Journal: Autophagy

Article Title: Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells.

doi: 10.4161/auto.22805

Figure Lengend Snippet: Figure 5. Upregulation of MCL1 contributed to the prosurvival property of UA-induced, autophagy-de- pendent EIF2AK3 activation. (A) Effects of UA treatments on MCL1 expression. The cells were treated with various concentrations of UA for 24 h and MCL1 expression was analyzed by western blot- ting. (B) Effects of EIF2AK3 inactivation by RNAi on MCL1 induction by UA. EIF2AK3 was silenced by siRNA and MCL1 expression was analyzed by western blotting. (C) Effects of BECN1 and ATG5 knock- down on MCL1 induction by UA. BECN1 and ATG5 were simultaneously silenced by a siRNA approach and MCL1 was measured by western blotting. (D) Effects of ATG5 knockdown on phosphoryla- tion of EIF2AK3-EIF2S1 and MCL1 induction. ATG5 was silenced by siRNA approach and phosphorylation of EIF2AK3-EIF2S1 and MCL1 expression were measured by western blotting. (E) Effects of 3-MA on MCL1 induction by UA. The cells were treated with 20 μM UA in the pres- ence or absence of 3-MA for 24 h, MCL1 was analyzed by western blotting. (E) Effects of MCL1 inhibition by siRNA on UA-induced apoptosis measured by sub-G1 analysis (n = 3, **p < 0.01). (The blots shown are representative of three independent experiments).

Article Snippet: Antibodies specific for MCL1 (CST, 4572), HSPA5 (CST, 3183), EIF2AK3 (CST, 3192), ERN1 (CST, 3294), MAPK1/3 (CST, 4695), MAPK8/9/10 (CST, 9258), MAPK11/12/14 (CST, 8690), AKT (CST, 9272), phospho-EIF2S1 (CST, 3597), phosphoMAPK1/3 (CST, 4094), phospho-MAPK8/9/10 (CST, 4668), phospho-JUN (CST, 5464), phospho-MAPK11/12/13/14 (CST, 4511), phospho-AKT (CST, 9271), phospho-RPS6KB1/2 (CST, 9205), BECN1 (CST, 3738), ATG5 (CST, 8540), BCL2 (CST, 2870), DDIT3 (CST, 2895) and cleaved poly (ADP-ribose) polymerase(PARP1; p89) (CST, 9548) were purchased from © 20 13 L an de s B io sc ie nc e. D o no t d is tri bu te . www.landesbioscience.com Autophagy 207

Techniques: Activation Assay, Expressing, Western Blot, Knockdown, Phospho-proteomics, Inhibition

Journal: Autophagy

Article Title: Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells.

doi: 10.4161/auto.22805

Figure Lengend Snippet: Figure 7. Activation of MAPK1/3 contributed to UA-induced autophagy. (A) Effects of UA treatment on MAPKs. The cells were treated with various concentrations of UA for 24 h. The total and phosphorylation of MAPK1/3, MAPK8/9/10, MAPK11/12/13/14, the expression of BCL2 and DDIT3 were analyzed by western blotting. (B) Effects of MAPK1/3 inhibitor on UA-induced changes of key parameters of autophagy, ER stress and apoptosis. The cells were treated with 20 μM UA in the presence or absence of PD98059 for 24 h, phosphorylation of MAPK1/3, LC3-I to LC3-II conversion, EIF2S1 phosphorylation, MCL1 and PARP1 cleavage were analyzed by western blotting. (C) Effects of MAPK8/9/10 inhibitor on UA-induced LC3-I to LC3-II conversion. The cells were treated with 20 μM UA in the presence or absence of SP600125 for 24 h, LC3 was analyzed by western blotting. (D) Effects of MAPK1/3 inhibition by its inhibitor on UA-induced apoptosis. The cells were treated with 20 μM UA in the presence or absence of PD98059 for 24 h, then apoptosis was assessed by sub-G1 analysis (n = 3, **p < 0.01). (The blots shown are representative of three independent experiments).

Article Snippet: Antibodies specific for MCL1 (CST, 4572), HSPA5 (CST, 3183), EIF2AK3 (CST, 3192), ERN1 (CST, 3294), MAPK1/3 (CST, 4695), MAPK8/9/10 (CST, 9258), MAPK11/12/14 (CST, 8690), AKT (CST, 9272), phospho-EIF2S1 (CST, 3597), phosphoMAPK1/3 (CST, 4094), phospho-MAPK8/9/10 (CST, 4668), phospho-JUN (CST, 5464), phospho-MAPK11/12/13/14 (CST, 4511), phospho-AKT (CST, 9271), phospho-RPS6KB1/2 (CST, 9205), BECN1 (CST, 3738), ATG5 (CST, 8540), BCL2 (CST, 2870), DDIT3 (CST, 2895) and cleaved poly (ADP-ribose) polymerase(PARP1; p89) (CST, 9548) were purchased from © 20 13 L an de s B io sc ie nc e. D o no t d is tri bu te . www.landesbioscience.com Autophagy 207

Techniques: Activation Assay, Phospho-proteomics, Expressing, Western Blot, Inhibition

Journal: Autophagy

Article Title: Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells.

doi: 10.4161/auto.22805

Figure Lengend Snippet: Figure 8. Signaling pathways underlying UA-induced cytoprotective autophagy in human MCF-7 breast cancer cells. UA at relatively low concentrations induced autophagy which in turn led to activation of UPR including EIF2AK3 pathway. Activation of EIF2AK3 suppressed UA-mediated apoptosis through upregulation of MCL1. UA-induced cytoprotective autophagy was attributed to MAPK1/3 activation but not associated with MTOR inhibition.

Article Snippet: Antibodies specific for MCL1 (CST, 4572), HSPA5 (CST, 3183), EIF2AK3 (CST, 3192), ERN1 (CST, 3294), MAPK1/3 (CST, 4695), MAPK8/9/10 (CST, 9258), MAPK11/12/14 (CST, 8690), AKT (CST, 9272), phospho-EIF2S1 (CST, 3597), phosphoMAPK1/3 (CST, 4094), phospho-MAPK8/9/10 (CST, 4668), phospho-JUN (CST, 5464), phospho-MAPK11/12/13/14 (CST, 4511), phospho-AKT (CST, 9271), phospho-RPS6KB1/2 (CST, 9205), BECN1 (CST, 3738), ATG5 (CST, 8540), BCL2 (CST, 2870), DDIT3 (CST, 2895) and cleaved poly (ADP-ribose) polymerase(PARP1; p89) (CST, 9548) were purchased from © 20 13 L an de s B io sc ie nc e. D o no t d is tri bu te . www.landesbioscience.com Autophagy 207

Techniques: Protein-Protein interactions, Activation Assay, Inhibition

Journal: Apoptosis

Article Title: Molecular mechanism underlying differential apoptosis between human melanoma cell lines UACC903 and UACC903(+6) revealed by mitochondria-focused cDNA microarrays

doi: 10.1007/s10495-008-0231-8

Figure Lengend Snippet: Consistent RNA levels measured by microarray and qRT-PCR. ( a ) Relative RNA ratios of 10 genes, each at 3 time points, between UACC903 and UACC903(+6) cell lines measured by microarrays. ( b ) Relative RNA levels of the same 10 genes, each at the same 3 time points, measured by qRT-PCR. The ‘+’ sign indicates P < 0.05, and the ‘*’ indicates P < 0.01 for a pair within measurement. Out of these 30 gene-and-time-point comparisons, 28 (93.3%) are consistent between these two types of measurements, including 21 (70%) with agreement in both change (down, no, or up) and P -value, and 7 (23.3%) consistent with the changes but not with P -value (underlined). Two (6.7%) was inconsistent and included BAK1 at 0-h time point and MCL1 at 3-h time point (arrowhead)

Article Snippet: TaqMan probes and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 4352934E); Fanconi anemia complementation group D2 (FANCD2, Hs00276992_m1); DEAH (Asp-Glu-Ala-His) box polypeptide 16 (DHX16, Hs00374356_m1); adenosylmethionine decarboxylase 1 (AMD1, Hs00750876_s1); BCL2-like 1 (BCL2L1, Hs99999146_m1); uracil-DNA glycosylase (UNG, Hs00422172_m1); Fas TNF receptor superfamily member 6 (FAS, Hs00531110_m1); BCL2-antagonist/killer 1 (BAK1, Hs00832876_g1); caspase 3 apoptosis-related cysteine peptidase (CASP3, Hs00234387_m1); protein phosphatase 2 catalytic subunit alpha isoform (PPP2CA, Hs00427259_m1); and myeloid cell leukemia sequence 1 (MCL1, Hs03043898_m1) were purchased from Applied Biosystems.

Techniques: Microarray, Quantitative RT-PCR

Journal: Apoptosis

Article Title: Molecular mechanism underlying differential apoptosis between human melanoma cell lines UACC903 and UACC903(+6) revealed by mitochondria-focused cDNA microarrays

doi: 10.1007/s10495-008-0231-8

Figure Lengend Snippet: Western blots and quantification. ( a ) Western blot of cell lysate from UACC903(+6) and UACC903 cell lines before (0) and at 1.5-, 3-, 6-, and 12-h after the UV treatment, using the antibodies against BAK1, BCL2L1, FAS, MCL1 and GAPDH proteins. GAPDH was used as a control for loading error. ( b ) Bar graph for the quantitative comparison among protein expression levels. The signal intensities of a protein band and its surrounding background were scanned from images derived from two independent Western experiments for each protein and quantified by using Scanalytics IPLAB 3.6 (Rockville, MD). The resultant background-subtracted values of protein expression were normalized to those of GAPDH and then plotted as the relative protein levels for each protein at each time point in each cell line with or without UV treatment

Article Snippet: TaqMan probes and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 4352934E); Fanconi anemia complementation group D2 (FANCD2, Hs00276992_m1); DEAH (Asp-Glu-Ala-His) box polypeptide 16 (DHX16, Hs00374356_m1); adenosylmethionine decarboxylase 1 (AMD1, Hs00750876_s1); BCL2-like 1 (BCL2L1, Hs99999146_m1); uracil-DNA glycosylase (UNG, Hs00422172_m1); Fas TNF receptor superfamily member 6 (FAS, Hs00531110_m1); BCL2-antagonist/killer 1 (BAK1, Hs00832876_g1); caspase 3 apoptosis-related cysteine peptidase (CASP3, Hs00234387_m1); protein phosphatase 2 catalytic subunit alpha isoform (PPP2CA, Hs00427259_m1); and myeloid cell leukemia sequence 1 (MCL1, Hs03043898_m1) were purchased from Applied Biosystems.

Techniques: Western Blot, Expressing, Derivative Assay

Journal: Cancer cell

Article Title: Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy

doi: 10.1016/j.ccell.2017.11.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies for immunoblotting were purchased from the following sources: p53 (sc-126), MDM2 (sc-5304), Bcl-2 (sc-7382), Mcl-1 (sc-819), Puma (sc-28226), ERK (sc-1647), N-Ras (sc-31) and GSK3 (sc-7291) from Santa Cruz Biotechnology (Dallas, TX, USA); pERK (T202/Y204, 4370), pMEK (S217/S221, 9121), MEK (4694), AKT (2920), pAKT(S473, 9271), pGSK3 (S21/S9, 8566), PARP-1 (9542), Bim (2819), Bak (12105), caspase-3 (9664), caspase-9 (9508), pMcl-1 (T163, 14765), B-Raf (9433), pB-Raf (S445, 2696), JNK (9252) and pJNK (T183Y185, 9255) from Cell Signaling Technology; Bax (B8554) and β-Actin (A2228) from Sigma-Aldrich; Noxa (ab13654) and pMcl-1 (T163S159, ab111574) from Abcam; p21 (OP64) from EMD Millipore (Darmstadt, Germany).

Techniques: Recombinant, Imaging, Staining, Mutagenesis, Transfection, shRNA, Negative Control, Plasmid Preparation, Software, In Vivo Imaging, Microscopy, Flow Cytometry

Journal: Molecular Cancer

Article Title: Loss of FBXW7-mediated degradation of BRAF elicits resistance to BET inhibitors in adult T cell leukemia cells

doi: 10.1186/s12943-020-01254-x

Figure Lengend Snippet: FBXW7 mutants selectively target c-Myc, CyclinE and MCL1 for degradation. a GSK3 phosphates NOTCH1, cyclin E, MCL1 and c-MYC at specific phosphodegrons. The phosphorylation is essential for FBXW7-mediated proteasome degradation. Oncoprotein specific inhibitors are indicated the Figs. b FBXW7 mutations are correlated with poor prognosis in TCGA pan-cancer analysis. c FBXW7 W425R increased the half-life of cyclin E and c-MYC but had no effect on MCL1 half-life. The effect of FBXW7 on the half-life of oncoproteins was determined by Western blot after cells treated with 100 μg/mL cycloheximide (CHX) for 0, 2, 4, and 6 h. d FBXW7 mutations impaired the degradation of oncoproteins. FBXW7-mediated degradation of oncoproteins was determined by Western blot. FBXW7 WT and R505C were used as positive and negative controls, respectively. e The interaction between FBXW7 and oncoproteins was analyzed by co-immunoprecipitation. 293 T cells co-transfected with FBXW7 and oncoprotein were lysed and immunoprecipitated with oncoprotein. Western blot was used to analyze the interaction between FBXW7 and oncoproteins. f FBXW7-mediated ubiquitination of cyclin E and c-MYC was deficient by FBXW7 mutations. 293 T cells were co-transfected with indicated FBXW7, K48-Ub and cyclin E (upper panel) or c-MYC (lower panel). Cells were treated with MG132 for 6 h before harvest. Immunoprecipitated cyclin E or c-MYC and Western blot Ub showed the ubiquitination level of cyclin E (upper panel) and c-MYC (lower panel). g Table summarizing FBXW7 wild-type and FBXW7 mutants

Article Snippet: Endogenous expression of proteins was performed using antibodies for CyclinE (Santa-Cruz #sc-198), Mcl-1 (Santa-Cruz #sc-12,756), c-myc (9E10), FBXW7 (Proteintech #28424–1-AP), BRAF (Santa-cruz #sc-5284), p-MEK-1/2 (Calbiochem), p-ERK-1/2, and STAT3 (Santa-Cruz #sc-482).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Transfection, Ubiquitin Proteomics