Journal: Cell death discovery

Article Title: L-asparaginase induces IP3R-mediated ER Ca 2+ release by targeting µ-OR1 and PAR2 and kills acute lymphoblastic leukemia cells.

doi: 10.1038/s41420-024-02142-9

Figure Lengend Snippet: Fig. 6 Stimulation of AC with forskolin or treatment with exogenous 8-CPT-cAMP in PAR2-knockdown aLL cells blocks L-asparaginase-induced ER Ca2+ release. #+shPAR2 cells loaded with Mag-Fluo-4 AM were subjected to Ca2+ tracing via single-cell Ca2+ imaging. After obtaining stable baseline ER Ca2+ levels, the cells were pretreated (or not pretreated) with forskolin A or 8-CPT-cAMP B and then treated with L-asparaginase to analyze ER Ca2+ release. Left panels show the average Ca2+ tracing measured per second in 10 individual cells after forskolin A or 8-CPT-cAMP B treatment. Data are from one of three independent experiments (n = 3) showing similar results. Charts on the right show the difference in ER Ca2+ release following treatment with L-asparaginase pretreated (or not pretreated) with forskolin A or 8-CPT-cAMP B. An F/F0 value of 5 s after L-asparaginase addition (left panel) was used to determine F/F0 reduction. Values are means ± SEMs from the three independent experiments. *p < 0.05.

Article Snippet: Forskolin (66575-29-9) was from Alomone Labs (Jerusalem, Israel).

Techniques: Knockdown, Imaging

Journal: Cell death discovery

Article Title: L-asparaginase induces IP3R-mediated ER Ca 2+ release by targeting µ-OR1 and PAR2 and kills acute lymphoblastic leukemia cells.

doi: 10.1038/s41420-024-02142-9

Figure Lengend Snippet: Fig. 8 L-asparaginase-induced ER Ca2+ release in aLL cells is associated with the downregulation of PLCβ3 at Ser1105 and BAD at Ser118 phosphorylations. Lysates of #+shPAR2 cells pretreated (or not pretreated: A with PTx (B, lanes 3 and 4), forskolin (B, lanes 6 and 7) or 14–22 amide (myr; lanes 9 and 10) then stimulated with L-asparaginase for 120 s were subjected to SDS‒PAGE and immuno- blotting for pSer1105-PLCβ3 and total PLCβ3, and pSer118-BAD and total BAD. Numbers under pSer1105-PLCβ3 and pSer118-BAD bands represent relative intensity ratios of the pSer1105-PLCβ3 or pSer118- BAD vs total PLCβ3 or BAD bands, respectively, with values at time 0 normalized to 1.

Article Snippet: Forskolin (66575-29-9) was from Alomone Labs (Jerusalem, Israel).

Techniques:

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: