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: Gingipains play important roles in Pg -aggravated oxidative stress. Flow cytometry analyses of the ROS production ( a ) and the ratio of PI + cells ( b ) in ox-LDL (60 μg/mL) loaded macrophages infected with Pg or KDP136 (MOI = 100). n = 4 per group. c Western blot analyses of RIPK3, p-MLKL, MLKL, and GAPDH expression in ox-LDL (60 μg/mL)-loaded macrophages infected with Pg or KDP136 (MOI = 100) for 24 h. GAPDH was used as the loading control. Flow cytometry analyses of the ox-LDL uptake ( d ), the ROS production ( e ), and the ratio of PI + cells ( f ) in ox-LDL-loaded macrophages exposed to RgpA, RgpB, or Kgp (1 μg/mL). n = 5 per group in ( d ), n = 4 per group in ( e , f ). g Western blot analyses of RIPK3, p-MLKL, MLKL, and GAPDH expression in ox-LDL (60 μg/mL)-loaded macrophages treated with RgpA, RgpB, or Kgp (1 μg/mL) for 24 h. GAPDH was used as the loading control. h The co-staining of RgpA and CD68 (macrophage marker) in the coronary plaques of human. Scale bar = 20 μm. i H&E, Oil Red O staining, and CD45 and F4/80 co-staining of the aortic root plaques from Apoe −/− mice infected with or without Pg or KDP136 for 8 weeks. Scale bar = 200 μm in H&E, scale bar = 100 μm in the rest images. j Quantitative analyses of plaque size, necrotic area, Oil Red O + area, CD45, and F4/80-positive areas of the plaques in i . n = 6 per group. Results were presented as mean ± SD ( a , b , d , e , f ) or mean ± SEM ( j ). All data were analyzed by one-way ANOVA. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: 207.9 μmol/L recombinant human FOXO3 (CSB-EP008836HU1, Cusabio) was incubated with 2.1 μmol/L RgpA (ab225982, Abcam), RgpB (CSB-EP310587EYA, Cusabio) or Kgp (CSB- EP690409PQP1, Cusabio) (37 °C, 60 min), followed by SDS-PAGE.

Techniques: Flow Cytometry, Infection, Western Blot, Expressing, Control, Staining, Marker

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: 207.9 μmol/L recombinant human FOXO3 (CSB-EP008836HU1, Cusabio) was incubated with 2.1 μmol/L RgpA (ab225982, Abcam), RgpB (CSB-EP310587EYA, Cusabio) or Kgp (CSB- EP690409PQP1, Cusabio) (37 °C, 60 min), followed by SDS-PAGE.

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: 207.9 μmol/L recombinant human FOXO3 (CSB-EP008836HU1, Cusabio) was incubated with 2.1 μmol/L RgpA (ab225982, Abcam), RgpB (CSB-EP310587EYA, Cusabio) or Kgp (CSB- EP690409PQP1, Cusabio) (37 °C, 60 min), followed by SDS-PAGE.

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