Fig 2: Knockdown of Fer activates AMPK and downregulates ERK1/2 in PDAC cells. (A) SU.86.86. cells were transfected with control siRNA (siRNAc), or fer-targeting siRNA (siRNAfer) and incubated in MEM supplied with 2 mM L-glutamine, for 72 hr. Cells were then harvested, and their lysates were resolved in SDS-PAGE and subjected to: anti-Fer (SH2), anti-pospho-ERK1/2 (Thr.202/Tyr204), anti-ERK1/2, anti-phspho-TSC2 (Ser 664), anti-TSC, anti-phospoh-AMPK (Thr172), anti-AMPK, anti-phospho-mTOR (Ser 2448), anti-mTOR, and anti-Tubulin, in a WB analysis. One out of three independent experiments that gave similar results is presented. (B) Summarizing scheme depicting the two regulatory pathways affected by E260 and converging to the downregulation of mTORC1 and mitochondrial function, in PDAC cells.

Fig 3: TMEM176B drives GC progression via the PI3K-Akt-mTOR signaling pathway. A Differentially expressed genes (DEGs) were subjected to KEGG pathway enrichment analysis. B Gene set enrichment analysis (GSEA) of DEGs. C, D WB determined the expression levels of ASNS proteins related to the PI3K-Akt-mTOR signaling pathway in GC cell lines after TMEM176B knockdown or overexpression. E HGC27 cell line with TMEM176B knockdown was treated with cycloheximide (CHX) to detect ASNS expression at various time points. F HGC27 cell line with TMEM176B knockdown was treated with dimethyl sulfoxide (DMSO), rapamycin, 3-methyladenine (3-MA), and MG132 to detect ASNS expression

Fig 4: In vivo experiment corroborating the role of TMEM176B in GC progression. A, B Tumorigenesis was compared between the control and TMEM176B knockdown groups. C, D Differences in tumor volume and mass between control and TMEM176B knockdown groups were measured. E, F WB determined the expression levels of ASNS and proteins related to the PI3K-Akt-mTOR signaling pathway in the control and TMEM176B knockdown groups. G IHC and H&E staining were used to compare proliferation and apoptosis between the control and TMEM176B knockdown groups. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: Suggested molecular signaling events contributing to rFlaA:Betv1-mediated activation of BMDMs. Stimulation of BMDM with aggregated rFlaA:Betv1 results in both TLR5-dependent and -independent uptake, leading to the activation of both MyD88− and MAPK-mediated signaling cascades. rFlaA:Betv1-induced pro-inflammatory IL-6 and TNF-α secretion were shown to be MyD88− and MAPK-dependent, likely promoting the induction of IFN-γ-producing Th1 cells in BMDM:TC co-cultures. Western blot analysis confirmed rFlaA:Betv1 to trigger STAT3 activation, increasing the expression of HIF-1a, which is likely to be responsible for the observed IL-1β secretion. Metabolically, we found a MyD88-dependent phosphorylation of the mTOR target protein p70 S6 kinase, which together with HIF-1a likely mediated the observed shift towards enhanced glycolysis with increased levels of the glucose transporter Glut-1 and PFKFB3, and the induction of the Warburg effect while being paralleled by a decreased expression of mitochondrial proteins such as ACO2 and reduced oxygen consumption rates, suggesting a disrupted Krebs cycle in these cells. Finally, TLR5, MyD88, and MAPK activation were shown to be involved in the rFlaA:Betv1-induced production of the anti-inflammatory cytokine IL-10 that likely caused the observed suppression of rBet v 1-induced Th1 and Th2 responses in BMDM:TC co-cultures.