Fig 2: Knockdown of endogenous miR-181a inhibits adipogenesis in BM-MSCs. (A) Graph showing miR-181a levels at days 0 and 21 of adipogenic differentiation for Scp-1 cells overexpressing either control vector (cmiR) or miR-181a specific antagomiR (antimiR). (B) Oil red O staining images of Scp-1 cmiR and antimiR cells on day 21 of adipogenic differentiation with representative micrographs (top) and quantification of Oil red O staining (bottom). (C) Graphs depicting decreased mRNA expression of PPARG and CEBPA at day 0 and decreased expression of adipogenesis target genes at day 21 in Scp-1 antimiR vs cmiR cells. (D) Representative western blot images showing decreased expression of CEBPA and PPARG in Scp-1 antimiR vs cmiR cells at day 0 with quantification (right) (Full blots are shown in Supplemental Fig. 9A). (E) Representative western blot images showing decreased expression of CEBPA, PPARG, and FABP4 at day 21 in Scp-1 antimiR vs cmiR cells with quantification (right) (Full blots are shown in Supplemental Fig. 9B). (F) Graph showing PER3 mRNA levels at days 0 and 21 of adipogenic differentiation in Scp-1 cmiR and antimiR cells. (G) Western blot showing PER3 protein levels at days 0 and 21 of adipogenic differentiation in Scp-1 cmiR and antimiR cells (Full blots are shown in Supplemental Fig. 9C,D). Data are representative of 3 independent experiments. * is p = 0.05, ** is p = 0.005, *** is p = 0.0005.

Fig 3: Knockdown of C/EBP? impairs epithelial homeostasis.a The results from qRT-PCR analysis showing CEBPA mRNA levels in HMLE cells expressing shRNA control (SCR) or HMLE cells expressing two independent shRNAs targeting CEBPA (shCEBPA_1 and shCEBPA_2). Data represented as mean ± SD of three independent experiments. p-values were calculated using unpaired two-tailed Student’s t test. *p < 0.05, ***p < 0.001. b Bright-field microscopy images of HMLE cells expressing shRNA control (SCR) or shRNA targeting CEBPA in two different magnifications. Data are representative of three independent experiments. Scale bar: 200 µm. c qRT-PCR data showing the expression levels of known epithelial and mesenchymal markers upon CEBPA knockdown in HMLE cells. Data represented as mean ± SD of three independent experiments. p-values were calculated using unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. ns not significant. d The results from immunoblotting analysis displaying protein levels of well-characterized EMT markers upon CEBPA knockdown in HMLE cells. Data are representative of three independent experiments. e The results from qRT-PCR analysis showing the mRNA levels of transcription factor ZEB1 upon CEBPA knockdown in HMLE and MCF10A cells. Data represented as mean ± SD of three independent experiments. p-values were calculated using unpaired two-tailed Student’s t test. **p < 0.01, ***p < 0.001. f ZEB1 mRNA levels in HMLE or MCF10A cells expressing EV or constitutive CEBPA. Data represented as mean ± SD of three independent experiments. p-values were calculated using unpaired two-tailed Student’s t test. **p < 0.01, ***p < 0.001. ß2m beta-2-microglobulin.

Fig 4: miR-181a is induced during BMSC/ASC adipogenesis. (A) Graphs depicting increasing expression of miR-181a during the induction of adipogenesis at days 0, 1, 3, and 7 for Scp-1 and PASC-1 cells. (B) Graphs depicting increasing mRNA expression of PPARG, CEBPA, and terminal adipogenesis markers during the induction of adipogenesis at days 0, 1, 3, and 7 for Scp-1 and PASC-1 cells. (C) Representative western blot images showing increasing protein expression of PPARG, CEBPA, and FABP4 during the induction of adipogenesis at days 0, 1, 3, and 7 for Scp-1 cells with respective quantifications below (Full blots are shown in Supplemental Fig. 5A). (D) Representative western blot images showing increasing protein expression of PPARG, CEBPA, and FABP4 during the induction of adipogenesis at days 0, 1, 3, and 7 for PASC-1 cells with respective quantifications below (Full blots are shown in Supplemental Fig. 5B). (E) Micrographs showing Oil red O staining at days 0 and 14 for Scp-1 and PASC-1 cells. Data are representative of 3 independent experiments. * is p = 0.05, ** is p = 0.005, *** is p = 0.0005.

Fig 5: Restoration of C/EBPa rescues epithelial phenotype.a Schematic representation of the experimental set up. HMLE cells were treated with 2.5 ng/ml of TGF-ß for 15 days, and posteriorly C/EBPa expression was induced upon 200 ng/ml of doxycycline for 64 h. b The results from qRT-PCR showing the expression levels of known EMT markers in EV or CEBPA-inducible HMLE cells treated with TGF-ß, doxycycline or both. Data represented as mean ± SD of four independent experiments. p-values were calculated using two-way ANOVA with Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns not significant. c Immunoblotting analysis of the expression levels of well-stablished EMT markers in TGF-ß-treated HMLE cells in the absence or presence of C/EBPa overexpression. Data are representative of three independent experiments. d Confocal microscopy visualization of MCF10A–epithelial spheroids treated with 5 ng/ml of TGF-ß for 4 days. Data are representative of three independent experiments. Scale bar: 45 µm. e Schematic representation of the 3D spheroid-formation assay. MCF10A cells expressing empty vector (EV) or doxycycline-inducible C/EBPa were stimulated with TGF-ß (5 ng/ml), doxycycline (200 ng/ml), or both as indicated. f Confocal microscopy visualization of epithelial spheroids using MCF10A cells expressing empty vector or doxycycline-inducible C/EBP? in the presence of TGF-ß, doxycycline (DOX), or both. Cells were stained for C/EBPa (green) and phalloidin (white). DAPI was used to visualize the nucleus (blue). Data are representative of three independent experiments. Scale bar: 45 µm. g Quantification of the number of spheres formed in MCF10A cells expressing empty vector or doxycycline-induced C/EBP? upon the treatment of TGF-ß, doxycycline, or both. The number of spheres are relative to empty vector untreated. Data are represented as mean ± SD of three independent experiments. p-values were calculated using two-way ANOVA with Tukey’s multiple comparisons test. **p < 0.01, ****p < 0.0001. ß2m beta-2-microglobulin, EV empty vector, UNT untreated.