Extended Data Fig. 1: SMC-specific inactivation of Tet3 induces pathological changes in airways.
From: Spurious transcription causing innate immune responses is prevented by 5-hydroxymethylcytosine
a, Schematic depiction of the conditional Tet3 allele. b, Whole-mount LacZ staining of Tet3+/+, Tet3floxLacZ/+embryos at E9.5 (n = 10). c, Immunofluorescence analysis of OCT4 in mESC and mESC derived SMCs (mESC-SMCs) (n = 3). Scale bar: 50 μm. DNA was stained by DAPI. d, Immunofluorescence analysis of α-SMA and MYH11 in mESC and mESC-SMCs (n = 3). Scale bar: 50 μm. DNA was stained by DAPI. e, RT-qPCR analysis of Sox2, Nanog, Acta2, Myh11, Tet1, Tet2, Tet3 in mESC and mESC-SMCs (n = 4 independent experiments; two-tailed unpaired t-test: *P< 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). f, Outline of the strategy to generate inducible SMC-specific Tet3 knockout mice (mice were 8 weeks old at first tamoxifen injection). g, RT-qPCR analysis of Tet3 expression in aortas from control and Tet3smKO mice, 8 weeks after tamoxifen injection. β-actin was used for normalization (control n = 6; Tet3smKO n = 9; two-tailed unpaired t-test: ****P < 0.0001). h, Immunofluorescence staining for α-SMA and TET3 on cryosections from control and Tet3smKO lungs, 8 weeks after tamoxifen injection (n = 3). DNA was stained by DAPI. Scale bar: 50 μm. i, Body weights of control and Tet3smKO mice at different time points after tamoxifen injection (n = 6 independent animals). Tamoxifen injections started when mice were 8-weeks-old. j, k, H&E staining of cryosections from lungs (j) and intestines (k) of control and Tet3smKO mice 8 weeks after tamoxifen injection to demonstrate normal tissue architecture (n = 3). Scale bar: 50 μm. l, Immunofluorescence staining for α-SMA and 5hmC on cryosections from control and Tet3smKO lungs, 8 weeks after tamoxifen injection (n = 3). DNA was stained by DAPI. Scale bar: 50 μm. m, FACS gating strategy to isolate SMCs from control and Tet3smKO:T lungs. n, RT-qPCR analysis of Tet3 expression in sorted SMCs from control and Tet3smKO:T mice, 8 weeks after tamoxifen injection (n = 6 independent animals; two-tailed unpaired t-test: ***P = 0.0001). β-actin was used for normalization. o, PCR of genomic DNA using primers located at 5’ upstream and 3’-downsteam of floxed exon 10 of the Tet3 gene. Genotyping was performed with 5 litters of animals. p, Quantification of the Western blot analysis of TET3 in sorted lung control and Tet3smKO:T SMCs (n = 3 independent animals; two-tailed unpaired t-test: **P < 0.01). Data (in e, g, i, n, p) are presented as mean values ± s.e.m.
