Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: ApoC3 expression levels were increased in calcified arteries and VSMCs. (a – c) Humans renal arteries were obtained from the patients with or without CRF who underwent unilateral nephrectomy. All the data were collected from human renal arteries. (a) Correlation between APOC3 mRNA expression levels and RUNX2 mRNA expression levels in human renal arteries (n = 12). (b) Representative von Kossa staining (left) and immunohistochemical staining of APOC3 (right) in human renal arteries. Bar = 100 μm. (c) Western blotting analysis of human renal arteries. (d – i) A 5/6 nephrectomy-induced CRF mouse model was established in C57BL/6 mice that were fed a 1.8% high-phosphate diet for 8 weeks. All the data were collected from WT mice. (d) Alizarin red S staining of aortic arch sections. Bar = 100 μm. Quantification of calcium deposition (e) and ALP activity (f) in aortas. (g) Western blotting analysis of aortas. (h) Quantitative analysis of RT‒PCR data from the aortas. (i) Immunofluorescence staining and quantitative analysis of α-SMA (green) and ApoC3 (red) in aortic arch sections. Nuclei were counterstained with DAPI (blue). Bar = 100 μm. (j – o) The calcification of primary rat VSMCs was induced with a high-Pi concentration (2.6 mM). All the data were collected from primary rat VSMCs. (j) Representative images of Alizarin red S staining of VSMCs. Quantification of calcium deposition (k) and ALP activity (l) in VSMCs. (m) Western blotting analysis of VSMCs. (n) Quantitative analysis of RT‒PCR results from VSMCs. (o) Immunofluorescence staining and quantitative analysis of DAPI, α-SMA and ApoC3 levels in VSMCs. Bar = 50 μm. n = 6 per group. ∗∗ P < 0.01, ∗∗∗ P < 0.001. Con, Control; CRF, chronic renal failure; Pi, phosphate medium.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: Expressing, Staining, Immunohistochemical staining, Western Blot, Activity Assay, Immunofluorescence, Concentration Assay, Control

Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: ApoC3 exacerbated the calcification of aortas and VSMCs in vivo and in vitro . A 5/6 nephrectomy-induced CRF mouse model was established in mice that were fed a 1.8% high-phosphate diet. (a – e) All the data were collected from WT and Tg mice. (a) Alizarin red S staining of aortic arch sections. Bar = 200 μm. Quantification of calcium deposition (b) and ALP activity (c) in aortas. (d, e) Western blotting analysis of aortas. (f – i) The calcification of primary rat VSMCs was induced with a high-Pi concentration (2.6 mM). All the data were collected from VSMCs infected with either Ad-EGFP or Ad-ApoC3. (f) Alizarin red S staining of VSMCs. Quantification of calcium deposition (g) and ALP activity (h) in VSMCs. (i) Western blotting analysis of VSMCs. (j – n) All the data were collected from WT and KO mice. (j) Alizarin red S staining of aortic arch sections. Bar = 200 μm. Quantification of calcium deposition (k) and ALP activity (l) in aortas. (m, n) Western blotting analysis of the aortas. (o – r) All the data were collected from VSMCs transfected with either scramble siRNA or ApoC3 siRNA. (o) Alizarin red S staining of VSMCs. Quantification of calcium deposition (p) and ALP activity (q) in VSMCs. (r) Western blotting analysis of VSMCs. n = 6 per group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CRF, chronic renal failure; WT, wild type mice; Tg, ApoC3 transgenic mice; KO, ApoC3 knockout mice; Con, Control; Pi, phosphate medium; VSMC, vascular smooth muscle cell; Ad, adenovirus; EGFP, enhanced green fluorescent; siApoC3, ApoC3 siRNA.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: In Vivo, In Vitro, Staining, Activity Assay, Western Blot, Concentration Assay, Infection, Transfection, Transgenic Assay, Knock-Out, Control

Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: ApoC3 exacerbated ferroptosis with decreases of SLC7A11/GPX4 in calcified aortas and VSMCs in vivo and in vitro. A 5/6 nephrectomy-induced mouse model of CRF was established in mice that were fed a 1.8% high-phosphate diet. (a – e) All the data were collected from WT and Tg mice. (a) Immunofluorescence staining and quantitative analysis of 4-HNE (green) and DAPI (blue) in aortic arch sections. Bar = 100 μm. Quantification of MDA (b) and GSH (c) in aortas. (d) Quantitative analysis of RT‒PCR data from aortas. (e) Western blotting analysis of aortas. (f – l) The calcification of primary rat VSMCs was induced with a high-Pi concentration (2.6 mM). All the data were collected from VSMCs infected with either Ad-EGFP or Ad-ApoC3. (f) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (g) Quantification of the MDA contents in VSMCs. (h) Quantitative analysis of RT‒PCR results in VSMCs. (i) Cell viability. (j) Quantification of the GSH contents in VSMCs. (k, l) Western blotting analysis of the levels in VSMCs. (m – q) All the data were collected from WT and KO mice. (m) Immunofluorescence staining and quantitative analysis of 4-HNE (green) and DAPI (blue) in aortic arch sections. Bar = 100 μm. Quantification of MDA (n) and GSH (o) in the aortas. (p) Quantitative analysis of RT‒PCR data from aortas. (q) Western blotting analysis of aortas. (r – x) All the data were collected from VSMCs transfected with either scramble siRNA or ApoC3 siRNA. (r, s) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (t) Quantification of the MDA contents in VSMCs. (u) Quantitative analysis of RT‒PCR results in VSMCs. (v) Cell viability. (w) Quantification of the GSH contents in VSMCs. (x) Western blotting analysis of VSMCs. n = 6 per group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CRF, chronic renal failure; WT, wild type mice; Tg, ApoC3 transgenic mice; KO, ApoC3 knockout mice; Con, Control; Pi, phosphate medium; Ad, adenovirus; EGFP, enhanced green fluorescent; siApoC3, ApoC3 siRNA; HA, hemagglutinin.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: In Vivo, In Vitro, Immunofluorescence, Staining, Western Blot, Concentration Assay, Infection, Transfection, Transgenic Assay, Knock-Out, Control

Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: ApoC3 inhibited the AMPK/NRF2 pathway by activating TLR2 in calcified aortas and VSMCs. (a) Co-IP analysis to detect the interaction between APOC3 and TLR2 in VSMCs. (b) Immunofluorescence staining of DAPI (blue), TLR2 (green) and ApoC3 (red) levels in primary human VSMCs. (c) Western blotting analysis of aortas in WT and Tg mice with CRF. (d) Western blotting analysis of VSMCs infected with either Ad-EGFP or Ad-ApoC3. (e, f) Western blotting analysis of aortas in WT and KO mice with CRF. (g) Western blotting analysis of VSMCs infected with either scramble siRNA or ApoC3 siRNA. Primary rat VSMCs were infected with Ad-ApoC3 or HA-ApoC3 plasmids and subsequently treated with a high-Pi concentration (2.6 mM). (h – p) All the data were collected from VSMCs transfected with either scramble siRNA or TLR2 siRNA. (h) Alizarin red S staining of VSMCs. Quantification of the calcium deposition (i) and ALP activity (j) in VSMCs. (k) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (l) Quantification of the MDA contents in VSMCs. (m) Quantitative analysis of RT‒PCR data from VSMCs. (n) Cell viability. (o) Quantification of the GSH contents in VSMCs. (p) Western blotting analysis of VSMCs. n = 6 per group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CRF, chronic renal failure; WT, wild type mice; Tg, ApoC3 transgenic mice; KO, ApoC3 knockout mice; Con, Control; Pi, phosphate medium; VSMC, vascular smooth muscle cell; n-NRF2, Nuclear NRF2; Ad, adenovirus; EGFP, enhanced green fluorescent; siTLR2, TLR2 siRNA; HA, hemagglutinin; siApoC3, ApoC3 siRNA.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Western Blot, Infection, Concentration Assay, Transfection, Activity Assay, Transgenic Assay, Knock-Out, Control

Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: Activation of AMPK or NRF2 decreased ApoC3-induced ferroptosis and calcification in VSMCs. Primary rat VSMCs were infected with Ad-ApoC3 or HA-ApoC3 plasmids and then treated with a high-Pi concentration (2.6 mM). (a – i) All the data were collected from VSMCs treated with either DMSO or the AMPK agonist AICAR (1 mM). (a) Alizarin red S staining of VSMCs. Quantification of the calcium deposition (b) and ALP activity (c) in VSMCs. (d) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (e) Quantification of the MDA contents in VSMCs. (f) Quantitative analysis of RT‒PCR data from VSMCs. (g) Cell viability. (h) Quantification of the GSH contents in VSMCs. (i) Western blotting analysis of VSMCs. (j – r) All the data were collected from VSMCs treated with either DMSO or the NRF2 agonist SFN (10 μM). (j) Alizarin red S staining of VSMCs. Quantification of the calcium deposition (k) and ALP activity (l) in VSMCs. (m) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (n) Quantification of the MDA contents in VSMCs. (o) Quantitative analysis of RT‒PCR data from VSMCs. (p) Cell viability. (q) Quantification of the GSH contents in VSMCs. (r) Western blotting analysis of VSMCs. n = 6 per group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Con, Control medium; Pi, phosphate medium; AICAR, acadesine; SFN, sulforaphane; n-NRF2, Nuclear NRF2; Ad, adenovirus; HA, hemagglutinin.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: Activation Assay, Infection, Concentration Assay, Staining, Activity Assay, Western Blot, Control

Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: Inhibition of SLC7A11 or GPX4 reduced the protective effects of ApoC3 silencing on ferroptosis and calcification in VSMCs. Primary rat VSMCs were transfected with ApoC3 siRNA and then treated with a high-Pi concentration (2.6 mM). (a – i) All the data were collected from VSMCs treated with either DMSO or HG106 (an inhibitor of SLC7A11) (1 μM). (a) Alizarin red S staining. Quantification of the calcium deposition (b) and ALP activity (c) . (d) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (e) Quantification of the MDA contents in VSMCs. (f) Quantitative analysis of RT‒PCR data from VSMCs. (g) Cell viability. (h) Quantification of the GSH contents in VSMCs. (i) Western blotting analysis of VSMCs. (j – r) All the data were collected from VSMCs treated with either DMSO or RSL3 (an inhibitor of GPX4) (1 μM). (j) Alizarin red S staining of VSMCs. Quantification of the calcium deposition (k) and ALP activity (l) in VSMCs. (m) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (n) Quantification of the MDA contents in VSMCs. (o) Quantitative analysis of RT‒PCR results in VSMCs. (p) Cell viability. (q) Quantification of the GSH contents in VSMCs. (r) Western blotting analysis of VSMCs. n = 6 per group. ∗∗ P < 0.01, ∗∗∗ P < 0.001. Con, Control medium; Pi, phosphate medium; siApoC3, ApoC3 siRNA.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: Inhibition, Transfection, Concentration Assay, Staining, Activity Assay, Western Blot, Control

Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: The ferroptosis inhibitors attenuated ApoC3-induced ferroptosis and calcification in aortas and VSMCs. The vitamin D 3 (VitD 3 ) overload-induced mouse model of calcification was established in Tg mice. (a – h) All the data were collected from Tg mice. (a) Alizarin red S staining of aortic arch sections. Bar = 300 μm. Quantification of the calcium deposition (b) and ALP activity (c) in aortas. (d) Immunofluorescence staining and quantitative analysis of 4-HNE (green) and DAPI (blue) in aortic arch sections. Bar = 100 μm. Quantification of MDA (e) and GSH (f) in aortas. (g) Quantitative analysis of RT‒PCR data from aortas. (h) Western blotting analysis of aortas. (i – q) The calcification of primary rat VSMCs was induced by a high-Pi concentration (2.6 mM). All the data were collected from VSMCs infected with Ad-ApoC3 or transfected with the HA-ApoC3 plasmid. (i) Alizarin red S staining of VSMCs. Quantification of the calcium deposition (j) and ALP activity (k) in VSMCs. (l) Representative images and quantitative analysis of intracellular Fe 2+ and ROS staining in VSMCs. Bar = 200 μm. (m) Quantification of the MDA contents in VSMCs. (n) Quantitative analysis of RT‒PCR data from VSMCs. (o) Cell viability. (p) Quantification of the GSH contents in VSMCs. (q) Western blotting analysis of VSMCs. n = 6 per group. ∗∗ P < 0.01, ∗∗∗ P < 0.001. Tg, ApoC3 transgenic mice; VitD 3 , vitamin D 3 ; Con, Control; Pi, phosphate medium; Ad, adenovirus; HA, hemagglutinin.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: Staining, Activity Assay, Immunofluorescence, Western Blot, Concentration Assay, Infection, Transfection, Plasmid Preparation, Transgenic Assay, Control

Journal: Redox Biology

Article Title: Apolipoprotein C3 exacerbates vascular calcification by promoting ferroptosis via the TLR2/AMPK pathway in vascular smooth muscle cells

doi: 10.1016/j.redox.2026.104088

Figure Lengend Snippet: ApoC3 exacerbated ferroptosis and calcification in aortic rings from mice and inhibition of ApoC3 improved ferroptosis and calcification in renal arterial rings from humans. Aortic rings were isolated from mice. The calcification of aortic rings was induced by a high-Pi concentration (2.6 mM). (a-f) All the data were collected from the aortic rings in WT and Tg mice. (a) Alizarin red S staining of aortic rings. Bar = 300 μm. Quantification of the calcium deposition (b) and ALP activity (c) in aortic rings. (d) Representative images and quantitative analysis of ROS staining (red) in aortic rings. Bar = 100 μm. Quantification of MDA (e) and GSH (f) in aortic rings. (g – l) All the data were collected from the aortic rings of WT and KO mice. (g) Alizarin red S staining of aortic rings. Bar = 300 μm. Quantification of the calcium deposition (h) and ALP activity (i) in aortic rings. (j) Representative images and quantitative analysis of ROS (red) staining in aortic rings. Bar = 100 μm. Quantification of MDA (k) and GSH (l) in aortic rings. Human renal arterial rings were exposed to a high-Pi concentration (2.6 mM). (m – p) All the data were collected from human renal arterial rings. (m) Representative von Kossa staining, immunofluorescence staining and quantitative analysis of APOC3 (red), α-SMA (green) and DAPI (blue) in human renal arteries rings. Bar = 300 μm. Quantification of the calcium deposition (n) and ALP activity (o) in human renal arterial rings. (p) Western blotting analysis of human renal arterial rings. (q) Representative images and quantitative analysis of ROS staining (red) in human renal arterial rings. Bar = 200 μm. Quantification of MDA (r) and GSH (s) in human renal arterial rings. n = 6 per group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. WT, wild type mice; Tg, ApoC3 transgenic mice; KO, ApoC3 knockout mice; Con, Control medium; Pi, phosphate medium; siApoC3, ApoC3 siRNA.

Article Snippet: To study the effects of ferroptosis, ApoC3 Tg mice were subcutaneously injected with a ferroptosis inhibitor ferrostatin-1 (Fer-1) (1 mg/kg, HY-100579, MedChemExpress, USA) daily for 7 days in VitD 3 -induced calcification model [ ].

Techniques: Inhibition, Isolation, Concentration Assay, Staining, Activity Assay, Immunofluorescence, Western Blot, Transgenic Assay, Knock-Out, Control

Journal:

Article Title: Apolipoprotein CIII promotes Ca 2+ -dependent ? cell death in type 1 diabetes

doi: 10.1073/pnas.0403551101

Figure Lengend Snippet: Amounts of apoCIII in T1D serum and effects on [Ca2+]i and cell death. (A) Relative levels of apoCIII1 + 2 in T1D and control serum, given as area under the curve (AUC). **, P < 0.01 (n = 5). (B) Pancreatic β cells were incubated with apoCIII or apoCIII plus antibodies against human apoCIII (n = 63, 35, and 33). ***, P < 0.001 versus control. (C) β cells were incubated with a control or a T1D serum and T1D serum plus anti-apoCIII (n = 18, 17, and 20). ***, P < 0.001 versus T1D serum. (D) Mouse β cells were incubated with apoCIII0, apoCIII1, apoCIII2, or vehicle (control; n = 54, 40, 48, and 37, respectively). ***, P < 0.001 versus control. (E) The insulin-secreting cell line RINm5F was exposed to control and T1D sera and T1D serum with the addition of two concentrations of anti-apoCIII, and finally control serum with apoCIII (n = 5). *, P < 0.05 and **, P < 0.01, versus control.

Article Snippet: Coincubation of β cells with 100 μg/ml of a polyclonal antibody against human apoCIII (Academy BioMedical, Houston) blocked the activity of both the commercial apoCIII and the T1D serum ( ).

Techniques: Incubation

Journal:

Article Title: Apolipoprotein CIII promotes Ca 2+ -dependent ? cell death in type 1 diabetes

doi: 10.1073/pnas.0403551101

Figure Lengend Snippet: Interaction of apoCIII with the voltage-gated L-type Ca2+ channel. (A) Summary graph of current density–voltage relationships. ApoCIII-treated cells (filled circles, n = 56) and control cells (open circles, n = 55) were depolarized to potentials between -60 and 50 mV, in 10-mV increments, from a holding potential of -70 mV. *, P < 0.05. (B) Sample whole-cell Ca2+ current traces from a control cell (cell capacitance: 4.3 pF) and a cell incubated with apoCIII (cell capacitance: 4.2 pF). Cells were depolarized by a set of voltage pulses (100 ms, 0.5 Hz) between -60 and 50 mV, in 10-mV increments, from a holding potential of -70 mV.

Article Snippet: Coincubation of β cells with 100 μg/ml of a polyclonal antibody against human apoCIII (Academy BioMedical, Houston) blocked the activity of both the commercial apoCIII and the T1D serum ( ).

Techniques: Incubation

Journal: International Journal of Molecular Sciences

Article Title: CD14 + -Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor

doi: 10.3390/ijms24032223

Figure Lengend Snippet: Expression of TNFα, IL-1β and IL-6 mRNAs and protein release by human CD14 + -monocytes exposed to ApoCIII. Human CD14 + -monocytes, purified as described in , were incubated for 3 h ( A – C ) or 20 h ( D – F ) in the absence (medium) or presence of 50 µg/mL ultrapure ApoCIII or, as a control, in 100 ng/mL LPS. Total RNA was then extracted and examined for TNFα ( A ), IL-1β ( B ) and IL-6 ( C ) mRNA expression by RT-qPCR. Gene expression is depicted as mean normalized expression (MNE) units after normalization to GAPDH mRNA (mean ± SEM, n = 7–9). Monocyte-derived supernatants were collected, and TNFα ( D ), IL-1β ( E ) and IL-6 ( F ) levels were measured by ELISA. Results are expressed as the mean value ± SEM of 3 independent experiments. Statistical analysis by unpaired t-test with Welch correction refers to ApoCIII-treated versus untreated monocytes, * p < 0.05.

Article Snippet: Ultrapure ApoCIII from human plasma was purchased from Academy Bio-Medical Company, Inc. (#33P-UP201, Houston, TX, USA), namely lots #110565, #10306 and #020377.

Techniques: Expressing, Purification, Incubation, Quantitative RT-PCR, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: CD14 + -Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor

doi: 10.3390/ijms24032223

Figure Lengend Snippet: Expression of tissue factor (TF) mRNA in human CD14 + -monocytes and autologous neutrophils exposed to ApoCIII. Human CD14 + -monocytes ( A , B ) and autologous neutrophils ( C ), purified as described in , were incubated for 3 h ( A – C ) and/or 20 h ( B , C ) in the absence (medium) or presence of 50 µg/mL ApoCIII, 100 ng/mL LPS ( A , B ), 1 µg/mL LPS ( C ) or 5 µM R848 ( B , C ). Total RNA was then extracted and examined for TF mRNA expression by quantitative RT-qPCR. Gene expression is depicted as MNE units after normalization to GAPDH mRNA. For panel A , data are represented as mean ± SEM, n = 8–9, and were analyzed by unpaired t-test with Welch correction. * p < 0.05. Panels B and C show a representative experiment from a single donor (out of 2 performed with similar results). Data are represented as mean ± SEM of experimental replicates.

Article Snippet: Ultrapure ApoCIII from human plasma was purchased from Academy Bio-Medical Company, Inc. (#33P-UP201, Houston, TX, USA), namely lots #110565, #10306 and #020377.

Techniques: Expressing, Purification, Incubation, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: CD14 + -Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor

doi: 10.3390/ijms24032223

Figure Lengend Snippet: Expression of surface TF in human CD14 + -monocytes and autologous neutrophils exposed to ApoCIII. Human CD14 + -monocytes ( A – C ) and autologous neutrophils ( B ), purified as described in , were incubated for 3 h in the absence (medium) or presence of 50 µg/mL ApoCIII, 5 µΜ R848 or 0.1 and 1 µg/mL LPS for CD14 + -monocytes and neutrophils, respectively. In selected experiments, the stimuli were preincubated with 20 µg/mL polymyxin B (PMX) before their addition to the cells ( C ). Cells were then stained for surface TF expression by FACS as described in . Panel A depicts the percentage of TF + CD14 + -monocytes (mean ± SEM, n = 8). Data were analyzed by one-way ANOVA followed by the Holm–Sidak post hoc test. * p < 0.05; *** p < 0.001. Panel B displays representative histograms showing the expression of surface TF by CD14 + -monocytes and neutrophils incubated with ApoCIII, LPS and R848 as compared to isotype control antibodies. Panel C displays representative flow cytometry plots showing the percentage of TF + CD14 + -monocytes (represented as red dots) incubated with ApoCIII, LPS and R848, in the absence or presence of PMX, as compared to the isotype control antibodies (represented as black dots).

Article Snippet: Ultrapure ApoCIII from human plasma was purchased from Academy Bio-Medical Company, Inc. (#33P-UP201, Houston, TX, USA), namely lots #110565, #10306 and #020377.

Techniques: Expressing, Purification, Incubation, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: CD14 + -Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor

doi: 10.3390/ijms24032223

Figure Lengend Snippet: Effect of sera with measured concentrations of ApoCIII on surface TF expression by CD14 + -monocytes. Panel A shows the percentages of CD14 + -monocytes expressing TF from PBMCs incubated for 3 h with or without 10% sera obtained from 19 individuals displaying different serum ApoCIII levels ranging from 1.5 to 29.6 mg/dL. Data are expressed as mean ± SEM ( n = 11–19) and were analyzed by unpaired t -test with Welch correction. *** p < 0.001. Panel B displays a scatter plot with a linear regression curve showing the relationship between the percentages of CD14 + -monocytes expressing TF and the ApoCIII concentrations of the sera used to stimulate PBMCs. The r-value determined by Pearson correlation is displayed. Panel C reports CD14 + -monocytes expressing TF from PBMC samples incubated for 3 h with either 50 µg/mL ApoCIII, sera with known concentrations of ApoCIII (ApoCIII-sera) or 100 ng/mL LPS in the presence or absence (as 100%) of 60 µg/mL ApoCIII-neutralizing rAbs. Statistics of data refer to the effects of anti-ApoCIII-recombinant antibodies (αApoCIII-rAbs) on each treatment, and were evaluated by one sample t-test. * p < 0.05; ** p < 0.01; n = 3–10.

Article Snippet: Ultrapure ApoCIII from human plasma was purchased from Academy Bio-Medical Company, Inc. (#33P-UP201, Houston, TX, USA), namely lots #110565, #10306 and #020377.

Techniques: Expressing, Incubation, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: CD14 + -Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor

doi: 10.3390/ijms24032223

Figure Lengend Snippet: Effect of sera with measured concentrations of ApoCIII on TNFα and IL-6 release by PBMCs. Panel A : PBMCs isolated from buffy coats as reported in were cultured for 3 h with the addition of ApoCIII-sera in the presence or absence of 60 µg/mL ApoCIII-neutralizing rAbs. After treatment, sera with known concentrations of ApoCIII (ApoCIII-sera) and PBMC-derived supernatants were collected and analyzed for TNFα and IL-6 content by ELISA. Data are expressed as mean± SEM (n = 8) and were analyzed by the Kruskal–Wallis test followed by Dunn’s multiple comparison test.. Panel B displays two scatter plots showing the correlation between TNFα or IL-6 release by PBMCs and the ApoCIII concentrations of the sera that were used as stimuli. The r-value determined by Pearson correlation is displayed together with the p -value.

Article Snippet: Ultrapure ApoCIII from human plasma was purchased from Academy Bio-Medical Company, Inc. (#33P-UP201, Houston, TX, USA), namely lots #110565, #10306 and #020377.

Techniques: Isolation, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay