. A: The ability of tPA to convert plasminogen to plasmin in the presence of ANGPTL4/8 was examined after addition of ANGPTL3/8, ANGPTL3, ANGPTL4, ApoC3, or ApoC2, with the molar ratio of each protein to ANGPTL4/8 being the same as in the LPL activity assays. Generation of plasmin was measured us- ing colorimetric plasmin activity assays, with absorbances recorded spectrophotometrically. Results shown are repre- sentative of three independent experiments. B: LPL stable–expressing cells were preincubated with ANGPTL4/8, followed by incubation in the absence or presence of tPA + plasminogen. Cells were washed, and heparin was added to release membrane-bound LPL, which was measured via Western blotting. Results are presentative of 6 replicates from 2 independent experiments. C: Following removal of membrane-bound LPL, cells were solubilized using RIPA buffer, and LPL present within the cells was analyzed via Western blotting. Results are presentative of six replicates from two independent experiments. ANGPTL, angiopoietin- like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Article Snippet: Human plasminogen (1939-SE), anti-ApoC2 rabbit antibody (MAF4497), anti-ANGPTL3 rabbit polyclonal antibody (AF3829), and anti-LPL antibody (AF7197) were purchased from R&D systems.

Techniques: Activity Assay, Expressing, Incubation, Membrane, Western Blot

Journal: Journal of lipid research

Article Title: Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

doi: 10.1016/j.jlr.2023.100441

Figure Lengend Snippet: Fig. 6. An updated model for adipose tissue LPL regulation in the postprandial state. After feeding, increased ANGPTL4/8 (a) binds LPL, protects it from inactivation by ANGPTL4, but also partially inhibits LPL (light blue color) (b). Partially active ANGPTL4/8-LPL binds GPIHBP1 on abluminal endothelial surfaces (c) and is translocated (d). Concurrently, tPA secreted by the endothelium and plasminogen present on endothelial plasminogen receptors (PLG-R) bind the LPL-ANGPTL4/8 complex on luminal capillary surfaces (e). ANGPTL4/8 increases tPA-mediated generation of plasmin, which cleaves ANGPTL4/8 (f) to restore LPL activity (dark blue color). The plasmin generated (g) also protects LPL from inhibition by circulating ANGPTL3/8, ANGPTL3, and ApoC3 as well as from any localized ANGPTL4 that may be present (for the purpose of clarity only ANGPTL3/8 is shown in the figure) (h), while permitting ApoC2-mediated LPL stimulation to occur (i). ANGPTL, angiopoietin-like protein; LPL, lipoprotein lipase; tPA, tissue plasminogen activator.

Article Snippet: Human plasminogen (1939-SE), anti-ApoC2 rabbit antibody (MAF4497), anti-ANGPTL3 rabbit polyclonal antibody (AF3829), and anti-LPL antibody (AF7197) were purchased from R&D systems.

Techniques: Activity Assay, Generated, Inhibition