Journal: PLOS One

Article Title: Ozone ultrafine bubble water sterilizes Porphyromonas gingivalis and neutralizes its virulence factors

doi: 10.1371/journal.pone.0334259

Figure Lengend Snippet: (A) Recombinant RgpA (rRgpA), (B) rRgpB, and (C) rKgp were added to distilled water (DW), air ultrafine bubble water (AUFBW), or 3.50 ppm ozone ultrafine bubble water (OUFBW) followed by SDS-PAGE and silver staining. (D–F) P. gingivalis culture supernatant was exposed to DW, AUFBW, or OUFBW and incubated for 60 min. (D) RgpA, (E) RgpB, and (F) Kgp were detected by western blotting. Representative images are shown. Intensities of western blotting signals for RgpA, RgpB, and Kgp were quantified by densitometry. The data represented means ± SD of triplicate experiments and were evaluated by one-way analysis of variance with Tukey’s multiple comparisons test. †, significant difference compared to DW group at P < 0.05. *, significant difference between the indicated groups at P < 0.05.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST), rinsed with TBST, and incubated with anti-RgpA antibody (CUSABIO, Cat# CSB-PA338957LA01PQP, 1:1000), anti-RgpB antibody (CUSABIO, Cat# CSB-PA310587LA01EYA, 1:1000), anti-Kgp antibody (MyBioSource, Inc., San Diego, CA, USA, Cat# MBS7103929, 1:1000), anti-E-cadherin antibody (Thermo Fisher Scientific, Cat# 20874-1-AP, 1:1000), or anti-IL-6 antibody (Abcam, Cat# ab6672, 1:1000) overnight at 4 °C, followed by incubation with a HRP-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, Beverly, MA, USA, Cat# 7074, 1:3000).

Techniques: Recombinant, SDS Page, Silver Staining, Incubation, Western Blot

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