Fig. 6 | Signal Transduction and Targeted Therapy

Fig. 6

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

Fig. 6The alternative text for this image may have been generated using AI.

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

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