Fig 2: CLB-inhibited NF-κB and ERK1/2 signaling pathways. A, B IKKα/β, p-IKKα/β, NF-κB p65, and p-NF-κB p65 expressions were increased significantly after 48 h of differentiation and decreased after CLB (100 μM) treatment (F = 45.315, 9.154, 123.457, and 13.449, all P < 0.001). C, D The JNK, p38 MAPK, and ERK1/2 levels were increased significantly after 48 h of differentiation and decreased after CLB (100 μM) treatment (F = 247.631, 154.638, and 14.324, all P < 0.001)

Fig 3: Erastin activates ferroptosis by targeting NF-κB and MAPK signaling (A) Western blotting showed that erastin affected NF-κB signal-related proteins IKK/p-IKK, p-IκBα, and P65/p-P65 (A, B) and MAPK signal-related proteins p-JNK/JNK, p-ERK/ERK and p-P38/P38 (D, E). Overexpression of FOXO3 inhibited erastin-activated NF-κB (C) and MAPK (D) signaling (G) Effect of intracellular overexpression of FOXO3 on NF-κB/MAPK signaling pathway. (H) Protein levels of GPX4 and SLC7A11 in ATDC5s treated with erastin (10 ​μM) and MAPK-IN-1 (10 ​μM) (I) Semiquantitative analysis of GPX4 and SLC7A11 expression based on WB analysis. (J) Protein levels of GPX4 and SLC7A11 in ATDC5s treated with erastin (10 ​μM) and NF-κB-IN-1 (50 ​μM) (K) Semiquantitative analysis of GPX4 and SLC7A11 expression based on WB analysis. (L, N) MDA levels in ATDC5 cells were quantitatively determined using an MDA assay kit. (M, O) GSH levels in ATDC5 cells were quantitatively determined using an GSH assay kit. ∗P ​< ​0.05; ∗∗P ​< ​0.01; ∗∗∗P ​< ​0.001; ∗∗∗∗P ​< ​0.0001. All data are from n ​= ​3 independent experiments.

Fig 4: OXCT1 promoted GEM resistance through the NF-κB signaling pathway. (A) The TCGA dataset was subjected to GSEA on the basis of OXCT1 expression. (B) The expression levels of p-IKKβ, p-Ikb-α, and p-P65 in MIA PaCa-2 cell lines with OXCT1 overexpression and knockdown were detected using western blot analysis. (C) Western blot analysis of the fractionated BxPC-3 and MIA PaCa-2 cells stably transfected with the empty vector or OXCT1. (D–F) BxPC-3 and MIA PaCa-2 cell lines with or without OXCT1 overexpression were treated with GEM or the combination of GEM and BAY 11-7082. Cell cytotoxicity was analyzed using RTCA (D). Cell cloning capability was analyzed using the colony formation assay (E). Apoptosis was detected via flow cytometry (F). The data are expressed as mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way ANOVA). NS, not significant.

Fig 5: PARP1 and NFκB interaction is important for transcriptional control of CCL2. (A) Quantitative real-time PCR for relative expression of CCL2 mRNA after NFκB p65 inhibitor Bay 11-7081 treatment in MB-231 cells. Cells were rhCCL2-treated at the indicated doses for 4 h. * p = 0.001, ** p = 0.0002, one way ANOVA and Tukey’s comparison with untreated (B) Left: Western blot showed activation of NFκB pathway probed with an antibody against phosphorylated P65 S536. MB-231 cells were pretreated with PJ34 overnight, and TNFα was added for 45 min. Right: Electrophoretic mobility shift assay (EMSA) with p65 probe. The nuclear lysate was prepared from PJ34 treated or untreated cells, incubated with p65 specific biotinylated DNA probe. Bound and unbound probes were visualized using HRP-conjugated streptavidin chemiluminescence. (C) PARP1 and NFκB interact in MB-231cells. Immunoprecipitation was done with the P65 antibody using nuclear lysate. Lysate and IP fractions were western blotted and probed with PARP1 and P65 specific antibodies. (D) Chromatin IP with PARP1 and P65 specific antibody. Fixed chromatin was immunoprecipitated with the antibody, as mentioned earlier, after overnight treatment with 10 µM PJ34. We performed PCR on a region flanking an NFκB binding motif at the CCL2 promoter (Dotted arrows showing the approximate location of the primers). Percent of input shows enrichment. Average of two independent experiments shown. t-test * p < 0.01. Whole western blots for 4B and 4C are in Figure S10.