Fig 2: Knockout of DOT1L inhibits ALV-J replication. (A) HD11 and DOT1L-KO cells were infected with ALV-J (MOI 2). After 48 h, the viral RNA levels were measured by qRT-PCR. GAPDH mRNA level was measured as an internal control (left panel). The supernatants were collected to measure the viral titers by a standard TCID50 method (right panel). (B) Immunofluorescence analyses of ALV-J Env protein. HD11 cells and DOT1L-KO cells were infected with ALV-J at indicated MOI for 48 h, the cells were then visualized with the inverted fluorescence microscope and a specific antibody to the ALV-J Env protein (green), and nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI; blue). Images show a representative image. All the data were shown as mean ± SD (error bars) from three independent experiments. **p < 0.01; unpaired, two-tailed Student’s t-test.

Fig 3: Disruptor of telomeric silencing 1-like (DOT1L) expression and H3K79me2 levels are upregulated during avian leukosis virus subgroup J (ALV-J) infection in chicken macrophage cell line HD11. (A,B) Western blot (A) and intensity (B) for DOT1L protein and DOT1L-mediated H3K79me2 levels in HD11 cells at the indicated time points post infection with ALV-J (MOI 2). a-tubulin and total H3 were used as a loading control. (C) The relative DOT1L mRNA levels in HD11 cells at the indicated time points post infection with ALV-J, measured by quantitative real-time reverse transcription PCR (qRT-PCR). GAPDH was used as a normalizing control. (D) The relative ALV-J RNA level in HD11 cells at the indicated time points post infection, normalized to chicken GAPDH mRNA, was measured by qRT-PCR. All the data were shown as mean ± SD (error bars) from three independent experiments. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; unpaired, two-tailed Student’s t-test.

Fig 4: Dot1L is upregulated in TGF-ß1-induced adult rat CFs and fibrotic mice hearts. A Dot1L, Col 1, FN, and CTGF protein expression level in cardiac fibroblasts (CFs) stimulated by different doses of TGF-ß1 (0, 2, and 5 ng/mL) for 48 h. B Col 1, CTGF, Col 3, FN, and Dot1L protein expression level in cardiac fibroblasts stimulated by TGF-ß1 (5 ng/mL) for the dedicated time (0, 12, 24, and 48 h). The data are presented as mean ± SD. Student’s t-test, *p < 0.05, **p < 0.01, and ***p < 0.001 versus control group, each acquired from three individual experiments. C Representative images of immunofluorescence staining of Dot1L as well as CTGF in CFs stimulated by 5 ng/mL TGF-ß1 for 48 h. D Dot1L, Col 3 and CTGF protein expression around infarct area were measured in mice undergoing 2 weeks of left descending anterior coronary artery (LAD) ligation. The data are presented as mean ± SD. Student’s t-test, ***p < 0.001 versus Sham group, n = 6/group

Fig 5: Knockdown or inhibition of Dot1L attenuates TGF-ß1-induced ECM deposition in adult rat CFs. A, B Knockdown of Dot1L attenuates ECM deposition in TGF-ß1 treated CFs. Dot1L siRNA and control siRNA were transfected into CFs, followed by 5 ng/mL TGF-ß1 stimulation for 48 h. The protein expressions of Dot1L, Col 3, FN and MMP9 were analyzed by western blot (A); Representative images of immunofluorescence staining of Dot1L and CTGF (B). C, D EPZ5676 attenuates ECM deposition in TGF-ß1-treated CFs. CFs were pretreated with 10 and 20 µmol/L EPZ5676 for 4 h and then co-incubated with 5 ng/mL TGF-ß1 for 48 h. H3K79me3, Dot1L, CTGF, Col 1, MMP9 and FN were analyzed by western blot (C); Representative images of immunofluoresence staining of CTGF or Col 1 were shown in 20 µmol/L EPZ5676-treated CFs (D). All data are presented as mean ± SD. ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001 versus Ctrl or Ctrl + siCtrl, #p < 0.05, ##p < 0.01, ###p < 0.001 versus TGF-ß1 or TGF-ß1 + siCtrl, each acquired from three individual experiments