Journal: Frontiers in Oncology

Article Title: Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers

doi: 10.3389/fonc.2021.738078

Figure Lengend Snippet: Erianin induces cell cycle arrest at the M phase in PC3 cells. (A, B) PC3 cells were treated with erianin at indicated concentrations for 24 h. (A) Cell cycle phase was determined by flow cytometry with propidium iodide (PI) staining. G 0 /G 1 , S and G 2 /M phase populations were estimated using Flowjo software and are indicated in purple, yellow and green, respectively. (B) Percentages of cells at each phase was quantified by Flowjo software. Data are expressed as mean ± SD. n=5. * p < 0.05; *** p < 0.001, versus untreated control. (C) PC3 cells were treated with erianin at 50 nM for indicated times. Cyclin b1 and phosphorylated CDK1 substrates were examined by Western blotting. (D, E) Cells were treated with S-trityl-L-cysteine (STLC) at 10 µM for 16 h to synchronize cells at the prometaphase. Then STLC was withdrawn and cell cycle progression to metaphase, telophase and interphase were monitored in the presence ot absence of erianin at 100 nM for 1 h. Confocal images were captured. Bar = 10 µm (D) . Percentages of cells at each cell cycle phase were quantified (E) .

Article Snippet: Immunoblot analyses were conducted according to standard protocol with the following antisera: caspase-3 (#9662), PARP (#9542), β-actin (#3770), IRE1α (#3294), p-eIF2α (#3597), t-eIF2α (#2103), p-P38 MAPK (#4511), t-P38 MAPK (#8690), p-JNK1/2 (#4668), t-JNK1/2 (#9252), p-ERK1/2 (#9101), t-ERK1/2 (#9102), CHOP (#5554), Bim (#2933), Bcl-2 (#4223), Bax (#5023), p-Akt (#4060), t-Akt (#9272), GAPDH (#5174), cyclin b1 (#12231), p-CDK1 substrates (#9477) and FLAG (#14793) from Cell Signaling Technology; p62 (#ab56416) and Mcl-1 (#ab31948) antibodies were purchased from Abcam, while LC3 antibody (#NB100-2220) was obtained from Novus Biologicals.

Techniques: Flow Cytometry, Staining, Software, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cyclin-dependent kinase–mediated phosphorylation of the exocyst subunit Exo84 in late G 1 phase suppresses exocytic secretion and cell growth in yeast

doi: 10.1074/jbc.RA119.008591

Figure Lengend Snippet: Exocytosis is inhibited in late G1 phase. A, conditional cdc34-2 mutants exhibit Bgl2 secretion defects at the restrictive temperature. Internal (“In”) and external (“Ex”) pools of Bgl2 in WT, cdc34-2 (late G1 phase), and cdc53-1 (late G1 phase) cells were examined by Western blot analysis. Cells were grown at 25 °C or shifted to 37 °C for 2 h. Alcohol dehydrogenase (Adh1) levels were probed as a protein loading control. The conditional mutants and cell cycle arrest points are listed to the right. B, quantification of Bgl2 accumulation in A. n = 3. *, p < 0.01. C, Cdk1 is required for the reduction in Bgl2 secretion in metaphase-arrested cdc34-2 cells. cdc34-2 and cdc34-2 cdk1-as1 cells were grown to early log phase at 25 °C and shifted to 37 °C for 1.5 h. Cells were then treated with DMSO (mock) or 15 μm 1NM-PP1 for 30 min. Internal and external pools of Bgl2 in cdc34-2 and cdc34-2 cdk1-as1 cells were examined by Western blotting. Corresponding immunoblots of alcohol dehydrogenase serve as a protein loading control. D, quantification of Bgl2 accumulation in C. n = 3; *, p < 0.01. E, invertase secretion was not changed in cdc34-2 and cdc28-4 mutants. Cells were grown at 25 °C or shifted to 37 °C for 1.5 h, and secretion of invertase was examined. The percentage of external invertase (secreted) versus total invertase was measured. Error bars represent S.D. (n = 3). AU, arbitrary unit.

Article Snippet: To confirm this result in vivo , we immunoprecipitated Exo84 from yeast and probed for Cdk1 phosphorylation using an antibody specific for Cdk1-phosphorylated peptides (Cell Signaling Technology, catalog number 2325) in cdc34-2 mutant arrested at late G 1 phase at 37 °C.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cyclin-dependent kinase–mediated phosphorylation of the exocyst subunit Exo84 in late G 1 phase suppresses exocytic secretion and cell growth in yeast

doi: 10.1074/jbc.RA119.008591

Figure Lengend Snippet: Exo84 is phosphorylated by Cdk1 at late G1 phase. A, Exo84-myc was immunoprecipitated from the WT and cdc34-2 mutant cells at the permissive or restrictive temperature and then probed for Cdk1 phosphorylation by immunoblotting. B, quantification of Exo84 phosphorylation level in A. Error bars represent S.D. (n = 3). *, p < 0.01. C, Exo84 is phosphorylated by Cdk1 in vitro. GST and GST-Exo84 were purified from E. coli and incubated with Cln2–Cdk1 or Clb5–Cdk1 in the presence of [γ-32P]ATP. Exo84 phosphorylation was detected by autoradiography (top). Corresponding Coomassie Blue–stained gels are shown on the bottom. D, in vitro Cln2–Cdk1 kinase assays with recombinant Exo84-A, which lacks the five Cdk1 phosphorylation sites. The phosphorylation of Exo84-A by Cln2–Cdk1 is barely detectable. AU, arbitrary unit.

Article Snippet: To confirm this result in vivo , we immunoprecipitated Exo84 from yeast and probed for Cdk1 phosphorylation using an antibody specific for Cdk1-phosphorylated peptides (Cell Signaling Technology, catalog number 2325) in cdc34-2 mutant arrested at late G 1 phase at 37 °C.

Techniques: Immunoprecipitation, Mutagenesis, Western Blot, In Vitro, Purification, Incubation, Autoradiography, Staining, Recombinant