Journal: Scientific Reports

Article Title: Tcf21 modulates fibroblast activation and promotes cardiac fibrosis after injury via Pdgfrb signaling

doi: 10.1038/s41598-025-13102-3

Figure Lengend Snippet: Tcf21 knockout (KO) fibroblasts show attenuated proliferation capacity and reduced Pdgfrb signaling. ( a,b ) 5-bromo-2′-deoxyuridine (BrdU) uptake decreased in Tcf21 KO fibroblasts (biologically n = 4). Scale bar, 200 µm. ( c ) Western blots of Smad2 and phosphorylated Smad2 (p-Smad2), with TGF-β (10 ng/mL) stimulation for 15 min (biologically n = 3). ( d ) Quantitative analysis of p-Smad2/Smad2. ( e ) Western blots of Erk and phosphorylated Erk (p-Erk), with PDGF-BB (20 ng/mL) stimulation for 15 min (biologically n = 3). ( f ) Quantitative analysis of p-Erk/Erk. ( g ) qRT-PCR, ( h,i ) Western blot analysis of Pdgfrb (biologically n = 3). ( j ) qRT-PCR analysis using human cardiac fibroblasts with TCF21 knockdown by CRISPR-CAS9 showing decreased expression of TCF21 , POSTN , ACTA2 , COL1A1 , COL3A1 , and PDGFRB (biologically n = 3).

Article Snippet: We transferred the Tcf21 Mouse ORF Clone (OriGene, MR201596) to the pTRE3G vector and electroporated it into pEFa-Tet3G-stable expressing 3T3-L1 cells (Tet-On 3G Inducible Expression System, Clontech).

Techniques: Knock-Out, Western Blot, Quantitative RT-PCR, Knockdown, CRISPR, Expressing

Journal: Scientific Reports

Article Title: Tcf21 modulates fibroblast activation and promotes cardiac fibrosis after injury via Pdgfrb signaling

doi: 10.1038/s41598-025-13102-3

Figure Lengend Snippet: Tcf21 regulates extracellular matrix, proliferation, and inflammation-associated genes. ( a,b ) Results of gene expression cluster analysis obtained from DAVID 6.8 and Metascape showing altered expression of extracellular matrix and proliferation signaling genes in fibroblasts obtained from the heart of Tcf21 KO mice compared to those obtained from control mice (biologically n = 3). ( c ) Volcano plot shows that the expression of 440 genes was significantly altered by Tcf21. Upregulated genes in Tcf21 WT fibroblasts contain many extracellular matrix genes. Red dots represent significant genes upregulated in Tcf21 WT ( P < 0.05). Blue dots represent significant genes downregulated in Tcf21 WT ( P < 0.05). ( d ) Results of quantitative RT-PCR showing decreased expression of extracellular matrix and fibroblast activation genes in Tcf21 KO fibroblasts (biologically Control n = 15, iKO n = 24).

Article Snippet: We transferred the Tcf21 Mouse ORF Clone (OriGene, MR201596) to the pTRE3G vector and electroporated it into pEFa-Tet3G-stable expressing 3T3-L1 cells (Tet-On 3G Inducible Expression System, Clontech).

Techniques: Gene Expression, Expressing, Control, Quantitative RT-PCR, Activation Assay

Journal: Scientific Reports

Article Title: Tcf21 modulates fibroblast activation and promotes cardiac fibrosis after injury via Pdgfrb signaling

doi: 10.1038/s41598-025-13102-3

Figure Lengend Snippet: Isoproterenol (ISO) injection leads to cardiac fibrosis and expansion of Tcf21-expressing cells. (a ) Overview of the ISO injection schedule. ( b ) Cardiac fibrosis area of Masson trichrome-stained sections after ISO injection. Dunnett’s test was performed for post-hoc analysis (biologically n = 3). ( c ) Tcf21 expression before and after ISO injection assessed by qPCR. Dunnett’s test was performed for post-hoc analysis (biologically n = 3). ( d ) Tcf21-positive area calculated based on β-galactosidase staining (biologically n = 4). The detected area was normalized to the cardiac cross-sectional area. ( e,f ) Tcf21-positive cells accumulated in the fibrotic area after 3 or 7 days of ISO injection. Tcf21-positive cells were calculated in the Masson trichrome (MT) positive or negative area. Scale bar, 100 µm (biologically n = 3). ( g ) Immunostaining of a section of heart from Tcf21 LacZ/+ transgenic mice. Tcf21 expression as assessed by β-galactosidase (green) and periostin (red) staining coexisted after 3 days of ISO injection. Scale bar, 100 µm. ( h ) Immunostaining of Ki67 (brown) and Tcf21 expression by β-galactosidase (blue). Ki67-positive cells accumulated in the Tcf21-positive area. Scale bar, 100 µm. ( i ) Proliferation capacity of Ki67-positive cells. Each sample was counted in a high-power microscopic field more than three times, and the average values were used for analysis. Unpaired two-tailed t -tests with Bonferroni correction were used for post-hoc analysis (biologically n = 4). ( j ) Fibroblast-associated gene expression increased after ISO injection. Dunnett’s test was performed for post-hoc analysis (biologically n = 3).

Article Snippet: We transferred the Tcf21 Mouse ORF Clone (OriGene, MR201596) to the pTRE3G vector and electroporated it into pEFa-Tet3G-stable expressing 3T3-L1 cells (Tet-On 3G Inducible Expression System, Clontech).

Techniques: Injection, Expressing, Staining, Immunostaining, Transgenic Assay, Two Tailed Test, Gene Expression

Journal: Scientific Reports

Article Title: Tcf21 modulates fibroblast activation and promotes cardiac fibrosis after injury via Pdgfrb signaling

doi: 10.1038/s41598-025-13102-3

Figure Lengend Snippet: Lack of Tcf21 suppresses cardiac fibrosis and maintains cardiac function. ( a ) Overview of isoproterenol (ISO) injection schedule. ( b ) Cardiac fibrosis observed at each time point. Masson trichrome staining was perfoemed. Before ISO injection and 3, 7, and 28 days after the injection. Scale bar, 1 mm. Yellow arrowheads indicated fibrotic area. ( c ) Fibrosis area from Masson trichrome staining normalized with cardiac cross-sectional area (biologically n = 3 ~ 4). ( d ) Immunostaining for Ki67. Scale bar, 50 µm. Arrows indicate Ki67-positive cells. ( e ) Change in the number of Ki67-positive cells, 7 days after ISO injection (biologically n = 5). ( f ) M-mode echocardiographic view, one week after ISO injection. ( g ) Changes in left ventricular fractional shortening (FS) after ISO injection. Dunnett’s multiple comparison test was performed to compare baseline data (biologically Control n = 14, iKO n = 7). ( h ) Protocol for hyperglycemic and hyperlipidemic injury. ( i ) Masson trichrome-stained heart Sections 16 weeks after diabetes induction with HFD. Arrows show fibrosis. Scale bar, 50 µm. ( j ) Fibrosis area calculated from Masson trichrome staining normalized by the cardiac cross-sectional area (biologically n = 4 and 3). Student’s t-test was performed.

Article Snippet: We transferred the Tcf21 Mouse ORF Clone (OriGene, MR201596) to the pTRE3G vector and electroporated it into pEFa-Tet3G-stable expressing 3T3-L1 cells (Tet-On 3G Inducible Expression System, Clontech).

Techniques: Injection, Staining, Immunostaining, Comparison, Control