Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: (A) Anti-GPR64 C-terminal antibody detects GPR64 C-terminal fragments. A673 cells were transfected with control siRNA or GPR64 siRNA, and the protein levels of GPR64 were analyzed by immunoblotting. (Left) Anti-GPR64 N-terminal antibody immunoblotting. (Right) Anti-GPR64 C-terminal antibody immunoblotting, which detected approximately 33 and 17 kDa fragments, both silenced by GPR64 siRNA. (B) Ceramide induces GPR64 C-terminal fragments. A673 cells were left untreated or treated with 1 μM C18 ceramide for 16 h. The levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting. (C) Exogenously expressed GPR64 C-terminal intracellular domain (ICD) is located in the nucleus. A673 cells were infected with lentiviruses expressing the FLAG-tagged GPR64 ICD (879–1017) or empty vector, and the subcellular location of FLAG-ICD was examined by anti-FLAG immunofluorescence. The nuclei were stained with DAPI. Scale bars: 10 μm. (D) The GPR64 C-terminal antibody detects a nuclear signal in A673 cells, which is abolished by GPR64 siRNA knockdown. Scale bars: 10 μm. (E) GPR64 ICD rescues growth arrest induced by GPR64 knockdown. A673 cells were infected with lentiviruses expressing FLAG-ICD or empty vector, followed by transfection with control siRNA or GPR64 siRNA. (Left) The protein levels of FLAG-ICD, endogenous full-length GPR64, and tubulin were assessed by immunoblotting. Note that the GPR64 cDNA clone is codon optimized and harbors numerous silent nucleotide substitutions, making the ICD expressed from GPR64 ICD cDNA resistant to silencing by GPR64 siRNA. (Right) Proliferation of cells was assessed by the IncuCyte. (F) GPR64 ICD rescues growth arrest induced by SMPD1 knockdown. A673 cells were infected with lentiviruses expressing FLAG-ICD or empty vector, followed by transfection with control siRNA or SMPD1 siRNA. (Left) The protein levels of FLAG-ICD, SMPD1, and tubulin were assessed by immunoblotting. (Right) Proliferation of cells was assessed by the IncuCyte. (G) Forskolin and bromo-cAMP induce the GPR64 C-terminal fragments. A673 cells were treated with the indicated concentration of forskolin or bromo-cAMP for 16 h, and the levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting. (H) The suppression of cAMP-PKA signaling blocks the induction of the GPR64 C-terminal fragments by ceramide. A673 cells were treated with 1 μM C18 ceramide for 16 h, followed by treatment with the indicated concentration of NKY80 (adenylate cyclase inhibitor) or H-89 (PKA inhibitor) for 48 h. The levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting. (I) A γ-secretase inhibitor, DAPT, blocks the induction of the GPR64 C-terminal fragments by ceramide. A673 cells were treated with 1 μM C18 ceramide for 16 h, followed by treatment with the indicated concentration of DAPT for 48 h. The levels of GPR64 C-terminal fragments were assessed by anti-GPR64 C-terminal antibody immunoblotting.

Article Snippet: Forskolin , Thermo Scientific , J63292.MA.

Techniques: Transfection, Control, Western Blot, Infection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Knockdown, Concentration Assay

Journal: Cell reports

Article Title: Ceramide-induced cleavage of GPR64 intracellular domain drives Ewing sarcoma

doi: 10.1016/j.celrep.2024.114497

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Forskolin , Thermo Scientific , J63292.MA.

Techniques: Control, Virus, Recombinant, Transfection, SYBR Green Assay, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Plasmid Preparation

Journal: iScience

Article Title: The nature of sex differences in catecholamine-induced lipolysis in subcutaneous fat cells

doi: 10.1016/j.isci.2025.113988

Figure Lengend Snippet: Effect on lipolysis of agents acting at specific steps in the catecholamine-induced lipolysis cascade in the investigated abdominal subcutaneous fat cells DOBU (dobutamine) is a beta-1 adrenoceptor selective agonist used in (A) and D). TER (terbutaline) is a beta-2 adrenoceptor selective agonist used in (B and E). CLO (clonidine) is an alpha-2A selective adrenoceptor agonist used in (C and F). FOR (forskolin) is a selective activator of adenylyl cyclase used in (G). dcAMP (dibutyryl cyclic AMP) is a phosphodiesterase-resistant cyclic adenosine monophosphate analog, which selectively activates the protein kinase A complex and is used in (H). Basal, spontaneous lipolysis; ADA, adenosine deaminase, which selectively breaks down adenosine and is added to basal lipolysis in the clonidine experiments to remove traces of endogenous antilipolytic adenosine. Results are expressed as boxplots with Tukey whiskers where ∗ indicates the mean and compared by unpaired t test. N , number of subjects.

Article Snippet: Forskolin , Selleck Chemicals , S2449.

Techniques: