Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Apolipoprotein C-III itself stimulates the Syk/cPLA2-induced inflammasome activation of macrophage to boost anti-tumor activity of CD8 + T cell

doi: 10.1007/s00262-023-03547-8

Figure Lengend Snippet: PLA2/NOX-induced inflammasome activation in macrophagesTG. A DEGs of macrophages by pairwise comparison. B Multiple genes that were upregulated in macrophagesTG whereas downregulated in macrophagesTG were sorted out. C Genes that were associated with arachidonic acid metabolism and had the most changes. D Western blot confirmed elevated expression of PLA2 G4A/G4F and NOX2 in macrophagesTG. E Transcriptional variations in genes associated with alternative inflammasome activation. F Confirmed alternative inflammasome activation by Western blot. G Upregulated pyroptosis of macrophagesTG identified by LDH releasing assay. H–I Inhibition of Cas8, NLRP3, or Cas1 decreased IL-1β production or secretion of macrophagesTG. J NAC was used to deplete cellular ROS, and NAC treatment decreased NLRP3, c-Cas1, c-Cac8, and RIPK1 protein levels. K–L IL-1β production or secretion by the NAC treatment. M MacrophagesTG lost stimulatory effects on CD8+ T cells after ROS depletion. N Inhibited cPLA2 (CAY10650) on NOX2, inflammasome activation, and CPT1 expression in macrophagesTG. O IL-1β production of macrophagesTG treated with CAY10650 or a sPLA2 inhibitor (Varespladid). P Release of IL-1β by rest or activated macrophagesTG after treatment with CAY10650. Experiments were repeated twice. Ns, no significance. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Anti-RIPK1 (38/RIP) antibody was obtained from BD Biosciences (NJ, USA), and anti- NADPH oxidase 2 (NOX2; 19013-1-AP) antibody was obtained from Proteintech (Illinois, USA).

Techniques: Activation Assay, Comparison, Western Blot, Expressing, Inhibition

Journal: Pharmacological research

Article Title: Cardiac PDK4 promotes neutrophilic PFKL methylation and drives the innate immune response in diabetic myocardial infarction.

doi: 10.1016/j.phrs.2025.107731

Figure Lengend Snippet: Fig. 2. Diabetes upregulated cardiac PDK4 and aggravated NETosis in MI. (A) Reanalysis of PDK4 gene expression in the GSE12639 dataset. (B) Myocardial infarction autopsy sample grouping schematic diagram. (C) Myocardial tissue from autopsy samples was stained with HE and PDK4 (n = 5). (D) Patients were clinically grouped: control = 30, MI = 30, and DM+MI = 30. (E) Scanning electron microscopy of peripheral blood neutrophils from patients. (F-H) cit-H3, MPO- DNA and cfDNA levels in the peripheral blood of patients (n = 30). (I) Diagram of the diabetic myocardial infarction mouse model. (J) H&E-stained images of mouse heart tissues and quantification of neutrophils were performed according to the extent of pathological injury (n = 6). (K) Colocalization assay for PDK4 (red) and the NET marker MPO (green) in the infarcted area. Nuclei were stained with DAPI (blue). Quantification of MPO+ cells in the infarcted area (n = 4). (L–N) cit-H3, MPO- DNA and cfDNA levels in mouse peripheral blood (n = 6). (O) Quantitative polymerase chain reaction analysis of PDK4, MPO, NE, PADI4 and CD177 mRNA expression in the hearts of MI or diabetic MI mice (n = 5). (P) Immunoblot analysis of PDK4 and MPO expression in mouse heart tissue (n = 3). Statistical analysis was performed by using two-way ANOVA, followed by Tukey’s multiple comparison test, and p-values < 0.05 were considered statistically significant. Data are presented as mean ± SEM.

Article Snippet: PDK4 (Proteintech, 12949–1-AP), β-actin (CST, 5174), LDHA (Proteintech, 19987–1-AP), Bax (Abcam, ab32503), Bcl-2 (Abcam, ab1196495), and NOX2 (Cat No: 19013–1-AP) were used.

Techniques: Gene Expression, Staining, Control, Electron Microscopy, Marker, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Comparison

Journal: Pharmacological research

Article Title: Cardiac PDK4 promotes neutrophilic PFKL methylation and drives the innate immune response in diabetic myocardial infarction.

doi: 10.1016/j.phrs.2025.107731

Figure Lengend Snippet: Fig. 3. Cardiomyocyte-specific PDK4 deficiency alleviates NETosis and cardiac dysfunction in diabetic MI. (A) A cardiac-specific PDK4 deficiency mouse model of diabetic MI was constructed. (B) Representative immunohistochemistry image of PDK4 (dark brown) in infarction heart tissue from nondiabetic and diabetic patients. (C) Representative images of H&E-stained hearts from PDK4fl/fl and PDK4fl/flMyh6Cre mice in each group. The infarct size was defined and calculated according to the extent of pathological injury (n = 3). (D) M-mode echocardiography images and cardiac function were measured by LVEF (n = 3). (E) Colocalization assay for LDH (green) and the NET marker MPO (red) in the infarcted area. Nuclei were stained with DAPI (blue). Quantification of MPO+ cells in the infarcted area (n = 3). (F-G) MPO-DNA and cfDNA levels in mouse peripheral blood (n = 6). (H) Immunoblot analysis of NETosis-related protein expression in the heart tissue of PDK4fl/fl and PDK4fl/flMyh6Cre mice (n = 3). (I) Coculture of H9C2 cells and rat neutrophils. (J) Quantitative analysis of PDK4 mRNA expression in H9C2 cells after PDK4 knockdown (n = 6). (K) MPO-DNA content of the neutrophil supernatant in the coculture system (n = 5). (L-M) Images of immunofluorescence staining with SYTOX Green and Hoechst 33342 (blue) (n = 3). Statistical analysis was performed by using two-way ANOVA, followed by Tukey’s multiple comparison test, and p-values < 0.05 were considered statistically significant. Data are presented as mean±SEM.

Article Snippet: PDK4 (Proteintech, 12949–1-AP), β-actin (CST, 5174), LDHA (Proteintech, 19987–1-AP), Bax (Abcam, ab32503), Bcl-2 (Abcam, ab1196495), and NOX2 (Cat No: 19013–1-AP) were used.

Techniques: Construct, Immunohistochemistry, Staining, Marker, Western Blot, Expressing, Knockdown, Immunofluorescence, Comparison

Journal: Pharmacological research

Article Title: Cardiac PDK4 promotes neutrophilic PFKL methylation and drives the innate immune response in diabetic myocardial infarction.

doi: 10.1016/j.phrs.2025.107731

Figure Lengend Snippet: Fig. 4. Cardiac PDK4 drives NETosis by promoting LA production. (A) The metabolic pathway of PDK4 regulates glycolytic LA production. (B) Spatiotemporal profiling of metabolic signatures and delineation of ischemia using MSI. Images of metabolites related to glycolysis metabolism (n = 3). (C) Targeted metabolomics was used to measure the lactate content in the infraction zone (n = 6). (D) Peripheral blood LA content in clinical patients (n = 30). (E) Peripheral blood LA content in clinical patients PDK4fl/fl and PDK4fl/flMyh6Cre mice (n = 6). (F) Seahorse analysis of the effect of shPDK4 on H9C2 glycolysis under hypoxic and HG conditions (n = 3). (G) HE staining was performed on the hearts of diabetic MI mice treated with lactate or AZD3965. (H) Infarct size was defined and calculated according to the extent of pathological injury (n = 4). (I) Measurement of the LVEF of cardiac function indices from echocardiographic data (n = 5). (J-K) Immunofluorescence analysis of MPO (red) in the infarct area. Quantification of MPO+ cells in the infarcted area (n = 3). (L–N) cit-H3, MPO-DNA and cfDNA levels in mouse peripheral blood (n = 6). (O) Scanning electron microscopy was used to observe the effect of an MCT1 inhibitor (AZD3965) on LA-induced NETosis. (n = 3). (P) Western blot analysis of NETosis-related protein expression in the LA and LA+AZD3965 groups (n = 3). Statistical analysis was performed by using two-way ANOVA, followed by Tukey’s multiple comparison test, and p-values < 0.05 were considered statistically significant. Data are presented as mean ± SEM.

Article Snippet: PDK4 (Proteintech, 12949–1-AP), β-actin (CST, 5174), LDHA (Proteintech, 19987–1-AP), Bax (Abcam, ab32503), Bcl-2 (Abcam, ab1196495), and NOX2 (Cat No: 19013–1-AP) were used.

Techniques: Staining, Immunofluorescence, Electron Microscopy, Western Blot, Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: FGL2 deficiency alleviates maternal inflammation-induced blood-brain barrier damage by blocking PI3K/NF-κB mediated endothelial oxidative stress

doi: 10.3389/fimmu.2023.1157027

Figure Lengend Snippet: Increased endothelial expression of FGL2 was associated with the reduced TJ proteins. (A) Experimental scheme of WT mice. WT dams were randomly intraperitoneally injected with LPS (200 µg/kg) or PBS at E17. Pups were sacrificed and their brains were collected at P1 and P7. (B) The protein expressions of Claudin-5, Occludin, and ZO-1 in brain at P1 and in cortex at P7 ( n = 5). (C) The mRNA levels of FGL2 in brain at P1 and in cortex at P7 ( n = 4:6:6:5). (D) The protein expressions of FGL2 in brain at P1 and in cortex at P7 ( n = 3:4:3:3). (E) FGL2 expression in CD31+ vessels by immunofluorescence staining in cortex at P7. The fluorescence intensity of FGL2 in CD31+ vessels was analyzed by the ImageJ software and normalized to controls ( n = 4). Representative images and quantitative results were shown. Scale bar=50 μm. (F) The mRNA levels of FGL2 in BMECs ( n = 6). BMECs were treated with LPS (5μg/ml) or PBS for 24h. (G) The protein expressions of FGL2 in BMECs ( n = 5). (H) The mRNA levels of Claudin-5, Occludin, and ZO-1 in BMECs ( n = 4). The data was expressed as mean ± SEM and was the representative of at least three independent experiments. Statistical comparisons were assessed by unpaired two-tailed Student’s t test. ns P >0.05, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The primary antibodies used were as follows: FGL2 (1:500, Abnova Cat# H00010875-M01), NOX2 (1:100, Proteintech Cat# 19013-1-AP), iNOS (1:100, Proteintech Cat# 18985-1-AP), vWF (1:100, Abcam Cat#ab11713), CD31 (1:2000, Abcam Cat# ab182981; 1:400, Abcam Cat# ab56299).

Techniques: Expressing, Injection, Immunofluorescence, Staining, Fluorescence, Software, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: FGL2 deficiency alleviates maternal inflammation-induced blood-brain barrier damage by blocking PI3K/NF-κB mediated endothelial oxidative stress

doi: 10.3389/fimmu.2023.1157027

Figure Lengend Snippet: FGL2 deficiency attenuated maternal inflammation-induced BBB damage and neuroinflammation. (A) Experimental scheme of FGL2 +/- mice. FGL2 +/- dams were randomly injected with LPS (200 µg/kg) or PBS at E17. FGL2 +/+ and FGL2 -/- pups were sacrificed and their brains were collected at P1 and P7. (B) The permeability of BBB measured by NaF in pups at P1 ( n = 9:12:7:6). (C) The permeability of BBB measured by NaF in pups at P7 ( n = 5:4:5:4). (D) The protein expressions of Occludin, Claudin-5, and ZO-1 in brain at P1 ( n = 6). (E) The protein expressions of Occludin, Claudin-5, and ZO-1 in cortex at P7 ( n = 6). (F) The protein expression of FGL2 in BMECs ( n = 5). BMECs were infected by Ad-FGL2-miRNA or Ad-GFP for 48h. (G) The mRNA levels of Claudin-5 , Occludin , and ZO-1 in BMECs ( n = 5). BMECs were infected by adenovirus for 48 h before treatment of LPS (5 μg/ml) or PBS for 24h. (H) The mRNA levels of TNF-α , IL-6 , and IL-10 in brain at P1 ( n = 6:5:4:5). (I) The mRNA levels of TNF-α , IL-6 , and IL-10 in cortex at P7 ( n = 4:3:3:3). The data was expressed as mean ± SEM and was the representative of at least three independent experiments. Statistical differences were assessed by unpaired two-tailed Student’s t test for two groups and one-way ANOVA with Tukey’s post-hoc test for four groups. ns P >0.05, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The primary antibodies used were as follows: FGL2 (1:500, Abnova Cat# H00010875-M01), NOX2 (1:100, Proteintech Cat# 19013-1-AP), iNOS (1:100, Proteintech Cat# 18985-1-AP), vWF (1:100, Abcam Cat#ab11713), CD31 (1:2000, Abcam Cat# ab182981; 1:400, Abcam Cat# ab56299).

Techniques: Injection, Permeability, Expressing, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: FGL2 deficiency alleviates maternal inflammation-induced blood-brain barrier damage by blocking PI3K/NF-κB mediated endothelial oxidative stress

doi: 10.3389/fimmu.2023.1157027

Figure Lengend Snippet: FGL2 deficiency decreased the maternal inflammation-induced generation of ROS and NO. (A) The level of ROS by DHE staining in cortex at P7 ( n = 4). Representative images were shown and quantitative results were normalized to controls. Scale bar=50 μm. (B) The NO contents in brain at P1 ( n = 6:7:5:6). (C) The NO contents in cortex at P7 ( n = 4:6:4:5). (D) The level of ROS by DHE staining in BMECs ( n = 6). FGL2 +/+ and FGL2 -/- BMECs were treated with LPS (5 μg/ml) or PBS for 24 h. Representative images were shown, and quantitative results were normalized to controls. Scale bar=100 µm. (E) The concentration of NO in culture mediums of BMECs ( n = 6). The data was expressed as mean ± SEM and was the representative of at least three independent experiments. Statistical differences were assessed by one-way ANOVA with Tukey’s post-hoc test for multiple groups. ns P >0.05, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The primary antibodies used were as follows: FGL2 (1:500, Abnova Cat# H00010875-M01), NOX2 (1:100, Proteintech Cat# 19013-1-AP), iNOS (1:100, Proteintech Cat# 18985-1-AP), vWF (1:100, Abcam Cat#ab11713), CD31 (1:2000, Abcam Cat# ab182981; 1:400, Abcam Cat# ab56299).

Techniques: Staining, Concentration Assay

Journal: Frontiers in Immunology

Article Title: FGL2 deficiency alleviates maternal inflammation-induced blood-brain barrier damage by blocking PI3K/NF-κB mediated endothelial oxidative stress

doi: 10.3389/fimmu.2023.1157027

Figure Lengend Snippet: FGL2 deficiency decreased the maternal inflammation-induced endothelial expressions of NOX2 and iNOS. (A) The protein expressions of iNOS in brain at P1 and in cortex at P7 ( n = 7). (B) The protein expressions of NOX2 in brain at P1 and in cortex at P7 ( n = 7). (C) NOX2 expressions in CD31+ vessels by immunofluorescence staining in cortex at P7 ( n = 3:4:3:4). The number of NOX2+ and CD31+ cells were counted manually by the ImageJ software, and the percentage was calculated. Representative images and quantitative results were shown. White arrows points to the representative cells. Scale bar= 30 μm. (D) iNOS expressions in CD31+ vessels by immunofluorescence staining in cortex at P7 ( n = 3:3:3:4). The fluorescence intensity of iNOS in CD31+ vessels was analyzed by the ImageJ software and normalized to controls. Representative images and quantitative results were shown. Scale bar= 30 μm. (E) The protein expressions of iNOS and NOX2 in BMECs ( n = 5). FGL2 +/+ and FGL2 -/- BMECs were treated with LPS (5 μg/ml) or PBS for 24h. The data was expressed as mean ± SEM and was the representative of at least three independent experiments. Statistical differences were assessed by one-way ANOVA with Tukey’s post-hoc test for multiple groups. ns P >0.05, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The primary antibodies used were as follows: FGL2 (1:500, Abnova Cat# H00010875-M01), NOX2 (1:100, Proteintech Cat# 19013-1-AP), iNOS (1:100, Proteintech Cat# 18985-1-AP), vWF (1:100, Abcam Cat#ab11713), CD31 (1:2000, Abcam Cat# ab182981; 1:400, Abcam Cat# ab56299).

Techniques: Immunofluorescence, Staining, Software, Fluorescence

Journal: Frontiers in Immunology

Article Title: FGL2 deficiency alleviates maternal inflammation-induced blood-brain barrier damage by blocking PI3K/NF-κB mediated endothelial oxidative stress

doi: 10.3389/fimmu.2023.1157027

Figure Lengend Snippet: FGL2 deficiency alleviated endothelial oxidative stress via inhibiting the activation of PI3K/NF-κB pathway. (A) The protein expressions of PI3K-p85 in brain at P1 and in cortex at P7 ( n = 7). (B) The protein expressions of NF-κB p65 and its phosphorylated form in brain at P1 and in cortex at P7 ( n = 7). (C) The mRNA level of pik3r1 in BMECs ( n =4:4:5:5). FGL2 +/+ and FGL2 -/- BMECs were treated with LPS (5 μg/ml) or PBS for 24 h. (D) The protein expression of PI3K-p85 in BMECs ( n = 5). (E) The protein expressions of NF-κB p65 and its phosphorylated form in BMECs ( n = 5). (F) The protein levels of NOX2 and iNOS in LY294002-pretreated BMECs ( n = 4). Before LPS (5 μg/ml) stimulation for 24 h, FGL2 +/+ BMECs were pretreated with or without LY294002 (20 μM) for 2 h. (G) The concentration of NO in culture mediums of LY294002-pretreated BMECs ( n = 6). (H) The levels of ROS by DHE staining in LY294002-pretreated BMECs ( n = 3). Representative images were shown, and quantitative results were normalized to controls. Scale bar=100 µm. (I) The protein expressions of flag, NOX2, iNOS, p-p65, and t-p65 in FGL2 -/- BMECs ( n = 4). FGL2 -/- BMECs were infected by Lenti-PI3K-flag or Lenti-ZsGreen for 48 h before treatment of LPS (5 μg/ml) for 24 h. (J) The protein levels of p-p65 and t-p65 in LY294002-pretreated BMECs ( n = 3). The data was expressed as mean ± SEM and was the representative of at least three independent experiments. Statistical differences were assessed by unpaired two-tailed Student’s t test for two groups and one-way ANOVA with Tukey’s post-hoc test for four groups. Welch’s ANOVA with Dunnett’s T3 post-hoc test was used to compare the protein expression of p-p65/t-p65 among four groups in brain at P1. ns P >0.05, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The primary antibodies used were as follows: FGL2 (1:500, Abnova Cat# H00010875-M01), NOX2 (1:100, Proteintech Cat# 19013-1-AP), iNOS (1:100, Proteintech Cat# 18985-1-AP), vWF (1:100, Abcam Cat#ab11713), CD31 (1:2000, Abcam Cat# ab182981; 1:400, Abcam Cat# ab56299).

Techniques: Activation Assay, Expressing, Concentration Assay, Staining, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: FGL2 deficiency alleviates maternal inflammation-induced blood-brain barrier damage by blocking PI3K/NF-κB mediated endothelial oxidative stress

doi: 10.3389/fimmu.2023.1157027

Figure Lengend Snippet: FGL2 deficiency attenuated blood-brain barrier disruption in maternal inflammation-exposed offspring. FGL2 deficiency downregulates the maternal inflammation-induced expressions of NOX2 and iNOS in BMECs via inhibiting PI3K/NF-κB pathway, which decreases the accumulation of ROS and NO. The reduced oxidative stress subsequently alleviates the declines of tight junction proteins (Claudin-5, Occludin, and ZO-1), thereby restoring permeability of blood-brain barrier and alleviating neuroinflammation in maternal inflammation-exposed offspring.

Article Snippet: The primary antibodies used were as follows: FGL2 (1:500, Abnova Cat# H00010875-M01), NOX2 (1:100, Proteintech Cat# 19013-1-AP), iNOS (1:100, Proteintech Cat# 18985-1-AP), vWF (1:100, Abcam Cat#ab11713), CD31 (1:2000, Abcam Cat# ab182981; 1:400, Abcam Cat# ab56299).

Techniques: Disruption, Permeability

Journal: Signal Transduction and Targeted Therapy

Article Title: Porphyromonas gingivalis aggravates atherosclerotic plaque instability by promoting lipid-laden macrophage necroptosis

doi: 10.1038/s41392-025-02251-6

Figure Lengend Snippet: Pg -enlarged oxidative stress mostly results from enhanced MSR1-mediated ox-LDL uptake. a TEM images of the aortic arch plaque of Apoe −/− mice infected with or without Pg for 8 weeks. Scale bar = 2 μm on the left, and 500 nm on the right. b Flow cytometry analyses of the ratio of PI + macrophages and the ox-LDL accumulation in PI + macrophages under ox-LDL (60 μg/mL) and Pg (MOI = 100) challenge for 24 h. n = 4 per group. c qRT-PCR analyses of Cd36 , Msr1 , Olr1 expression in macrophages treated with or without ox-LDL (60 μg/mL) and/or Pg (MOI = 100) for 24 h. β-actin was used as control. n = 4–6 per group. Western blot analyses of MSR1, CD36, OLR1, or GAPDH expression in ox-LDL (60 μg/mL)-loaded macrophages treated with Pg (MOI = 100) ( d ), or RgpA, RgpB, or Kgp (1 μg/mL) ( e ) for 24 h. GAPDH was used as the loading control. The co-localization of MSR1 and CD45-labled macrophages in the plaques of Apoe –/– mice aortic roots ( f ), rabbit aortic arches ( g ), and human coronary vessels ( h ). Scale bar = 50 μm. Flow cytometry analyses of the ox-LDL uptake ( i ), ROS production ( j ), and the ratio of PI + cells ( k ) in Msr1 -knockdown macrophages loaded with ox-LDL (60 μg/mL) and infected with Pg (MOI = 100). n = 4 per group. Flow cytometry analyses of the ox-LDL uptake ( l ), ROS production ( m ), and the ratio of PI + cells ( n ) in fucoidan (40 μg/mL) pretreated macrophages loaded with ox-LDL (60 μg/mL) and challenged by Pg (MOI = 100). n = 4 per group. o The co-staining of p-MLKL and F4/80 (macrophage marker) in the aortic root plaques from Apoe −/− mice treated with fucoidan and/or Pg for 8 weeks. Scale bar = 100 μm. p H&E and Oil Red O staining of aortic root plaques from Apoe −/− mice treated with fucoidan and/or Pg for 8 weeks. Quantitation of the plaque size, as well as the ratios of necrotic area and Oil Red O + area to plaque area were at right. n = 6 per group. Scale bar = 200 μm in H&E, scale bar = 100 μm in Oil Red O. Results were expressed as mean ± SD ( b , c , i – n ) or mean ± SEM ( p ). Data were analyzed by unpaired two-tailed student t -test ( b ), two-way ANOVA ( c ) or one-way ANOVA ( i – n , p ). **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: MSR1 (A2401, Abclonal), p-MLKL ( Ab196436 , Abcam), FOXO3 (10849-1-AP, Proteintech), NOX2 (19013-1-AP, Proteintech) expression, and gingipain (CSB-PA338957LA01PQP, Cusabio) existence in macrophages were assessed by immunofluorescence staining.

Techniques: Infection, Flow Cytometry, Quantitative RT-PCR, Expressing, Control, Western Blot, Knockdown, Staining, Marker, Quantitation Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Porphyromonas gingivalis aggravates atherosclerotic plaque instability by promoting lipid-laden macrophage necroptosis

doi: 10.1038/s41392-025-02251-6

Figure Lengend Snippet: Proteolysis of FOXO3 by gingipains upregulates the transcription of MSR1. a GO enrichment analysis of differential proteins between ox-LDL (60 μg/mL)-loaded macrophages treated with Pg (MOI = 100) versus PBS for 24 h ( n = 4 per genotype). P < 0.05 represented statistically significant difference, and upregulated or downregulated proteins were identified with a fold-change ( Pg /PBS) > 1.15 or <0.87. qRT-PCR ( b ) and Western blot ( c ) analyses of TPT1, BNIP3, FOXO3, CD74, HDAC4, PDCD4, and TAX1BP1 expression in ox-LDL (60 μg/mL)-loaded macrophages infected with Pg or KDP136 (MOI = 100) for 24 h. β-actin was used as control, and n = 4 per group ( b ). GAPDH was employed as the loading control in ( c ). Western blot analyses ( d ) and immunohistochemical staining ( e ) of FOXO3 in ox-LDL (60 μg/mL)-loaded macrophages infected with Pg , KDP136 (MOI = 100), RgpA, RgpB, Kgp, or Pg -LPS (1 μg/mL) for 24 h. GAPDH was employed as the loading control in ( d ). Scale bar = 10 μm in ( e ). The co-staining of FOXO3 and F4/80 (macrophage marker) in the plaques of rabbit aortic arches ( f ), and mouse aortic roots ( g ). Scale bar = 20 μm. h Silver staining of recombinant human FOXO3 (rFOXO3, 207.9 μM) incubated with RgpA, RgpB or Kgp (2.1 μM) for 60 min at 37 °C. Red arrowhead points to original rFOXO3. Blue arrowhead points to RgpA. Orange arrowhead points to RgpB. Green arrowhead points to Kgp. Dashed lines encircle rFOXO3 fragments. i Western blot analyses of MSR1 expression in Foxo3 siRNA (si- Foxo3 ) transfected macrophages loaded with ox-LDL (60 μg/mL) and stimulated with RgpA, RgpB, or Kgp (1 μg/mL) for 24 h. GAPDH was used as control. Flow cytometry analyses of the ox-LDL uptake ( j ), ROS production ( k ), and the ratio of PI + cells ( l ) in si- Foxo3 transfected macrophages treated with RgpA, RgpB, or Kgp (1 μg/mL) in the presence of ox-LDL (60 μg/mL). n = 4 per group. m Flow cytometry analyses of the ratio of PI + cells in si- Foxo3 transfected macrophages pre-administrated with or without fucoidan (40 μg/mL) and exposed to ox-LDL (60 μg/mL) for 24 h. n = 4 per group. n Flow cytometry analyses of the proportion of PI + cells in Msr1 and/or Foxo3 knockdown macrophages treated with ox-LDL (60 μg/mL) for 24 h. n = 4 per group. Results were expressed as mean ± SD. Data were analyzed by two-way ANOVA ( b ) or one-way ANOVA ( j–n ). **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: MSR1 (A2401, Abclonal), p-MLKL ( Ab196436 , Abcam), FOXO3 (10849-1-AP, Proteintech), NOX2 (19013-1-AP, Proteintech) expression, and gingipain (CSB-PA338957LA01PQP, Cusabio) existence in macrophages were assessed by immunofluorescence staining.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Infection, Control, Immunohistochemical staining, Staining, Marker, Silver Staining, Recombinant, Incubation, Transfection, Flow Cytometry, Knockdown

Journal: Signal Transduction and Targeted Therapy

Article Title: Porphyromonas gingivalis aggravates atherosclerotic plaque instability by promoting lipid-laden macrophage necroptosis

doi: 10.1038/s41392-025-02251-6

Figure Lengend Snippet: HDAC2 is recruited to the promoter by FOXO3 and inhibits MSR1 transcription. a Potential FOXO3 binding sites on mouse Msr1 promoter predicted by JASPAR database. b CHIP was conducted using FOXO3 antibody and IgG (negative control). c Vector information of the wild-type Msr1 promoter (−496 /−490) and its mutation/depletion mutants. d Dual luciferase reporter assay of the activities of the wild-type Msr1 promoter, and its mutation and depletion mutants in macrophages loaded with ox-LDL (60 μg/mL). n = 4 per group. e Dual luciferase reporter assay of the activities of the wild-type Msr1 promoter in macrophages loaded with ox-LDL (60 μg/mL) and infected with Pg or KDP136 (MOI = 100). n = 4 per group. f , g CHIP-PCR analyses of the histone H3 and histone H4 acetylation level in ox-LDL (60 μg/mL)-loaded macrophages treated with Pg or KDP136 (MOI = 100). n = 4 per group. qRT-PCR analyses of Msr1 expression ( h ), flow cytometry analyses of the ox-LDL uptake ( i ) and the ratio of PI + cells ( j ) in ox-LDL (60 μg/mL)-loaded macrophages pre-administrated with TSA (1 μM) and challenged with Pg (MOI = 100) for 24 h. n = 4 per group. k FOXO3 and HDAC2 binding detection by Co-IP in ox-LDL (60 μg/mL) loaded macrophages infected with Pg or KDP136 (MOI = 100) for 24 h. l FOXO3 and HDAC2 binding detection by Co-IP in si- Foxo3 transfected macrophages upon ox-LDL (60 μg/mL) stimulation for 24 h. m Schematic diagram showing the mechanism of Pg -promoted macrophage necroptosis. In macrophages, gingipains secreted by Pg directly hydrolyze FOXO3 in the cytoplasm, thereby inhibiting its nuclear translocation. Additionally, Pg -LPS activates MAPK/ERK-mediated phosphorylation of FOXO3, promoting its nuclear export, and subsequent ubiquitination (Ub) and degradation. The reduction in FOXO3 levels diminishes HDAC2 binding to the MSR1 promoter and increases promoter acetylation, leading to upregulation of MSR1 expression. This enhanced MSR1 expression promotes lipid uptake and the following oxidative stress, ultimately triggering macrophage necroptosis. Diagram is generated by figdraw.com. Results were represented as mean ± SD. Data were analyzed by one-way ANOVA ( d , e , h , i , j ), or Kruskal–Wallis test ( f , g ). **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: MSR1 (A2401, Abclonal), p-MLKL ( Ab196436 , Abcam), FOXO3 (10849-1-AP, Proteintech), NOX2 (19013-1-AP, Proteintech) expression, and gingipain (CSB-PA338957LA01PQP, Cusabio) existence in macrophages were assessed by immunofluorescence staining.

Techniques: Binding Assay, Negative Control, Plasmid Preparation, Mutagenesis, Luciferase, Reporter Assay, Infection, Quantitative RT-PCR, Expressing, Flow Cytometry, Co-Immunoprecipitation Assay, Transfection, Translocation Assay, Phospho-proteomics, Ubiquitin Proteomics, Generated