Fig 2: Western blotting for proteins related to canonical and non-canonical Wnt-signaling.ST2 cells with serial periods of MG treatment were subjected to Western blotting. Hela cell lysate was used as a control. While Wnt5a/b, Ror1, Ror2, whole- and phospho-SAPK/JNK, whole- and phospho-c-jun (factors related to non-canonical Wnt-signaling) were almost constant, the β-catenin protein level decreased and the phospho-β-catenin protein level increased reciprocally at 12 hr after treatment.

Fig 3: SAPK/JNK is the effector molecule responsible for Cbx7 mediated delayed migration.(A) Total cellular protein was extracted from U373/VC and U373/Cbx7 and the expression level of indicated proteins was assayed using Western blot, asterisk (*) refers to a non-specific band obtained with anti-Cbx7 antibody. All PAGE gels were run under same experimental conditions. (B) U373 cells were treated with siNT (siRNA non targeting) and siCTGF, after 72 hours protein was extracted to assay the levels of phospho-SAPK/JNK. (C,D) A172 and LN229 cell lines were treated with SP600125 (50 μM) (SAPK inhibitor) followed by in vitro scratch assay, the scratch area were photographed at 0th hour and thereafter at regular intervals. The bar graphs represent the gap of the wound (average of 8 different locations from two wells with standard deviation) at the beginning and at the end of the experiment. Significance testing was performed using student’s t-test and the symbols are indicated, ***p ≤ 0.001.

Fig 4: Knockdown of Has2os affected the expression of JNK upstream and downstream factors. Cells Transfected with siRNAs were maintained in growth medium for two days and shifted into DM for another three days, then cells were harvested for analysis. (A). qRT-PCR assays of the key factors of the JNK/MAPK signaling (MEKK1, MEKK2, MKK4, MKK7, JNK1, JNK2, and C-JUN) in C2C12 cells that were transfected with si-Has2os or si-NC. The results are normalized to those of with si-NC. (B). Western blot analysis shows the protein expression of key factors of JNK/MAPK signaling (MEKK1, MEKK2, MKK4, MKK7, p-C-Jun, and C-Jun). (C). Relative expression in (B) were calculated. GAPDH was the internal control. The results are normalized to those of with si-NC. Values were presented as means ± SEM. The statistical significance was calculated by t-test.

Fig 5: 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.