Extended Data Fig. 2: Inactivation of Tet2 has no impact on 5hmC levels and tissue morphology.
From: Spurious transcription causing innate immune responses is prevented by 5-hydroxymethylcytosine
a, Expression of Tet1, Tet2 and Tet3 genes by RNA-seq in sorted control lung SMCs (n = 3 independent animals; one-way ANOVA with Tukey’s post hoc test: **P = 0.0017; ***P = 0.0007). b, Western blot analysis of TET2 in sorted lung SMCs (n = 3 independent animals; two-tailed unpaired t-test). The pan-actin loading control was run on a different gel due to differences in molecular weights. c, Localization of the sgRNA-targeting site in the Tet2 gene. The sgRNA-targeting sequence is underlined, the protospacer-adjacent motif (PAM) sequence is labeled in green. The restriction site at the target region is in bold and capitalized. PCR primers (p1, p2) used for genotyping are indicated. d, Genotyping of Tet2+/+ (Ctrl) and Tet2-/- (Tet2KO) mice with or without EcoRV digestion. Genotyping was performed with 5 litters of animals. e, Immunofluorescence staining for TET2 & α-SMA and 5hmC & α-SMA on cryosections of 8-weeks-old Tet2+/+ (Ctrl) and Tet2KO aortas (n = 3). DNA was stained by DAPI. Scale bar: 50 μm. f, Dot blot analysis of 5mC and 5hmC levels using genomic DNA from 8-weeks-old Tet2+/+ (Ctrl) and Tet2KO hearts and lungs (n = 3). Plasmid and brain tissue DNA from Tet2+/+ (Ctrl) mice were used as negative and positive controls, respectively. Methylene blue (MB) staining served as loading control. g, Schematic depiction of the excision of floxed exon 3 in the Tet2 gene by CMV-Cre. h, Western blot analysis of TET2 in lung tissues (n = 3 independent animals; two-tailed unpaired t-test: *P = 0.0189). i, H&E staining (upper panel) and trichrome staining (lower panel) of paraffin sections from 8-weeks-old Tet2fl/fl (Ctrl) and CMV-CreposTet2fl/fl (Tet2cKO) lungs (n = 3). Scale bar: 200μm, 50 μm. j, k, Immunofluorescence staining for 5hmC & α-SMA (h), CCSP & Mucin5AC and Vimentin & α-SMA on cryosections from 8-weeks-old Tet2fl/fl (Ctrl) and Tet2cKO lungs (n = 3). DNA was stained with DAPI. Arrows indicate 5hmC-positive SMCs. Scale bar: 50 μm. l, H&E staining of paraffin sections from control or Tet2/Tet3smKO lungs (n = 5). Scale bar: 200μm, 50 μm. m, Immunofluorescence staining for 5hmC and α-SMA on cryosections from Tet2/Tet3fl/fl (Ctrl), Tet3smKO, Tet2/Tet3smKO lungs (n = 3 independent animals). DNA was stained with DAPI. Arrows indicate 5hmC-positive SMCs. Inserts depict enlarged images of 5hmC-stained SMCs from the respective panels. Mean fluorescence intensity (MFI) of 5hmC signals was quantified by Image J and is shown in the lower panel (n = 3; two-tailed unpaired t-test: *P = 0.0187). Scale bar: 50 μm. The enlarged image was scaled to 150×150 pixels. Data in (a, b, h, m) are presented as mean values ± s.e.m.
