Fig 2: Green tea polyphenols-mediated apoptosis induced by death receptor pathway in prostate cancer LNCaPshV and LNCaPshp53 cells.[A] Cells were treated with 40 µg/ml concentration of GTP for 1, 5, 10, 15, 30 and 60 min and Western blotting was performed for SAPK/JNK, p-JNK, FAS, FADD, p-FADD, BID and t-BID proteins. A typical actin blot demonstrates internal loading control. [B] Cells were treated with 40 µg/ml concentration of GTP for 3, 6, 9, 12, 24 and 48 h and Western blotting was performed for cleaved PARP, procaspase-8, cleaved caspase-8, c-IAP and XIAP proteins. A typical actin blot demonstrates internal loading control. [C] Cells were treated with 20 µM concentration of JNK inhibitor SP600125 for 8 h and with 40 µg/ml GTP for 16 h alone, or SP600125 for 8 h followed by GTP treatment in combination followed by Western blotting for JNK, p-JNK, FAS and p-FADD proteins. The expression of native JNK protein was considered as loading control. [D] Cell death measurement was performed by photometric enzyme immunoassay Cell Death Detection ELISA kit. The bars represent mean±SD of at least two independent experiments each performed in duplicate, **p<0.001 represents significant differences as compared to control group. [E] Light microscopic images of LNCaPshV and LNCaPshp53 cells treated with SP600125 and GTP alone or in combination. The details are described in the materials and methods section.

Fig 3: Phosphorylation of BNIP3 at S60/T66 by JNK enhances mitophagy and impedes BNIP3 proteasomal degradation.a PC12 cells were transfected with negative control (NC), Jnk1 and Jnk2 siRNA for 48 h and then exposed to 20% O2 or 0.3% O2 for 6 h, and cell lysates were subjected to western blot analysis with the indicated antibodies. n = 3. b HeLa Cells stably expressing mt-Keima were co-transfected with plasmids encoding constitutively active (CA) or dominant negative (DN) HA-JNK1 and Flag-BNIP3 WT or S60/T666A, and mitophagy was identified and quantified by the ratio of acidic (590 nm, red) to normal mitochondria (440 nm, green). Scale bar, 10 µm. n ? 30. c HeLa cells were transfected with NC or JNK1 and JNK2 siRNA and plasmids encoding GFP-LC3. After 48 h, the cells were transfected with plasmids encoding wild-type (WT) or S60/66 A Flag-BNIP3 and HA-JNK1CA or HA-JNK1DN for an additional 24 h. Cell lysates were immunoprecipitated with an anti-Flag antibody and examined via western blotting with the indicated antibodies. n = 3. d, e HeLa cells were co-transfected with constitutively active or dominant negative HA-JNK1 and BNIP3 WT (d, left) or the S60/T66A mutants (e, right). After transfection for 48 h, 20 µg ml-1 CHX was added to the cultures for the indicated time, and the degradation of BNIP3 was detected via western blotting with the indicated antibodies and quantified, respectively. f HeLa cells were co-transfected with Flag-BNIP3, Myc-Ub and constitutively active or dominant negative HA-JNK1 for 48 h, and 10 µM MG132 was added 12 h before samples were collected. Cell lysates were boiled and immunoprecipitated with an anti-Flag antibody. The immune complexes were then analyzed via western blotting. n = 3. The data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group.

Fig 4: JNK1/2 is the kinase responsible for BNIP3 phosphorylation.a PC12 cells were treated with various kinase inhibitors, including 10 µM PD184352 (MEK inhibitor), 10 µM SP600125 (JNK inhibitor), 10 µM SB203580 (p38 inhibitor), 10 µM Roscovitine (CDK inhibitor), 100 µM TBB (CK2 inhibitor), 10 µM K252c and 1 µM Bis I (PKC inhibitor). Then, cell lysates were subjected to western blotting with the indicated antibodies. DMSO, dimethylsulfoxide. b The phosphorylation of BNIP3 was detected via western blotting after PC12 cells were treated with JNK inhibitor SP600125 (10 µM) or JNK-IN-8 (10 µM) and exposed to 20% O2 or 0.3% O2 for 6 h. n = 3. c PC12 cells stably expressing WT or S60/T66A mutant Flag-BNIP3 were treated with DMSO or SP6000125 and followed by detection of phosphorylation of BNIP3. n = 3. d After Jnk was knocked down with the indicated siRNA in PC12 cells, the levels of JNK protein and BNIP3 phosphorylation were measured by western blotting. n = 3. e HeLa cells were transfected with Flag-BNIP3 and HA-JNK1 or HA-JNK2 for 48 h. Cell lysates were then immunoprecipitated with an anti-Flag antibody and detected by western blotting with an anti-HA or anti-Flag antibody. f Immunoprecipitated constitutively active (CA) HA-JNK1 or dominant negative (DN) HA-JNK1 was incubated with WT or S60/T66A GST-BNIP3 protein at 30 °C for 30 min, and then the reaction products were subjected to western blotting with the indicated antibodies. g Jnk1 and Jnk2 knockdown PC12 cells were transfected with HA-JNK1CA or HA-JNK1DN mutants for 48 h, cell lysates were then analyzed via western blotting with the indicated antibodies. n = 3. The data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group.

Fig 5: Overexpression of MKK7AC to promote the phosphorylation of JNK2 inhibited the metastasis of 4T1 cells(A) Western blot results of MKK7, p-JNK, JNK2 and Fra1 in 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells. (B) Transwell assay results of 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells (n = 3). (C) Wound healing assay results of 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells (n = 8). (D) Fra1-reporter luciferase activity in 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells, n = 3. (E) Immunofluorescent staining of E-cad (red), N-cad (green) in 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells, the nuclei were counterstained with DAPI (blue). Scale bar: 50 µm.