Fig 2: PNM promotes DC maturation and relieves the immunosuppressive tumor microenvironment. (A and B) Representative flow cytometric plots of CD80+CD86+ cells in DLNs and CD4+Foxp3+ Treg cells in the DLNs and spleen in each group. (C and D) Quantification of CD80+CD86+ cells and CD4+Foxp3+ Treg cells in different group (n = 5). (E) Representative immunohistochemical images of Foxp3 and immunofluorescence of CD11c in tumor sections in different groups (scale bar = 25 µm). Data are shown as the mean ± SEM (*P < 0.05; ***P < 0.001).

Fig 3: Presenting melatonin at histochemical and RNA levels inhibits chronic inflammation in the lungs of mice exposed to LPS. (A–C) Significantly fewer CD45+ T cells, CD11b+ and CD11c+ and F4/80+ macrophages infiltrated into mouse lung bronchi and alveoli when LPS-exposed mice were given melatonin orally daily than non-treated mice. Luzindole (MT1/MT2 inhibitor) increased the infiltration of these inflammatory cells into the mouse lung, even when the mice were treated with melatonin. N = 5–7. (D) Mice treated with melatonin expressed lower levels of Il-6, Il-1β, Ifn-γ, and Tnf-α mRNAs as well as more Gpx, Ho-1, Sod1, and Sod2 mRNAs in the lungs than compared with LPS-exposed mice. Mice treated with luzindole (MT1/MT2 inhibitor) along with melatonin had higher levels of Il-6, Ifn-γ, and Tnf-α and decreased Gpx, Ho-1, and Sod1 mRNA expression in the lungs. N = 4–5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance.

Fig 4: Gemcitabine-cisplatin chemotherapy promotes an overwhelmingly high release of inhibitory DAMP to attenuate DC maturation.a Western blot analysis of COX-2 expression in G69 cell lysates treated with either vehicle or GC for 48 h. GAPDH expression was used as loading control (n = 3 biologically independent experiments). b ELISA for PGE2 release in the culture medium of G69 cells treated with either vehicle control or GC for 48 h (n = 4 independent biological samples). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test **p = 0.0016). c COX-2 expression in G69-tumor tissue lysates treated with either vehicle or GC for two chemotherapy cycles. GAPDH expression was used as a loading control (n = 3 biologically independent experiments). d ELISA for PGE2 release in the serum of G69-tumor-bearing mice treated with either vehicle or GC for 2 cycles (n = 5 and n = 4 biologically independent samples for vehicle and GC, respectively). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test ****p < 0.0001). e Immunofluorescence analysis of COX-2 expression in G69-tumor tissues treated with either vehicle or GC for two chemotherapy cycles (Scale bar: 100 µm; Images representative of n = 3 independent experiments). f Schematic representation of BMDC treatment with conditioned media in the presence or absence of iDAMP blockade. g Flow cytometric analysis of CD40, CD86, and MHCI expression in BMDCs treated with G69-conditioned media with or without iDAMP blockade. h Geometric mean fluorescence intensity (gMFI) of CD40, CD86, and MHCI molecule expression in BMDCs upon treatment with condition media as in g (n = 2 biologically independent samples). Data are presented as mean values. i Schematic representation of footpad vaccination assay. j Flow cytometric analysis of CD40, CD86 and MHCI expression in vaccination-draining lymph node CD11c + MHC2 + DCs upon injection of G69 cells treated with GC in the presence or absence of iDAMP blockade (n = 5 biologically independent samples). Non-vaccination-draining lymph node DCs were used as control (n = 3 biologically independent samples). Data are presented as mean values ± SEM. (One-way ANOVA-Tukey’s multiple comparisons test. CD40: GC vs GC+ Celex, p = 0.045; GC vs GC+ aPGE2, p = 0.036; CD86: GC vs GC+ Celex, p = 0.046; GC vs GC+ aPGE2, p = 0.0007; H2-K: GC vs GC+ Celex, p = 0.0004; GC vs GC+ aPGE2, p = 0.005). Source data are provided as a Source data file. ns not significant.

Fig 5: Nox4 suppresses activation of canonical TGF-β signaling in vivo. (A) Scheme representing the canonical TGF-β signaling pathway. (B) Reverse-transcription quantitative PCR analysis of the expression of indicated TGF-β regulatory genes in the WT or Nox4-/- mouse colon tissue; these mice differ from those used for RNA-seq (n = 3). Black dots represent untreated WT mice, and red dots represent untreated Nox4-/- mice. (C) Active TGF-β level in WT (black) or Nox4-/- (red) colon tissue lysates measured by enzyme-linked immunosorbent assay. (D) Immunoblotting of TGF-β and TGFβR1 protein levels in WT or Nox4-/- colon tissue extracts; β-actin was used as an internal control. The band size was quantified by ImageJ. (E) Immunoblotting of pSmad2/3, Smad2/3, and Smad4 protein levels in cytosolic and nucleic fraction extracts from WT and Nox4-/- colon tissues. Smad2/3 was used as the pSmad2/3 control proteins, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HDAC were used as the cytosol and nucleus control proteins, respectively. The band size was quantified by ImageJ. Representative immunofluorescence images of WT or Nox4-/- colon sections stained with (F) TGF-β (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue), or (G) TGF-β (green), CD11c (violet), and DAPI. Data are expressed as means ± SD. ∗∗P < .01, and ∗∗∗∗P < .001 compared with WT. Cy, Cytosel; KO, Knock-out; LP, Lamina propria; Nu, Nucleus; P, phosporelated.