Fig 2: Association of RelB and Tat binding to the HIV-1 LTR. a Schematic illustration of the HIV-1 LTR and location of oligonucleotide primers. ChIP primers for HIV-1 LTR analysis were designed to span the enhancer region, including the duplicated κB enhancers (− 104 to − 81) and specificity protein 1 binding sites (SP1, − 79 to − 47). Primers for analysis of initiated HIV-1 transcripts were directed against TAR. b–d TZM-bl cells were transfected with pFlag-Tat (6 μg) or vector plasmids. The relative expression of Tat and RelB in the transfected cells was monitored by a western blot assay (b). Chromatin of the fixed TZM-bl cells (1 × 107) was immunoprecipitated with the indicated antibodies. Samples were assessed for enrichment of HIV-1 LTR DNA by PCR (c). Relative levels of the LTR DNA that were co-precipitated with RNA Pol II, RelB, and p52 were quantified by real-time PCR performed in triplicate. The data are normalized to the input controls. Data are representative of three independent experiments (d). e–h Control and RELB−/− TZM-bl cells were transfected with pFlag-Tat (6 μg) or vector plasmids (e). The co-precipitated LTR DNA was assessed by PCR (f). Tat (g) and RNA Pol II (h) associated HIV-1 LTR was quantified by real-time PCR

Fig 3: Entinostat enhances the production of TNF-α through acetylation of TNF-α promoter region. (A) There are three NFkB binding sites and 6 SP3 binding sites on the TNF-α promoter region. (B) Chromatin-IP (ChIP) qPCR assay using anti-acetylated histone H3 (AcH3) antibody. All isotype controls (IgG) were under 0.01% of input. DNA samples we corrected from OVCAR8 cells after tolinapant (25 μM) alone or in combination with entinostat (2 μM) 18 h treatment. (* p < 0.05). (C) ChIP qPCR assay using anti-NFkB (RelA, p50, RelB, p52) antibodies. DNA samples we corrected from OVCAR8 cells after tolinapant (25 μM) alone or in combination with entinostat (2 μM) 18 h treatment. (D) ChIP qPCR assay using anti-SP3 antibody. DNA samples we corrected from OVCAR8 cells after tolinapant (25 μM) alone or in combination with entinostat (2 μM), 18 h treatment. (E) Genomic locations of transcription factor binding sites in the promoter region for the TNF-α gene.

Fig 4: NFκB2 is required for FAS transactivation. A) Gel shift assay was performed to observe the retardation of GST, GST‐p50 or GST‐p52 to mobility of DNA probe covering 3′‐2a regions of FAS promoter. Probe and protein concentration were 20 nm. B) Gel shift assay was performed with probe 3′‐2a, GST‐p52 protein and increasing doses of GST‐PIR protein. The final concentration of probe was 20 nm. C) Gel shift assay was performed with probe 3′‐2a and indicated proteins. The final concentration of probe was 20 nm. D) Flag‐tagged PIR and HA‐tagged RELB were transiently expressed in HEK293T cells. Co‐IP assay were performed with anti‐Flag agarose (Flag IP), followed by detection of Flag‐PIR and HA‐RELB in the immuno‐precipitates. E) Co‐IP assays were performed with endogenous proteins to determine the association of PIR with RELB in HCT116 cells. F) Flag‐PIR, Flag‐RELB and HA‐p52 were transiently expressed in HEK293T cells. Co‐IP were performed to determine whether PIR disrupts the interaction between RELB and p52. G) Flag‐RELB and Flag‐p52 were transiently expressed in HCT116 cells, followed by knockdown of PIR with shPIR and further reconstitution of PIR with rHA‐PIR. Chromatin immunoprecipitation (ChIP) was then performed to determine the binding of p52/RELB to FAS promoter using anti‐Flag antibody. H) HCT116 cells were transfected with Flag‐RELB and Flag‐p52, followed by treatment with DMSO or TphA (50 µm, 12 h). ChIP assays were then performed as in (G). I) HCT116 cells were firstly expressed for shGFP (as control) and shNFκB2 individually, to create cell lines with NFκB1 knockdown. 24 hours later, each group of cells were further expressed for shGFP or shPIR‐1. After cultured for another 72 hours, cells were evaluated for survival rate by flow cytometry (upper panel) and expression levels of indicated proteins by WB (lower panel). Data represent the mean±SD. (n = 5, unpaired Student's t‐test, **p < 0.01, ***p < 0.001). J) HCT116 cells were transfected with indicated plasmids. After 72 hours of transfection, cells were determined for survival rate (upper panel) and expression levels of proteins (lower panel). Date represents the mean±SD. (n = 4, unpaired Student's t‐test, ***p < 0.001). K) HEK293T cells were co‐transfected with luciferase reporter plasmids carrying FAS full‐length promoter (FAS‐Luc) or its 3′‐2a deletion mutants (FAS‐3′‐2a‐Luc), along with different doses of Flag‐p52/RELB complex. After 24 hours of transfection, luciferase activities were determined and normalized to the first column (upper panel). Protein levels were determined by WB (lower panel). Data are presented as mean±SD. (n = 3, unpaired Student's t‐test, **p < 0.01, ***p < 0.001, n.s.: no significant difference).

Fig 5: Targeting NF-κB attenuates the EGFR-TKI resistance driven by TET2 deficiency. a–c Relative survival curve describing the viability of indicated cell lines treated with the indicated concentrations of the osimertinib for 72 h. We generated different combinations of gene knockout cell lines in HCC827-OR to mimic the repression of the NF-κB pathway (a). Bortezomib was used to inhibit the NF-κB pathway in PC-9OR or PC9TET2-KO (b) and HCC827-OR or HCC827TET2-KO cells (c). d Growth curves of xenograft tumors derived from HCC827-OR cells with or without NFKB2 and RelB double knockout following osimertinib treatment (2.5 mg/kg, daily) or in combination with bortezomib (0.25 mg/kg, daily) (n = 6 mice per group). e Growth curves of xenograft tumors derived from HCC827-OR cells treated by osimertinib (25 mg/kg, oral, daily) or bortezomib (0.25 mg/kg, daily) or combined (n = 5 mice per group). right panel, body weight of mice in each group at the endpoint of treatment. Growth curves of PDX-1R (f), PDX-2R (g) and PDX-1S (h) in NCG mice following osimertinib treatment (5 mg/kg, daily) only or bortezomib (0.25 mg/kg, daily) only or combined (n = 6 mice per group). The administration strategy in mice was intraperitoneal injection (i.p.) unless stated otherwise. P values were calculated using Two way ANOVA (d–h). Data are shown as mean ± SEM