Triptolide

In vitro differentiated neutrophils from ECOMG cells were used as a model system to study nuclear architecture in response to calcium influx in neutrophils. ECOMG cells were cultured and differentiated as previously described (Zhu et al. 2017 (link)). For inhibitors, neutrophils were treated with 10 µM Triptolide (Cayman), 100 µM 5,6-dichloro-1-β-D-ribofuranosyl-1H-benzimidazole (Cayman) or 10 µM flavopiridol (Cayman) for 30 min, or 2 µM FK506 for 2 h before activation. Primary B cells were treated with 2 µM FK506 (Cayman) for 2 h before activation. Inhibitors were kept in culture during activation time course. Acute degradation was induced by treating the cells with 0.5 µM dTAG-13 (Tocris) before activation. dTAG-13 was kept in culture during activation time course. The time series degradation experiments were performed by inducing protein degradation at the beginning of the time course and harvesting the samples at different time points. RPMI-1640 (Gibco) contains 0.42 mM calcium. Additional CaCl₂ was supplied to the medium before activation to a final concentration of 1 mM. A23187 was purchased from Sigma and Cayman. Neutrophils were activated either using fast activation conditions (20 µM A23187 for 15 min), or using slow activation conditions (5 µM A23187 for 4 h). These two activation conditions were used throughout the manuscript unless otherwise mentioned.

The spt6l heterozygous seeds (SALK_016621), previously described (14 (link)), were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. Three formerly generated transgenic lines: ProREF6:REF6-GFP (16 (link)), ProACC1:ACC1-GFP (17 (link)) and 35S:GFP (16 (link)) were used. All Arabidopsis seeds used were in the Columbia (Col-0) background. Plants were grown either on half-strength Murashige and Skoog ( MS) medium (0.5× MS salts, 1.5% [w/v] sucrose, and 0.8% agar [pH 5.8]) or in soil under 16 h/8 h light/dark cycle at 23 °C. For the inhibitor treatment, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) (10010302, Cayman Chemical), flavopiridol (10009197, Cayman Chemical), or triptolide (11973, Cayman Chemical) was added to the media at a final concentration of 100, 10 and 10 μM, respectively. For the short-time treatment, 10-day-old seedlings were sprayed with 10 μM flavopiridol and grown on MS plates for 1 h. For the heat shock treatment, seeds were germinated and grown on MS plates for 7 days at 23 °C. The plates were moved to 17 °C for 3 days, after which time, the seedlings were subjected to a heat shock treatment for 1 h at 27 °C. The primers used for genotyping are listed in Supplementary Table S1.