Fig 2: SESN2 but not Beclin 1 contributed to TIIA-induced autophagic induction and anchorage-independent growth inhibition in human osteosarcoma cells.a 143B cells were treated with various concentrations of TIIA (1, 5, 10, and 20 μM) as indicated for 24 h, after which BECN1, class III PI3K, ATG5, ATG7, SESN2, AMPK-α, p-AMPK-α, JNK1, p-SAPK/JNK, c-Jun, and p-c-Jun were measured by western blot. b–d shRNA SESN2 or shRNA BECN1 was stably transfected into 143B cells and the stable transfectants were identified (b). Following treatment with TIIA (20 μM) in 143B SESN2KD (shSESN2) (c) and 143B BECN1KD (shBECN1) (d) for 24 h, total lysates were immunoblotted for LC3B and p-AMPK-α. β-actin served as loading control. e SESN2 mRNA was measured by qRT-PCR in 143B cells treated with various concentrations of TIIA (1, 5, 10, and 20 μM) for 12 h. f 143B cells were treated with or without TIIA 20 μM for 12 h and subjected to ChIP assay using c-Jun antibodies. The purified DNA was analyzed by semiquantitative PCR (left) and real-time PCR (right) using specific primers spanning the c-Jun-binding sites on SESN2 promoter. ChIP DNA level was normalized to the level of input DNA. g Representative images of colonies of 143B SESN2KD (shSESN2), 143B BECN1KD (shBECN1), and 143B-mock (nonsense) cells in a soft agar colony formation assay in the absence or presence of various concentrations of TIIA were captured using a microscope Scale bar: 500 μm (left). Results were expressed as average number of colonies counted (in six microfields) (right). The results were expressed as the means ± SD from three independent experiments (n ≥ 3, *P < 0.05, **P < 0.01 compared with untreated control)

Fig 3: MAPK8/9 activation phosphorylates BCL2L11 and dissociates it from BECN1 in melanoma cells undergoing ER stress. (A) Whole cell lysates from Mel-RM cells transduced with control or RIPK1 shRNA 1 cells treated with tunicamycin (TM) (3 μM) for 16 h were immunoprecipitated by BECN1 antibody. The resulting precipitates were subjected to western blot analysis of BECN1, BCL2L1, BCL2, and MCL1. The data shown are representative of 3 individual experiments. (B) Whole cell lysates from Mel-RM cells treated with TM (3 μM) for 16 h with or without pretreatment with the MAPK8/9 inhibitor SP600125 (10 μM) for 1 h were subjected to western blot analysis of RIPK1, pMAPK8/9, MAPK8/9, pBCL2L11 (using a phosphorylated BCL2L11-specific antibody), and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (C) Whole cell lysates from Mel-RM cells treated with TM (3 μM) for 16 h with or without pretreatment with the MAPK8/9 inhibitor SP600125 (10 μM) for 1 h were subjected to immunoprecipitation with an antibody against BECN1. The resulting precipitates were subjected to western blot analysis of BECN1 and BCL2L11. The data shown are representative of 3 individual experiments. (D) Whole cell lysates from Mel-RM and MM200 cells transduced with the control or BCL2L11 shRNA were subjected to western blot analysis of BCL2L11 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (E) Mel-RM and MM200 cells stably transduced with the control or BCL2L11 shRNA were transduced with RIPK1 shRNA 1 and treated with TM (3 μM) or thapsigargin (TG) (1 μM) for 16 h. Whole cell lysates were subjected to western blot analysis of SQSTM1, MAP1LC3A, or GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (F) Mel-RM and MM200 cells stably transduced with the control or BCL2L11 shRNA were treated with TM (3 μM) or TG (1 μM) for 16 h with or without pretreatment with the MAPK8/9 inhibitor SP600125 (10 μM) for 1 h were subjected to western blot analysis of SQSTM1, MAP1LC3A, or GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (G) Mel-RM and MM200 cells with BCL2L11 stably knocked down were transduced with the control or BECN1 shRNA. Twenty-four h later, whole cell lysates were subjected to western blot analysis of BECN1 or GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (H) Mel-RM and MM200 cells with or without BCL2L11 and BECN1 co-knocked down were treated with TM (3 μM) or TG (1 μM) for 16 h. Whole cell lysates were subjected to western blot analysis of SQSTM1, MAP1LC3A, and GAPDH (as a loading control). The data shown are representative of 3 individual experiments.

Fig 4: Induction of autophagy by RIPK1 in melanoma cells upon pharmacological ER stress was due to activation of BECN1 as a result of its disassociation from BCL2L11. A schematic illustration of the proposed pharmacological ER stress-XBP1-HSF1-RIPK1-MAPK8/9-autophagy pathway.