Journal: Clinical and Molecular Hepatology

Article Title: Modulation of phosphatase of regenerating liver-1 within placental mesenchymal stem cells instigates the transition between epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition subsequent to hepatic fibrosis

doi: 10.3350/cmh.2024.0741

Figure Lengend Snippet: Placenta-derived mesenchymal stem cells engineered to overexpress phosphatase of regenerating liver-1 (PD-MSCs PRL-1 ) downregulates the mesenchymal phenotype, repressing TGFB/SMAD signaling in fibrotic rat liver. (A) Serological TGFB1 and liver mRNA Tgfb1 expression in rats (n=4–5/group) and their correlation. (B) Histological mesenchymal morphology in hepatocytes (yellow dotted lines) of liver tissues. V, vein; H, hepatocytes; C, cholangiocytes. (C) qPCR of mesenchymal markers ( Vim , Snai1 ) in BDL-injured rat liver at 1, 2, 3, and 5 weeks (n=4–5/group). (D) Western blotting of mesenchymal markers and p-SMAD2 in total and nuclear liver protein extracts (n=4–5/group), normalized to GAPDH for total protein and LMNB1 for nuclear protein. (E) IF of p-SMAD2 in injured rat livers at 2 weeks post-transplantation (arrows indicate merge, red=p-SMAD2, blue=DAPI). (F) Schematic of reversed EMT by repressing p-SMAD2, leading to decreased SNAI1 and VIM. Scale bar=100 μm. Data represent mean±standard deviation, analyzed by one-way ANOVA. EMT, epithelial-to-mesenchymal transition; IF, immunofluorescence; ns, not significant. * P <0.05, ** P <0.01, **** P <0.0001.

Article Snippet: The following day, a sterile 1,000 μl tip was used to create a straight-line scratch across the monolayer and subsequently they were treated with TGFB1 (PeproTech) for 48 hours, followed by treatment with PRL-1 (Novus Biologicals) and Pentamidine (Sigma-Aldrich), which is a PRL-1 inhibitor, respectively.

Techniques: Derivative Assay, Expressing, Western Blot, IF-P, Transplantation Assay, Standard Deviation, Immunofluorescence

Journal: Clinical and Molecular Hepatology

Article Title: Modulation of phosphatase of regenerating liver-1 within placental mesenchymal stem cells instigates the transition between epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition subsequent to hepatic fibrosis

doi: 10.3350/cmh.2024.0741

Figure Lengend Snippet: Placenta-derived mesenchymal stem cells engineered to overexpress phosphatase of regenerating liver-1 (PD-MSCs PRL-1 ) coculture retains the epithelial phenotype of rat liver epithelial cells. (A) Schematic of PD-MSCs or PD-MSCs PRL-1 coculture with hepatocytes exposed to TGFB1 for 48 hours and siRNA-PRL-1 (siPRL-1; 50 nM) for 24 hours. Correlation of TGFB1 and BMP7 by ELISA. Western blotting of PRL-1 and ALB. IF showing CDH1 (green) and BMP7 (red) expression. Quantification of CDH1. (B) Western blotting of BMP7, CDH1, and t/p-SMAD1/5 in hepatocytes. (C) Exogenous BMP7 and TGFB1 in supernatant by ELISA. (D) qPCR of epithelial ( Bmp7 , Dsp , Cdh1 ) and mesenchymal ( Tgfb1 , Snai1 , Col1a1 ) markers. (E) Western blotting of COL1A1, SNAI1, and t/p-SMAD2. (F) Schematic of TGFB1/BMP7 balance regulating EMT/MET. Scale bar=50 μm. Data represent mean±standard deviation, analyzed by one-way ANOVA. ns, not significant. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: The following day, a sterile 1,000 μl tip was used to create a straight-line scratch across the monolayer and subsequently they were treated with TGFB1 (PeproTech) for 48 hours, followed by treatment with PRL-1 (Novus Biologicals) and Pentamidine (Sigma-Aldrich), which is a PRL-1 inhibitor, respectively.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Standard Deviation

Journal: Clinical and Molecular Hepatology

Article Title: Modulation of phosphatase of regenerating liver-1 within placental mesenchymal stem cells instigates the transition between epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition subsequent to hepatic fibrosis

doi: 10.3350/cmh.2024.0741

Figure Lengend Snippet: Phosphatase of regenerating liver-1 (PRL-1) regulates BMP7 expression. (A) Exogenous BMP7 level in the supernatant and western blotting for BMP7 and PRL-1 in the lysates of PD-MSCs or PD-MSCs PRL-1 in presence or absence of siPRL-1. BMP7 protein expression in placenta-derived mesenchymal stem cells (PD-MSCs) in presence of PRL-1 (5 pg/mL) for 24 hours. (B) Fluorescence image showing the co-localization of BMP7 (green) and PRL-1 (red) in WB-F344 cells (blue=DAPI). Western blotting for the interaction between BMP7 and PRL-1 using a Co-immunoprecipitation assay. (C) Western blotting of p-SMAD1/5 and p-SMAD2 in WB-F344 cells exposed to TGFB1 and cocultured with PD-MSCs or PD-MSCs PRL-1 with or without siRNA BMP7 (siBMP7; 50 nM) for 24 hours. (D) Wound healing assay and quantification in WB-F344 cells in response to TGFB1, followed by PRL-1 and/or pentamidine (PRL-1 inhibitor, 1 μg/mL) for 30 min. (E) Western blotting of p-SMAD1/5 and p-SMAD2 in WB-F344, normalized to t-SMAD1/5 and t-SMAD2, respectively. (F) Schematic of TGFB1/BMP7 balance by PRL-1 through SMAD1/2/5 in hepatocytes regulating epithelial-to-mesenchymal transition (EMT)/mesenchymal-to-epithelial transition (MET). Scale bar=50 μm. Data represent mean±standard deviation, analyzed by Student’s t -test or one-way ANOVA. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The following day, a sterile 1,000 μl tip was used to create a straight-line scratch across the monolayer and subsequently they were treated with TGFB1 (PeproTech) for 48 hours, followed by treatment with PRL-1 (Novus Biologicals) and Pentamidine (Sigma-Aldrich), which is a PRL-1 inhibitor, respectively.

Techniques: Expressing, Western Blot, Derivative Assay, Fluorescence, Co-Immunoprecipitation Assay, Wound Healing Assay, Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional responses to direct and indirect TGFB1 stimulation in cancerous and noncancerous mammary epithelial cells

doi: 10.1186/s12964-024-01821-5

Figure Lengend Snippet: Changes in the global transcriptional profiles of MCF10A and MCF7 cells directly stimulated with recombinant TGFB1 (TGF) or exposed to conditioned media (CM). ( a ) Number of genes induced (log2FC > 1.0, padj < 0.05) or repressed (log2FC < − 1.0, padj < 0.05) after TGF or CM treatment. ( b ) Overlap of genes with altered expression after 6 days of TGF and/or CM treatment in both cell lines (see also Figure ). ( c ) Heatmap with hierarchical clustering of normalized read counts from RNA-seq (row z-score) for selected genes (1,685) with altered expression after TGF treatment and their assignment to hallmark pathways (on the right). ( d ) Gene set enrichment analysis showing significant pathways from the hallmark gene set collection detected in MCF10A and MCF7 cells treated with TGF or CM, as well as differences between treatments and differences between cell types: untreated (Ctr, shown in green rectangle) and in response to TGF (first compared to corresponding Ctr). The sizes of the pie charts correspond to the effect size, while color intensity corresponds to the p value; blue and red indicate the fractions of downregulated and upregulated genes, respectively. The cells were treated according to the scheme shown in Figure a

Article Snippet: Two different recombinant human TGFB1 cytokine (#100–21 C, PeproTech EC Ltd., London, UK; at calculated final concentration 10 ng/ml) treatment protocols were applied depending on further analyses: (i) for continuous treatment, starting from day 1, half of the medium was replaced daily with fresh TGFB1 for 24 h; (ii) for pulsed treatment, each day, TGFB1 was administered in fresh medium for 2 h and then the medium was changed to fresh medium for another 22 h to obtain conditioned medium (CM).The CM from each treatment day was used to treat other cells to induce potential bystander effects (Figure a).

Techniques: Recombinant, Expressing, RNA Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional responses to direct and indirect TGFB1 stimulation in cancerous and noncancerous mammary epithelial cells

doi: 10.1186/s12964-024-01821-5

Figure Lengend Snippet: Differences in gene expression profiles between TGFB1-stimulated MCF10A and MCF7 cells. ( a ) Scatterplots of log2-fold changes on the sixth day of TGFB1 stimulation (vs. Ctr) in the genes associated with selected terms from the hallmark collection in MCF10A (X-axis) and MCF7 (Y-axis) cells. ( b ) Volcano plots of the RNA-seq results showing the differentially expressed genes in untreated cells (red color/up – higher in MCF10A cells, blue color/down – higher in MCF7 cells). Each dot represents one gene. Genes with the most significant differences are labeled

Article Snippet: Two different recombinant human TGFB1 cytokine (#100–21 C, PeproTech EC Ltd., London, UK; at calculated final concentration 10 ng/ml) treatment protocols were applied depending on further analyses: (i) for continuous treatment, starting from day 1, half of the medium was replaced daily with fresh TGFB1 for 24 h; (ii) for pulsed treatment, each day, TGFB1 was administered in fresh medium for 2 h and then the medium was changed to fresh medium for another 22 h to obtain conditioned medium (CM).The CM from each treatment day was used to treat other cells to induce potential bystander effects (Figure a).

Techniques: Gene Expression, RNA Sequencing, Labeling

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional responses to direct and indirect TGFB1 stimulation in cancerous and noncancerous mammary epithelial cells

doi: 10.1186/s12964-024-01821-5

Figure Lengend Snippet: Time trends in the expression of genes related to TGFβ signaling in MCF10A and MCF7 cells. ( a ) Genes involved in SMAD-mediated signaling. ( b ) Transcription factors involved in EMT. ( c ) Epithelial ( CDH1 ) and mesenchymal markers. The cells were treated according to the scheme shown in Figure a. TGF, direct TGFB1 treatment; CM, conditioned medium treatment. *** padj < 0.0001, ** padj < 0.001, * padj < 0.05 (significance of differences was marked for the entire run if the adjusted p value vs. Ctr reached a given value at least at one experimental point). See also Figure S5b

Article Snippet: Two different recombinant human TGFB1 cytokine (#100–21 C, PeproTech EC Ltd., London, UK; at calculated final concentration 10 ng/ml) treatment protocols were applied depending on further analyses: (i) for continuous treatment, starting from day 1, half of the medium was replaced daily with fresh TGFB1 for 24 h; (ii) for pulsed treatment, each day, TGFB1 was administered in fresh medium for 2 h and then the medium was changed to fresh medium for another 22 h to obtain conditioned medium (CM).The CM from each treatment day was used to treat other cells to induce potential bystander effects (Figure a).

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional responses to direct and indirect TGFB1 stimulation in cancerous and noncancerous mammary epithelial cells

doi: 10.1186/s12964-024-01821-5

Figure Lengend Snippet: Inhibition of estrogen signaling and induction of cell death after TGFB1 treatment in MCF7 cells. Time trends in the expression of genes related to estrogen signaling and apoptosis in MCF10A and MCF7 cells: ( a ) steroid receptors and EGFR ; ( c ) ESR1 targets; ( d ) prosurvival genes; ( e ) proapoptotic genes. The cells were treated according to the scheme shown in Figure a. TGF, direct TGFB1 treatment; CM, conditioned medium treatment. *** padj < 0.0001, ** padj < 0.001, * padj < 0.05 (significance of differences was marked for the entire run if the adjusted p value vs. Ctr reached a given value at least at one experimental point). ( b ) Response of MCF7 and MCF10A cells to TGFB1 treatment analyzed by Western blot. The cells were treated according to the scheme shown in Figure a. The levels of the indicated proteins were analyzed in total protein extracts. ACTB was used as a loading control

Article Snippet: Two different recombinant human TGFB1 cytokine (#100–21 C, PeproTech EC Ltd., London, UK; at calculated final concentration 10 ng/ml) treatment protocols were applied depending on further analyses: (i) for continuous treatment, starting from day 1, half of the medium was replaced daily with fresh TGFB1 for 24 h; (ii) for pulsed treatment, each day, TGFB1 was administered in fresh medium for 2 h and then the medium was changed to fresh medium for another 22 h to obtain conditioned medium (CM).The CM from each treatment day was used to treat other cells to induce potential bystander effects (Figure a).

Techniques: Inhibition, Expressing, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional responses to direct and indirect TGFB1 stimulation in cancerous and noncancerous mammary epithelial cells

doi: 10.1186/s12964-024-01821-5

Figure Lengend Snippet: Time trends in the expression of genes related to cell cycle progression in MCF10A and MCF7 cells. Examples of ( a ) transcriptional regulators, ( b ) cyclins, ( c ) cyclin-dependent kinases and ( d ) their inhibitors, ( e ) cell division cycle proteins, ( f ) cell division cycle associated proteins, ( g ) DNA polymerases, ( h ) proteins involved in chromosomal replication, ( i ) proteins involved in DNA repair and ( j ) markers of proliferation. The cells were treated according to the scheme shown in Figure a. TGF, direct TGFB1 treatment; CM, conditioned medium treatment. *** padj < 0.0001, ** padj < 0.001, * padj < 0.05 (significance of differences was marked for the entire run if the adjusted p value vs. Ctr reached a given value at least at one experimental point). See also Figures (cell cycle in KEGG pathways) and (time trends of other cell cycle-related genes)

Article Snippet: Two different recombinant human TGFB1 cytokine (#100–21 C, PeproTech EC Ltd., London, UK; at calculated final concentration 10 ng/ml) treatment protocols were applied depending on further analyses: (i) for continuous treatment, starting from day 1, half of the medium was replaced daily with fresh TGFB1 for 24 h; (ii) for pulsed treatment, each day, TGFB1 was administered in fresh medium for 2 h and then the medium was changed to fresh medium for another 22 h to obtain conditioned medium (CM).The CM from each treatment day was used to treat other cells to induce potential bystander effects (Figure a).

Techniques: Expressing