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: Journal of Biological Chemistry

Article Title: LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

doi: 10.1074/jbc.m114.563742

Figure Lengend Snippet: FIGURE 3. Ussing chamber experiments demonstrating that LMTK2 knockdown increases CFTR mediated Isc across CFBE41o- monolayers. CFBE41o- cells stably expressing WT-CFTR were plated on tissue culture plates and incubated with the optimized transfection mixture containing 50 nM of siRNA specific for LMTK2 (siLMTK2) or the siRNA negative control (siCTRL). After 24 h, cells were trypsinized and plated on collagen-coated Snapwell permeable supports and cultured for an additional 6 days to estab- lishpolarizedmonolayers(total7daysinculture).CFBE41o-cellswerebathed in solutions with apical-to-basolateral Cl gradient in the presence of amiloride (Amilo, 50 M) in the apical bath solution to inhibit Na absorption throughENaC.Iscwasstimulatedwithforskolin(FSK,20M)andIBMX(50M) added to the apical and basolateral bath solution. Thiazolidonone CFTRinh- 172 (5 M) was added to the apical bath solution to inhibit CFTR-mediated Isc. Data are expressed as net stimulated Isc, calculated by subtracting the base- line Isc from the peak stimulated Isc. siCTRL did not affect the forskolin/IBMX- stimulated Isc across CFBE41o-cells compared with the non-transfected cells (data not shown). Representative experiment (A) and summary of data (B) demonstrating that LMTK2 knockdown increased the forskolin/IBMX-stimu- lated Isc across CFBE41o- cells. siLMTK2 did not change the transepithelial resistance across the monolayers (C). *, p 0.05 versus siCTRL. 9 monolayers/ group from 2 different cultures. Error bars, S.E.

Article Snippet: The following reagents were used: Complete Protease Inhibitor Mixture and PhosSTOP phosphatase inhibitor mixture tablets (Roche Applied Sciences, Indianapolis, IN), the adenylate cyclase activator forskolin and the cAMP phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) (SigmaAldrich), and the inhibitor of protein serine/threonine phosphatases calyculin A (Cell Signaling Technology, Inc.; Danvers, MA).

Techniques: Knockdown, Stable Transfection, Expressing, Incubation, Transfection, Negative Control, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

doi: 10.1074/jbc.m114.563742

Figure Lengend Snippet: FIGURE 5. Immunoblots demonstrating that the anti-CFTR phosphosite antibodyAb-737recognizesspecificallyphosphorylationtheSer737site. A, CFBE41o- cells stably expressing WT-CFTR were treated with IBMX (1 mM) and forskolin (FSK, 20 M) added to the apical and basolateral medium at 37 °C for 10 min before experiments to raise the intracellular cAMP levels and promote PKA-mediated phosphorylation (34). To increase the phosphory- lated CFTR fraction we attenuated serine/threonine protein phosphatases by treating cells with calyculin A (CalA, 50 nM) added to the basolateral medium at 37 °C for 15 min before experiments (63, 64). Low level of CFTR was detected by the phosphosite antibody Ab-737 in WCL of the vehicle control (CTRL)-treated cells. By contrast, the phosphosite antibody detected more CFTR after treatment with either forskolin/IBMX or calyculin A or both. B, parental CFBE41o- cells were transfected with the WT-CFTR (WT) or the mutant CFTR-S737A (S737A). Unlike the WT-CFTR, the CFTR-S737A was not detected by the antibody Ab-737 in WCL. Antibody CFF596 was used to detect the total CFTR. Ezrin expression was used as a loading control. Experi- ments were repeated at least three times from different cultures with similar results.

Article Snippet: The following reagents were used: Complete Protease Inhibitor Mixture and PhosSTOP phosphatase inhibitor mixture tablets (Roche Applied Sciences, Indianapolis, IN), the adenylate cyclase activator forskolin and the cAMP phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) (SigmaAldrich), and the inhibitor of protein serine/threonine phosphatases calyculin A (Cell Signaling Technology, Inc.; Danvers, MA).

Techniques: Western Blot, Phospho-proteomics, Stable Transfection, Expressing, Control, Transfection, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

doi: 10.1074/jbc.m114.563742

Figure Lengend Snippet: FIGURE 10. Ussing chamber experiments demonstrating that LMTK2 knockdown facilitates the CFTR-mediated Isc across CFBE41o- monolay- ers expressing F508-CFTR rescued with VX-809. CFBE41o- cells stably expressing F508-CFTR and transduced with shRNA against the human LMTK2 gene (shLMTK2) or the shRNA negative control (shCTRL) as described in Fig. 10 were cultured on collagen-coated Snapwell filters at 1.0 106 in air-liquid interface for 7–9 days. VX-809 (10 M) was used for 48 h before experiments to facilitate the biosynthetic processing and rescue the abun- dance of F508-CFTR band C. CFBE41o- cells were bathed in solutions with apical-to-basolateral Cl gradient in the presence of amiloride (Amilo, 50 M) in the apical bath solution to inhibit Na absorption through ENaC. Isc was stimulated with forskolin (FSK, 20 M) and IBMX (50 M) added to the apical and basolateral bath solution. Thiazolidonone CFTRinh-172 (5 M) was added to the apical bath solution to inhibit CFTR-mediated Isc. Data are expressed as net stimulated Isc, calculated by subtracting the baseline Isc from the peak stimulated Isc. shCTRL did not affect the forskolin/IBMX-stimulated Isc across CFBE41o- cells compared with the non-transfected cells (data not shown). Representative experiment (A) and summary of data (B) demonstrating that LMTK2 knockdown increased the forskolin/IBMX-stimulated Isc across CFBE41o-cells after the VX-809 rescue. shLMTK2 did not change the transepi- thelial resistance across cell monolayers (C). *, p 0.05 versus siCTRL. 16–17 monolayers/group from two different cultures. Error bars, S.E.

Article Snippet: The following reagents were used: Complete Protease Inhibitor Mixture and PhosSTOP phosphatase inhibitor mixture tablets (Roche Applied Sciences, Indianapolis, IN), the adenylate cyclase activator forskolin and the cAMP phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) (SigmaAldrich), and the inhibitor of protein serine/threonine phosphatases calyculin A (Cell Signaling Technology, Inc.; Danvers, MA).

Techniques: Knockdown, Expressing, Stable Transfection, Transduction, shRNA, Negative Control, Cell Culture, Transfection

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: