Was excited at 340 and 380 nm (shift in excitation wavelength occurs upon
Was excited at 340 and 380 nm (shift in excitation wavelength occurs upon binding Ca2+), and fluorescent emission was detected at 510 nm. Intracellular Ca2+ concentration was expressed as a ratio of fluorescent emission intensity ( F340/F380). The fluorescence signal was expressed as a adjust in percentage soon after being normalized to basal intensity levels established prior to stimulation. For hormone secretion research, 5000 GFP+ cells from MIP-GFP mice, Venus+ cells from Glucagon-Venus mice or YFP+ cells from iADKO mice were collected by FACS and loaded onto the microfluidic device. To measure insulin secretion, YFP+ cells had been incubated in basal KRB with 2mM glucose for 30 mins after which stimulated with KRB containing 14 mM glucose for 30 mins followed by 2 mM glucose for ten mins. To measure glucagon secretion, YFP+ cells were incubated in KRB with 11.2 mM glucose for 30 min and then stimulated with KRB with 2 mM glucose for 30 minutes. Ultrasensitive Rodent Insulin or Glucagon ELISAs (Mercodia, Uppsala, Sweden) had been applied to measure perfusate insulin or glucagon levels.Supplementary MaterialRefer to Net version on PubMed Central for supplementary material.AcknowledgmentsWe thank Dr. H.E. Arda for discussions for FACS experiments, Dr. Y. Hang for aid with islet isolations, members with the Kim lab, specially Dr. S. Park, for advice, and Drs. J. A. Golden, and L. Jackson-Grusby for mouse strains.Cell Metab. Author manuscript; available in PMC 2018 March 07.Chakravarthy et al.Page 13 We also thank N. Neff and G. Mantalas for help with sequencing. H.C. was supported by a T32 coaching award for the Endocrinology Division, Dept. of Medicine, Stanford University College of Medicine, and fellowships from the Stanford Youngster Health Study Institute, plus the JDRF. C.D. was supported by Stanford University ViceProvost Undergraduate Education grants. N.D. was supported by a grant from the DKK-3, Human (HEK293, His) Institute of Genetics and Genomics of Geneva. Effort in the Quake group was supported by National Institutes of Overall health grants U01HL099999 and U01-HL099995, California Institute of Regenerative Medicine grant GC1R-06673, Center of Excellence for Stem Cell Genomics, The Wallenberg Foundation Postdoctoral Scholarship Program at Stanford along with the Howard Hughes Healthcare Institute (HHMI), in the Herrera group by grants from JDRF, the Swiss National Science Foundation, the U.S. NIH Beta-cell Biology Consortium (BCBC), and also the European Union (IMIDIA), and within the Kim group by HHMI, the H.L. Snyder Foundation, the Elser Trust, by grants from JDRF, the NIH BCBC (UO1DK089532 and UO1DK089572) and NIH Human Islet Resource Network (UC4DK104211).Author Manuscript Author Manuscript Author Manuscript Author Manuscript
CHEMMEDCHEM COMMUNICATIONSDOI: 10.1002/cmdc.A Cell-Permeable Ester Derivative on the JmjC Histone Demethylase Inhibitor IOXRachel Schiller,[a] Giuseppe Scozzafava,[b] Anthony Tumber,[b] James R. Wickens,[a] Jacob T. Bush,[a] Ganesha Rai,[c] Clarisse Lejeune,[a] SFRP2, Human (HEK293, His) Hwanho Choi,[a] Tzu-Lan Yeh,[a] Mun Chiang Chan,[a] Bryan T. Mott,[c] James S. O. McCullagh,[a] David J. Maloney,[c] Christopher J. Schofield,[a] and Akane Kawamura[a, d]The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family would be the biggest family members of histone lysine demethylases. There is certainly interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5Carboxy-8-hydroxyquinoline (IOX1) is the most potent broadspectrum inhibitor of 2OG oxygenases, includin.