Fig 2: (A) H&E staining was performed to assess pathological infiltration and ischemic areas of the myocardial tissues. (B) Tetrazolium chloride staining was used to detect the ischemic areas of the brain tissue. (C) Protein and (D) mRNA expression levels of CPNE3 and RACK1 in I/R-induced rat tissues. **P<0.01, ***P<0.001. CPNE3, copine 3; RACK1, receptor for activated C kinase 1; I/R, ischemia/reperfusion.

Fig 3: Transfection efficiency of Ov-CPNE3 was detected by (A) western blotting and (B) RT-qPCR. CPNE3 expression in H/R-induced H9c2 cell after transfection with Ov-CPNE3 was detected by (C) western blotting and (D) RT-qPCR. RACK1 expression in H/R-induced H9c2 cells after transfection with Ov-CPNE3 overexpression plasmid was detected by (E) western blotting and (F) RT-qPCR. (G) Relative enrichment of CPNE3 and RACK1 in H/R-induced H9c2 treated with anti-CPNE3 was detected by performing an immunoprecipitation assay. ***P<0.001. Ov, overexpression; CPNE3, copine 3; RT-qPCR, reverse transcription-quantitative PCR; H/R, hypoxia/reoxygenation; RACK1, receptor for activated C kinase 1; NC, negative control.

Fig 4: Schematic model by which RACK1 promotes the progression of OSCC. RACK1 inhibits the activation of NF‐κB, regulates the expression and secretion of proinflammatory factors and macrophage chemokines, inhibits the massive recruitment of macrophages and severe inflammatory reactions, induces a chronic smoldering inflammation microenvironment and promotes the development of tumors.

Fig 5: RACK1 inhibits macrophage activation but increases the proportion of M2 macrophages. (A) Immunoblots of p‐mTOR, CREB, p‐CREB, PPARγ, p‐PPARγ, p‐ERK, STAT1, p‐STAT1, c‐Jun, STAT3, NF‐κB and p‐NF‐κB in macrophages, induced from THP‐1 cells by 100 ng·mL−1 PMA for 24 h, cocultured with infected OSCC supernatants for the indicated times (0, 0.25, 0.5, 1 and 2 h). (B) Macrophages were induced from THP‐1 cells by PMA following incubation with transfected OSCC supernatants (sh‐NC, sh‐RACK1, OE‐Vec or OE‐RACK1) for 8 h. After centrifugation, the macrophages were stained with CD206 and CCR7 antibodies. The percentages and cell numbers of different macrophages were analyzed using flow cytometry. (C) Analysis of the M2/M1 ratio of different THP‐1‐induced groups detected by flow cytometry (mean ± SD; *P < 0.05). (D) Single cells were isolated from tumor tissues using collagenase IV and then stained with CD206, CD11b and F4/80 antibodies. The percentages and cell numbers of macrophages were analyzed using flow cytometry. R5: CD11b‐positive macrophages; E6: both CD206‐ and F4/80‐positive macrophages (M2). (E) Analysis of the numbers of M0 and M2 macrophages in tumor tissues from different groups detected by flow cytometry (mean ± SD; *P < 0.05, **P < 0.01).