Fig 2: CEBPA expression promotes a lipofibroblast phenotype.a-b) Oil Red O staining and quantification in the Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with (AD: adipogenic medium) or without adipogenic medium induction (c: control medium). Scale bar = 100 µm. c-f) qRT-PCR analysis of PLIN2, PPARA, PPARG1 and PPARGC1A transcript levels in Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with or without TGF-ß1 treatment for 48 h. Data are expressed as mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001)

Fig 3: Histone deacetylation is required for LMP1-induced CEBPA repression. a, b mRNA levels of CEBPA were determined by RT-qPCR. CNE1/HNE2-TetOn-Vector and CNE1/HNE2-TetOn-LMP1 cells were pretreated with 100 ng/ml Dox for 24 hours then followed by 1 µM TSA or 200 nM JNJ treatment for further 24 hours. c, d Protein levels of CEBPA were determined by immunoblotting. CNE1/HNE2-TetOn-Vector and CNE1/HNE2-TetOn-LMP1 cells were pretreated with 100 ng/ml Dox for 24 hours then followed by 1 µM TSA or 200 nM JNJ treatment for further 24 hours. e CNE1-TetOn-Vector and CNE1-TetOn-LMP1 cells were treated with 100 ng/ml Dox for 48 hours. ChIP assays were performed using anti-acetylated histone H3 and anti-acetylated histone H4 antibodies. f, g Protein levels of CEBPA were determined by immunoblotting. Knockdown of BRD4 by siRNA or 5 µM BRD4 inhibitor treatment in CNE1-TetOn-Vector and CNE1-TetOn-LMP1 cells then LMP1 expression were induced by 100 ng/ml Dox for 24 hours and exposed to 200 nM JNJ for further 24 hours. Statistics (a, b), significance: **P < 0.01, ***P < 0.001; two-tailed Student’s t-tests

Fig 4: STAT5A recruits HDAC1/2 to CEBPA Gene Locus. a, b Truncated CEBPA promoter luciferase activity in CNE1-TetOn-LMP1 and CNE1-TetOn-Vector cells were detected by dual-luciferase reporter assays. c Prediction of known transcription factor binding in the -588 to -403 region. d STRING analysis of known transcription factor binding in the -588 to -403 region and HDAC1/2. Image shows the physical and functional interactions. e CNE1-TetOn-LMP1 and CNE1-TetOn-Vector cells were transfected with siRNAs of indicated TFs. Then expression levels of CEBPA were examined by immunoblotting. f Western blot analysis of the expression and localization of STAT5A, HDAC1, and HDAC2 in CNE1-TetOn-LMP1 and CNE1-TetOn-Vector cells. g Western blot analysis of the co-immunoprecipitates between endogenous HDAC1 and STAT5A, and HDAC2 and STAT5A. h CNE1-TetOn-LMP1 and CNE1-TetOn-Vector cells were treated with 100 ng/ml Dox for 48 hours. Cells were collected and ChIP assays were performed using anti-STAT5A. ChIP signals were detected by PCR using primers for the CEBPA promoter region. i Biotinylated CEBPA promoter pull-down assay was performed. The associated proteins were subjected to immunoblot analysis with indicated antibodies. j STAT5A was knockdown by siRNA in CNE1-TetOn-LMP1 and CNE1-TetOn-Vector cells. Cells were collected and ChIP assays were performed using anti-HDAC1 and anti-HDAC2. ChIP signals were detected by qRT-PCR using primers for the CEBPA promoter region. k Wild-type or mutant CEBPA promoter luciferase activity in CNE1/HNE2-TetOn-LMP1 and CNE1/HNE2-TetOn-Vector cells was detected by dual-luciferase reporter assays. Statistics (b, j and k), significance: *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significance; two-tailed Student’s t-tests

Fig 5: CEBPA overcomes LMP1-induced dedifferentiation in NPC cells. a Western blot analysis with differentiation and stem-like markers in CNE1-TetOn-LMP1 and CEBPA-overexpressed CNE1-TetOn-LMP1 cells by 100 ng/ml Dox treatment for 48 hours. b Proliferation ability of CNE1-TetOn-LMP1 and CEBPA-overexpressed CNE1-TetOn-LMP1 cells was determined by CCK8 assay. c Colony formation in CNE1-TetOn-LMP1 and CEBPA-overexpressed CNE1-TetOn-LMP1 cells. d Immunofluorescence staining for Ki67 in CNE1-TetOn-LMP1 and CEBPA-overexpressed CNE1-TetOn-LMP1 cells. e SA-ß-gal staining in CNE1-TetOn-LMP1 and CEBPA-overexpressed CNE1-TetOn-LMP1 cells. f CNE1-TetOn-LMP1 and CEBPA-overexpressed CNE1-TetOn-LMP1 cells were injected into nude mice subcutaneously. After continuous Dox administration, tumor volume was determined. g CEBPA, differentiation, and stem-like markers were analyzed by immunoblot in tumor tissue and primary cultured tumor cells. h IHC staining of differentiation markers in CNE1-TetOn-LMP1 and CEBPA-overexpressed CNE1-TetOn-LMP1 tumor tissues. Representative images are shown. Statistics (b–f), significance: **P < 0.01, ***P < 0.001; two-tailed Student’s t-test