Fig 2: IKK2-mediated activation of JNK regulates c-fos and c-jun expression. (A) Phosphorylation of JNK after IL-3 stimulation. Following IL-3 deprivation for 6 h, Ba/F3 cells were stimulated with IL-3 for the indicated durations, and whole-cell lysates were subjected to immunoblot analysis using the indicated Abs. (B–D) Phosphorylation of JNK in IKK KO cells after IL-3 stimulation. Ba/F3 parental (WT), IKK1 KO, IKK2 KO, and IKK1/2 DKO cells were stimulated with IL-3 after IL-3 deprivation for 6 h. Immunoblot analysis was performed with whole-cell lysates using the indicated Abs. (E) Expression levels of c-fos, c-jun, and c-myc mRNAs 20 and 40 min after IL-3 stimulation in the presence of JNK-IN-8 or DMSO. Ba/F3 cells were pre-incubated with 0.5 µM JNK-IN-8 or DMSO for 6 h and then stimulated with IL-3. * p < 0.05; ** p < 0.01; n.s., not significant, as assessed by Mann-Whitney U-tests, for differences between JNK-IN-8-treated and control cells at each time point. Each dot represents the result from independent experiments (n = 5). Means are indicated by bars.

Fig 3: Involvement of nuclear factor kappa B (NF-?B)/mitogen-activated protein kinase (MAPK) and AP-1 signaling pathway by NF50E. (a) Immunoblotting analysis of NF-?B and p-c-Jun. (b) Relative band intensity of the expressions of NF-?B and p-c-Jun were quantified by Image lab software. UV(-)+Vehicle (G1), UV(+)+Vehicle (G2), UV(+)+EGCG (10 mg/mL) (G3), UV(+)+NF50E at a concentration of 10 mg/mL (G4), and UV(+)+NF50E at a concentration of 50 mg/mL (G5). #p < 0.05 versus the non-UV-irradiated group; **p < 0.05 versus the UV-irradiated group. (c) Effects of NF50E on MAPK which include p38 and JNK signaling pathways were confirmed by western blot analysis.

Fig 4: DPI inhibits multiple PB01-mediated cell death pathways in NSCLC cells. (a) Western blot of p-PERK, PERK, p-eIF2a, eIF2a, ATF4, p-IRE1a, IRE1a, p-JNK, JNK, CHOP, GADD45a, and cleaved caspase-3 levels from thapsigargin (3 µM, 24 h) and PB01 (100 nM, 24 h)-treated A549 and H460 cells. ß-actin was used as the protein loading control. (b) A549 and H460 cells were pretreated with SP600125 (1, 5, and 10 µM, for 4 h) and subsequently treated with PB01 (100 nM, 24 h). Cell viability was determined using a WST-1 assay; *p < 0.05. Western blot analysis was conducted on total lysates to identify the inhibition of apoptosis markers such as cleaved caspase-3 and -9. ß-actin was used as the protein loading control. (c) The FACS data indicates the change in fluorescence intensity of DCFDA dye in PB01 (100 nM, 4 and 8 h)-treated A549 and H460 cells. (d) A Western blot of cytochrome c and Bax from the cytosolic and mitochondrial fractions was performed in PB01 (100 nM, 0, 3, 6, and 20 h)-treated A549 and H460 cells. ß-actin and VDAC were used as protein loading controls for the cytosolic and mitochondrial fractions, respectively. (e) A549 and H460 cells were pretreated with DPI (1 µM) and NAC (100 µM) for 4 h and subsequently treated with PB01 (100 nM, 24 h). Cell viability was determined using a WST-1 assay; *p < 0.05. (f) A549 and H460 cells were pretreated with DPI (1 µM) for 4 h and subsequently treated with PB01 (100 nM, 24 h). Apoptotic cell death was determined using a Annexin V FACS assay. (g) Western blot showing cleaved caspase-3 and -9 levels was performed using these samples. ß-actin was used as the protein loading control.

Fig 5: Impaired activation of NF-?B and JNK in response to LPS stimulation in TAK1-deficient macrophages.BMDMs were stimulated with LPS (100 ng/ml) and nuclear/cytoplasmic extracts were collected at the indicated times. (A) NF-?B DNA-binding activity in nuclear extracts was determined by EMSA. The lysates used for EMSA were subjected to immunoblot analysis using a TFIIB-specific antibody as a loading control. (B) Phosphorylation of JNK (p-JNK) in cytoplasmic extracts was assessed by immunoblot with antibody specific for its phosphorylated form. The membrane was reprobed for total JNK. Results are representative of three independent experiments.