Fig 2: The p65 activation in CAC. (a) The phosphorylation of p65 detected in PFKFB3 overexpression and knockdown. (b) The nuclear translocation of p65 detected in PFKFB3 overexpression and knockdown; scar bar = 20 µm. (c)-(d) The expression of IL-1ß and TNF-a level by PFKFB3 overexpression and p65 knockdown in IECs. (e) The proliferation of CRC by PFKFB3 overexpression and p65 knockdown in IECs. (f) The invasion of CRC by PFKFB3 overexpression and p65 knockdown in IECs; scar bar = 50 µm. *P < 0.05, **p < 0.01.

Fig 3: ERK-mediated PFKFB3 phosphorylation at the O-GlcNAcylation site.a–c In vitro phosphorylation analyses were performed by mixing the purified ERK1 with the indicated purified GST-PFKFB3 proteins or mutant proteins in the presence of [?-32P]ATP (a, b). d The indicated purified PFKFB3 WT or mutant proteins were analysed by enzymatic activity assay. e, f The indicated purified GST–PFKFB3 protein, or pre-incubated with purified OGT (f), was mixed with purified His–G3BP2 protein with or without purified ERK1. GST pulldown analyses were performed. g SW1990 cells with indicated shRNA expression were pretreated with or without PUGNAc (20 µM) for 24 h and cultured for 12 h under hypoxia or normoxia. In a–c, and e–g, immunoblotting analyses were performed using the indicated antibodies. In d, the values are presented as mean ± s.e.m. (n = 3 independent experiments).

Fig 4: Macrophage GIT1 is critical to successful bone regeneration via regulating inflammatory responses in an ERK/NRF2-dependent way. In response to LPS, once GIT1 is involved, it promotes NRF2 to stabilize and translocate into the nucleus for genes transcriptionally regulation via phosphorylating ERK. In the absence of GIT1, inactivated ERK/NRF2 axis could lead to abnormal upregulation of ROS production and IL1ß secretion and glycolysis, which is detrimental to the osteogenic differentiation of BMSCs. Furthermore, macrophage GIT1-mediated activation of ERK/NRF2 controls glycolysis by limiting PFKFB3 expression.

Fig 5: IL1ß production and glycolysis of macrophage in response to LPS were governed by GIT1 in an ERK/NRF2-dependent manner. (A) Immunoblot images showing protein levels of NRF2 (nuclear), NRF2 (total), HO1, NQO1, IL1ß, and ß-Actin of BMDMs in indicated groups. NRF2-siRNA (si-1 and si-2) could effectively increase the expression level of IL1ß proteins, and DEM-treated cells limited IL1ß production. (B) LPS-induced ECAR in BMDMs after treatment with NRF2-siRNA or DEM, as indicated. (C, D) Quantification of glycolysis (C) and glycolytic capacity (D) were revealed in indicated groups (one-way ANOVA with post hoc test). (E) In response to LPS, altered protein expression levels of PFKFB3 in BMDMs and RAW264.7 cells when blocking ERK using SCH772984 were detected via Western blotting. (F) After treated with LPS, the protein expression patterns of PFKFB3 in GIT1fl/fl BMDMs with or without NRF2 silencing and GIT1 CKO BMDMs with or without DEM treatment were shown. (G) CHIP-qPCR analysis of NRF2-binding regions (peaks 1 and 2) in GIT1fl/fl BMDMs with or without LPS treatment (two-way ANOVA with post hoc test). DEM = diethyl maleate; CHIP-qPCR = chromatin immunoprecipitation with quantitative polymerase chain reaction.