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. 2021 Oct 7;9:671564. doi: 10.3389/fcell.2021.671564

FIGURE 6.

FIGURE 6

P16 deletion improved up-regulated proinflammatory level of macrophages induced by change of intestinal microbiota ameliorated in Bmi-1–/– mice. Fecal mixed bacteria (FB) were prepared using the fecal of 7-week-old WT and Bmi-1–/– mice to infect BMDMs from WT, Bmi-1–/–, and Bmi-1–/–p16–/– mice. (A) Representative micrographs showing immunofluorescence for F4/80 after Dil-labeled WT-FM–infected BMDMs, with DAPI staining the nuclei, red dots showing Dil-labeled WT-FB, and green dots showing F4/80. (B) Percentage of DiI and F4/80 double-positive cells. (C–I) Tumor necrosis factor (TNF-α), p16, interleukin (IL-1β), IL-6, NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) and monocyte chemoattractant protein 1 (MCP1) and engulfment and cell motility protein 1 (ELMO1) mRNA levels in BMDMs by real-time RT-PCR, calculated as the ratio to β-actin mRNA and expressed relative to WT. (J) Western blots of BMDM extracts showing TNF-α, NF-κB–p65, IκBα, and p-IκBα (Ser32); β-actin was used as the loading control. (K) Protein levels relative to β-actin were assessed by densitometric analysis. Cell experiments were performed with three biological repetitions per group. Statistical analysis was performed with one-way ANOVA test. Values are mean ± SEM from six determinations per group, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the WT group at the same treatment; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with the Bmi-1–/– group at the same treatment; &p < 0.05, &&p < 0.01, &&&p < 0.001 compared with the same genotyped BMDMs of vehicle-FM group; +p < 0.05, ++p < 0.01, +++p < 0.001 compared with the same genotyped BMDMs of the WT-FM group.