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. 2021 Dec 17;71(9):1843–1855. doi: 10.1136/gutjnl-2021-325180

Figure 2.

Figure 2

Reprogramming of activated PSCs and downregulation of antiapoptotic proteins in PDAC cells by NO. (A) The mRNA expression levels of a panel of 84 key fibrosis-associated genes in primary, culture-activated human PSCs after 24 hours of treatment with DNIC (0.5 µM) were measured with an RT2 Profiler PCR array. The results are expressed as the fold change relative to the corresponding level in the untreated control group (n=2). (B) Expression levels of myofibroblast activation markers in primary, culture-activated human PSCs after 24 hours of treatment with DNIC (0.5 µM) were measured by RT-qPCR. The results are expressed as the fold change relative to the corresponding level in the untreated control group (n=5). (C) Western blotting was used to analyse α-SMA and collagen I protein expression as well as downstream TGFβ signalling activation (phospho-AKT, phospho-NF-κB and phospho-IκBα levels) in primary, culture-activated human PSCs treated with or without increasing concentrations of DNIC. The experiments were repeated two times independently. (D) Expression levels of proinflammatory cytokines in primary, culture-activated human PSCs in a coculture system with PDAC cells (2×105 AsPC-1 cells) after 24 hours of treatment with DNIC (2 µM) were measured by RT-qPCR. The results are expressed as the fold change relative to the corresponding level in the untreated control group (n=3). (E) Western blotting was used to analyse p53, Bcl-xL and Bcl-2 expression in AK4.4 cells. The experiments were repeated two times independently. (F) Dox (2 or 4 µM) in combination with DNIC (2 µM) significantly enhanced the induction of apoptosis in murine AK4.4 and human AsPC-1 PDAC cells, as detected using annexin V staining (n=3). (G) Recombinant TRAIL (2000 ng/mL) in combination with DNIC (2 µM) significantly enhanced the induction of apoptosis in murine AK4.4 and human AsPC-1 PDAC cells, as detected using annexin V staining (n=4). DNIC, dinitrosyl iron complexes; ECM, extracellular matrix; IL, interleukin; NO, nitric oxide; PDAC, pancreatic ductal adenocarcinoma; PSC, pancreatic stellate cells; SMA, smooth muscle actin; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.