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Additional file 6 of Targeting CDK7 suppresses super enhancer-linked inflammatory genes and alleviates CAR T cell-induced cytokine release syndrome

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posted on 05.01.2021, 05:17 by Ye Wei, Chong Li, Huifang Bian, Wei Qian, Kairui Jin, Tingting Xu, Xiaomao Guo, Xueguan Lu, Fengtao Su
Additional file 6: Figure S5 Supplementary data related to Fig. 5. (A) Schematic of CAR T cell generation. CD25 and CD69 were detected on day 2 to verify the T cell activation. CD3, CD4, and CD8 were examined weekly to monitor the distribution of T subsets. (B) Western blot analysis of STAT1 and RNA Pol II phosphorylation in mTHP-1 cells stimulated by the supernatant of coculture with Raji and CAR T cells following 30 nM THZ1 pretreatment for 4 hours. (C, E) Effects of THZ1 on cell proliferation. CAR T or NCT cells were treated with indicated concentrations for the indicated times, and detected using the CCK8 kit. (D, F) Effects of THZ1 on cell apoptosis. CAR T or NCT cells were treated with indicated concentrations for 48 hours, and detected using flow cytometry. (G) Transcriptional levels of inflammatory genes in NCT or CAR T cells treated with 20 nM THZ1 at 24 hours. (H) The residual Raji cells were detected in coculture systems with E/T ratio increases from CAR T: Raji = 1: 10 to CAR T: Raji = 1: 2 at 24 hours. (I) The residual Raji cells were detected in coculture systems with the E/T ratio NCT: Raji = 1: 2 at 24 hours. Coculture of NCT and Raji cells was set as the control to calculate the elimination rate. (J) The residual Raji cells were detected in coculture systems with the E/T ratio CAR T: THP-1: Raji = 1: 1: 4 at 24 hours. (K) Proliferation of CAR T or NCT cells in the presence of THZ1 was measured by CFSE dilution after 24, 48 and 72 hours. (L) Human cytokines were detected in the supernatant of coculture of Raji and CAR T cells at 24 hours with 20 nM THZ1 or not. n.s.: no statistical significance. Data are the mean ± SD, n = 3 in (A) to (L).

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