Fig 2: Schematic model for BA-mediated acceleration of diabetic wound healing. BA Betulinic acid; eNOS endothelial nitric oxide synthase, GLUT4 glucose transporter type 4, NFκB p65 nuclear factor NFκB p65 subunit, Nrf2 nuclear factor erythroid 2-related factor 2

Fig 3: Inhibition of the NFκB pathway reduces the abnormal up-regulation of Gal3. a, b Primary microglia harvested from R6/2 mice and their littermate controls (WT) were cultured for 24 h and then treated with Bay11-702 (3 µM) or vehicle (0.1% DMSO) as indicated for 24 h and were then fixed for immunofluorescence staining of Gal3 (green), IbaI (gray), and p65 (red). Nuclei were stained with Hoechst (blue). The localization of nuclei in the right-most panels is outlined by dotted lines. The color bars labeled p65 intensity represent the level of p65 intensity, from low to high fluorescence signals (blue → red, respectively). c An NFκB transcription factor assay was performed on the nuclear extracts prepared from the indicated primary microglia (n = 4). d ELISA was performed on the supernatants collected from the indicated primary microglia to measure the levels of IL1β, IL6, TNFα, and IL10 released by the cells. One dot represents the mean value of each sample. e The levels of nitrite (NO) in the supernatants were measured using the Griess reagent (n = 5). The results were analyzed by two-way ANOVA followed by Tukey’s post hoc test. Data are presented as the means ± SEM. *Specific comparison between WT and R6/2 cells of the same treatment; #Specific comparison between the Bay11-treated and DMSO-treated groups of the same genotype; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Same P-value denotation for #. Scale bar: 10 µm. Source data is available as a Source Data File

Fig 4: Tumour-secreted IL-1β induced nuclear ESE3 (PSCs) expression by activating NF-κB.a Western blot analysis of IL-1β expression in SW1990, MIA-PaCa-2 and BxPC-3 cell lines treated with Gem (10 μM) for 24 h. b ELISA of IL-1β secretion in the CMs of SW1990, MIA-PaCa-2 and BxPC-3 cell lines treated with Gem (10 μM) for 24 h. c Western blot analysis of ESE3 in ihPSCs, hpPSCs and mpPSCs treated with recombinant human IL-1β (100 ng/mL, 24 h). d Analysis of NF-κB (p65) transcriptional activities in ihPSCs and hpPSCs treated with recombinant human IL-1β (100 ng/mL, 24 h) and/or NF-κB (p65) inhibitor (Bay11, 8 μM; 12 h) by commercial kit. e Western blot analyses of p-p65 and ESE3 in ihPSCs cultured with CM (SW1990 and MIA-PaCa-2 cell lines treated with Gem) and/or IL-1β-neutralising antibody for 24 h. f Western blot analyses of p-p65 and ESE3 expression in nucleus and cytoplasm separation from the whole protein of PSCs treated with recombinant human IL-1β and/or Bay11. g RT-PCR analysis of ESE3 mRNA expression in ihPSCs, hpPSCs and mpPSCs treated with recombinant human IL-1β and/or Bay11. h Schematic of the structure of the ESE3 gene promoter. Shown is one κB-binding site and its location (upper). ChIP analysis of NF-κB binding to the ESE3 promoter in ihPSCs (lower). i Luciferase assay-based promoter activity analysis of HEK293 (left) and ihPSCs (right) knockdown NF-κB (sip65) and control cells (siNC) transfected with pGL3-ESE3, pGL3-Empty Vector (pGL3-EV) and pGL3-Mutation (pGL3-MUT). The cells were subjected to dual-luciferase analysis 48 h after transfection. The results are expressed as fold induction relative to that in the corresponding cells transfected with the control vector after the normalisation of firefly luciferase activity according to Renilla luciferase activity. The data are expressed as means ± SD from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 5: Knockdown of CISD2 expression augmented activation of NFκB and enhanced apoptosis in non-stressed EOC microglial cells. (A) Expression levels of p65 (total) were determined by western blot analysis (n = 3 in each group). (B) Evaluation of P65-NFκB DNA binding activity. The nuclear proteins were prepared, and the NFκB DNA binding activity was analyzed by ELISA (n = 3 in each group). (C) The extent of anti-apoptosis reflecting BCL2 mRNA expression was determined by real-time qRT-PCR. Vertical bars indicate the mean ± (SEM) of mRNA or protein expression (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001 indicate statistically significant difference compared to scrambled control cells.