Extended Data Fig. 4: Characterization of Trmt61a KO T cells in vitro.
From: tRNA-m1A modification promotes T cell expansion via efficient MYC protein synthesis
a. The T cell activation was assessed by flow cytometry analysis of CD69 and CD44 staining. Quantification of the fraction of activated T cells (CD69hiCD44hi) was shown in the right graph. Error bars represent mean ± s.e.m., n = 4 biologically independent samples. NS: non-significant; two-tailed, unpaired t-test. b. Representative flow cytometry gating strategy for CellTrace dilution in CD4+ T cell. c. Apoptosis was assessed by flow cytometry after staining by Annexin V and 7-AAD. The percentage is listed in the right graph. Error bars represent mean ± s.e.m., n = 4 biologically independent samples. NS: non-significant; two-tailed, unpaired t-test. d. Naïve CD4+ T cells were differentiated into Th1 subset under defined optimal conditions. The percentage is listed in the right graph. Error bars represent mean ± s.e.m., n = 4 biologically independent samples. **** P < 0.0001; two-tailed, unpaired t-test. e. Naïve CD4+ T cells were differentiated into Th17 subset under defined optimal conditions. The percentage is listed in the right graph. Error bars represent mean ± s.e.m., n = 4 biologically independent samples. **** P < 0.0001; two-tailed, unpaired t-test. f. Naïve CD4+ T cells were differentiated into iTreg subset under defined optimal conditions. The percentage is listed in the right graph. Error bars represent mean ± s.e.m., n = 5 biologically independent samples. ** P = 0.0022; two-tailed, unpaired t-test. g. Retrovirus-mediated expression of TRMT61A-WT and TRMT61A-Dead in WT and Trmt61a-KO CD4+ T cells. The protein level of TRMT61A was quantified by immunoblot. G: EGFP, W: TRMT61A-WT, D: TRMT61A-Dead. Representative data of three independent experiments are shown. h. Flow cytometric analysis of cell counts. Data are shown as box plots (boxes show median, upper and lower quartiles, whiskers show 1.5× IQR on either side). n = 3 biologically independent samples. **** P < 0.0001, * P = 0.0267, NS: non-significant; two-tailed, unpaired t-test. i. Up left: CellTrace dilution in WT and Trmt61a KO CD4+ T cells before transfer; Up right: flow cytometric analysis of CellTrace dilution in different treatments; Down: flow cytometry shows CellTrace dilution in different treatments. Error bars represent mean ± s.e.m., n = 5 biologically independent samples. **** P < 0.0001, ** P = 0.0045, NS: non-significant; two-tailed, unpaired t-test.
