The dox-inducible plasmid, pLVET-tTR-KRAB, was obtained from Addgene (Cambridge, MA, USA; plasmid #11644). Based on the gene sequences retrieved from GenBank, the SV40 large T antigen (LT) fragment was synthesized and used to construct a lentiviral shuttle recombinant plasmid. Specifically, the human EEF1A1 promoter and enhanced green fluorescent protein (EGFP)-coding sequence in pLVET-tTR-KRAB were replaced with the collagen type I alpha 2 chain (COL1A2) promoter and LT gene, respectively. All plasmid construction and lentiviral packaging procedures were performed using VectorBuilder (Guangzhou, Guangdong, China).

pCFBs were isolated from ventricular tissue of a fetus at 14 weeks of gestation. The tissue was donated by the fetal parents with written informed consent. This study was approved by the Ethics Committee of Longgang District People’s Hospital of Shenzhen (approval number: 2025101). Cells were cultured in high-glucose Dulbecco’s modified Eagle medium (H-DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Catalog No. 11965118) supplemented with 10% fetal bovine serum (FBS; Gibco, Catalog No. A5256901), 1% penicillin-streptomycin (Gibco, Catalog No. 15140122), and 10 ng/mL recombinant human basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN, USA; Catalog No. 233-FB) and maintained at 37 °C in a humidified incubator with 5% CO₂. The culture medium was replaced every three days. Upon reaching confluence, pCFBs were passaged at a 1:4 ratio and cells at population doubling 4 (PD4) were used for subsequent experiments.

The pCFBs at approximately PD4 were transduced with lentiviral vectors carrying the dox-inducible LT expression system. After 24 h of initial culture in the primary cell medium, the medium was replaced with proliferation medium supplemented with 100 ng/mL dox (Sigma-Aldrich, St. Louis, MO, USA; Catalog No. D9891) to induce LT expression. Successfully transduced cells were designated conditional iCFBs and maintained in freshly prepared dox-containing proliferation medium, which was replaced every alternate day. When the cultures reached approximately 90% confluence, they were passaged in a 1:8 ratio.

For growth curve analysis, iCFBs and pCFBs were passaged at ratios of 1:8 and 1:4, respectively, upon reaching confluency. To evaluate the proliferation rates and their dependence on LT expression, the cells were seeded at a low density and cultured in medium supplemented with 100 ng/mL dox (iCFBs) or standard culture medium (pCFBs). The cell numbers were quantified at the indicated time points using a cytometer (BodBoge, Guangzhou, Guangdong, China).

Primary human foreskin fibroblasts (FFBs) were purchased from Jennio Biotech Co., Ltd. (Guangzhou, Guangdong, China; Catalog no. JNO-H0612) and cultured in H-DMEM supplemented with 10% FBS in a humidified incubator at 37 °C with 5% CO₂. The culture medium was replaced every three days. The cells were routinely passaged at a 1:4 ratio when the cultures reached approximately 80% confluence.

All primary cells were routinely tested for mycoplasma contamination using the BeyoDirect™ Mycoplasma qPCR Detection Kit (Beyotime, Shanghai, China; Catalog No. C0303S) and consistently tested negative, as defined by the kit’s criteria (sample Ct $>$35). Their identity was confirmed by marker gene expression and/or biological function analyses.

The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.5% Tween-20 in PBS, and blocked with PBS containing 1% bovine serum albumin (Sigma-Aldrich, Catalog No. A8022), and 0.05% Tween-20 (Sangon Biotech, Shanghai, China; CAS No. 9005-64-5). pCFB, iCFB, and FFB monolayers were incubated overnight at 4 °C with the following primary antibodies: mouse anti-SV40 LT (1:50; Santa Cruz Biotechnology, Dallas, TX, USA; Catalog No. sc-147), anti-Collagen type I (anti-COL-1) (1:200; Proteintech, Wuhan, Hubei, China; Catalog No. 66761-1-Ig), anti-$\alpha$-smooth muscle actin (anti-$\alpha$-SMA) (1:200; Proteintech, Catalog No. 67735-1-Ig), and anti-connexin 43 (anti-CX43) (1:50; Proteintech, Catalog No. 80543-1-RR).

After three washes with PBS containing 0.05% Tween-20, the bound primary antibodies were detected using Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L) (1:200; Thermo Fisher Scientific, Catalog No. A-11005). Nuclei were counterstained with Hoechst 33342 (10 µg/mL; Thermo Fisher Scientific, Catalog No. H3570). Fluorescence images were acquired using a Leica STELLARIS 5 confocal laser-scanning microscope (Leica Microsystems).

Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on 0.22 or 0.45 µm polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA; Catalog No. IPVH00010). After blocking with 5% skim milk in Tris-buffered saline containing Tween-20 (TBST), the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-SV40 LT (1:1000; Santa Cruz Biotechnology, Catalog No. sc-147) and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (1:5000; Proteintech, Catalog No. 60004-1-Ig).

The membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Proteintech, Catalog No. SA00001-0) for 1 h at room temperature. Protein bands were visualized using the Thermo iBright CL1000 imaging system (Thermo Fisher Scientific) with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Catalog No. 32209). Band intensities from at least three independent experiments were quantified using the ImageJ software (version 1.46r, National Institutes of Health, Bethesda, MD, USA).

Total RNA was extracted from the cultured cells using AG RNAex Pro Reagent (Accurate Biotechnology, Ningbo, Zhejiang, China) according to the manufacturer’s instructions. The isolated RNA was reverse transcribed into complementary DNA (cDNA) using Evo M-MLV RT Premix for qPCR (Accurate Biotechnology). Gene expression levels were determined by RT-qPCR using the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology). PCR amplification and fluorescence detection were performed using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Relative gene expression was calculated using the 2^{-Δ⁢Δ⁢Ct} method. The primer sequences are listed below. 5^′-CAGCCACTAGCCATTGTGGA-3^′ (F, gap junction protein alpha 1 [GJA1]); 5^′-GGCTGTTGAGTACCACCTCC-3^′ (R, GJA1); 5^′-TGACGAGACCAAGAACTGCC-3^′ (F, collagen type I alpha 1 chain [COL1A1]); 5^′-GCACCATCATTTCCACGAGC-3^′ (R, COL1A1); 5^′-GGAGGCAAACAGCTCAGAGT-3^′ (F, periostin [POSTN]); 5^′-GGTGTGTCTCCCTGAAGCAG-3^′ (R, POSTN); 5^′-GTTCCGCTCCTCTCTCCAAC-3^′ (F, actin alpha 2 [ACTA2]); 5^′-TAGTCCCGGGGATAGGCAAA-3^′ (R, ACTA2); 5^′-ACCATCCGGGTGTCCTTTTC-3^′ (F, T-box transcription factor 20 [TBX20]); 5^′-TGACCCTCGATTTGGGGTTG-3^′ (R, TBX20); 5^′-TTGCATCAGCCTGTGGATGT-3^′ (F, discoidin domain receptor tyrosine kinase 2 [DDR2]); 5^′-GAGTCCAGCCAAAGGTCTCC-3^′ (R, DDR2); 5^′-CTCTGGCACGTCTTGACCTT-3^′ (F, vimentin [VIM]); 5^′-TTGCGCTCCTGAAAAACTGC-3^′ (R, VIM); 5^′-AATGGGCAGCCGTTAGGAAA-3^′ (F, GAPDH); 5^′-GCGCCCAATACGACCAAATC-3^′ (R, GAPDH).

The collagen-based cell contraction assay was performed using a commercial kit (Cell Biolabs, San Diego, CA, USA; Catalog No. CBA-201) according to the manufacturer’s instructions. iCFBs and pCFBs (2.0 $\times$ 10⁵ cells per well) were seeded and cultured in the presence or absence of TGF-$\beta$1. After 48 h, the collagen gels were photographed and gel surface area was quantified using ImageJ software (version 1.46r, National Institutes of Health).

The iCFBs were cultured in doxycycline-free DMEM for 4 days to suppress LT expression. Thereafter, pCFBs and iCFBs were treated with 10 ng/mL TGF-$\beta$1 (R&D Systems, Minneapolis, MN, USA; Catalog No. 240-B), 1 µM Ang II (MedChemExpress, Monmouth Junction, NJ, USA; Catalog No. HY-13948) for 48 h, or 200 µM PA (MedChemExpress, Monmouth Junction, NJ, USA; Catalog No. HY-N0830) for 24 h to induce myofibroblast differentiation.

To evaluate the anti-fibrotic response of iCFBs, cells were treated for 48 h with medium containing 10 ng/mL TGF-$\beta$1 in combination with 10 µM N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-1H-benzimidazol-2-amine (NS8593; MedChemExpress, Monmouth Junction, NJ, USA; Catalog No. HY-137952) or 1 mg/mL pirfenidone (PFD; MedChemExpress, Monmouth Junction, NJ, USA; Catalog No. HY-B0673).

Data are presented as mean $\pm$ standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test was used for multiple group comparisons. All experiments were independently repeated at least thrice. Statistical significance was set at p 0.05.

To establish an iCFB line, pCFBs were transduced with lentiviral particles encoding a dox-inducible LT expression cassette under the control of the COL1A2 promoter (Fig. 1A). Successful immortalization was confirmed by LT expression, which was robustly expressed in transduced cells (iCFBs) cultured in dox-containing medium, whereas no LT signal was detected in pCFBs (Fig. 1B,C).

Fig. 1.

Generation of conditionally immortalized cardiac fibroblasts (iCFBs). (A) Schematic overview of the conditional immortalization strategy. (B) Immunofluorescence staining confirming LT expression in transduced cardiac fibroblasts cultured in the presence of dox. Scale bar, 50 µm. (C) Western blot analysis confirming LT expression in transduced CFB cultured in the presence of dox. (D) Growth curves of pCFBs and transduced CFBs cultured in the presence of dox. CFB, cardiac fibroblast; pCFB, primary cardiac fibroblast; LT, SV40 large T antigen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; dox, doxycycline; PD, population doubling; iCFB+dox, iCFB cultured in the presence of dox; DNA, deoxyribonucleic acid. Created in BioRender. Liu, J. (2026) https://BioRender.com/iuxys2w.

Growth curve analysis demonstrated that pCFBs ceased to proliferate after approximately 9 population doublings (PDs). In contrast, iCFBs cultured in the presence of dox maintained sustained proliferation for over 70 PDs without entering senescence, with an average population doubling period of approximately 24 h (Fig. 1D). Taken together, these results confirmed the successful establishment of an iCFB line.

To determine whether LT expression and the associated immortalization phenotype in iCFBs were strictly dox-dependent, they were cultured in a medium switched from dox-containing to dox-free conditions (Fig. 2A). Western blot analysis confirmed LT expression in iCFBs maintained in dox-containing medium (day 0), with a time-dependent decline to undetectable levels by day 6 after dox withdrawal (Fig. 2B,C), which was further validated by immunofluorescence staining (Supplementary Fig. 1). Consistent with the changes in LT expression, Ki-67 antigen (Ki-67) positivity was abundant in dox-cultured iCFBs, but decreased substantially after dox withdrawal (Fig. 2D). Accordingly, iCFBs proliferated continuously after dox treatment. In contrast, following dox removal, cell numbers plateaued after a modest initial increase within days 0–2 (Fig. 2E). Taken together, these findings demonstrate that dox tightly regulates both LT expression and proliferative capacity of iCFBs, enabling a reversible switch between an immortalized proliferative state and a non-immortalized phenotype.

Fig. 2.

Conditional proliferative capacity of conditionally immortalized cardiac fibroblasts (iCFBs). (A) Schematic illustration of iCFB preparation and dox withdrawal process. (B,C) Western blot analysis and corresponding quantification of LT protein expression in iCFBs before (day 0) and after (days 2, 4, 6, and 8) dox withdrawal. (D) Immunofluorescence staining of the proliferation marker Ki-67 in iCFBs before (day 0) and after (days 2, 4, 6, and 8) dox withdrawal. Scale bar, 50 µm. (E) Quantitative analysis of iCFB cell counts after culture in media with or without dox. iCFB, conditionally immortalized cardiac fibroblast; LT, SV40 large T antigen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; dox, doxycycline; Ki-67, Ki-67 antigen; DNA, deoxyribonucleic acid. Data were presented as mean $\pm$ standard deviation (SD; n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test. *p 0.05, **p 0.01.

Next, we examined whether the iCFBs retained their phenotypic characteristics. We compared the expression of canonical CFB markers in iCFBs maintained under proliferative conditions with dox (iCFB+dox), iCFBs cultured without dox for 4 days to revert to a non-immortalized state (iCFB), and pCFBs. No significant differences were observed in the expression levels of key CFB marker genes, including the fibroblast-associated markers COL1A1, DDR2, VIM, and POSTN, as well as cardiac markers GJA1 and TBX20 between pCFBs and iCFBs (Fig. 3A). Notably, sustained exposure to dox (iCFB+dox) reduced the expression of these CFB-associated markers compared with that in both pCFBs and iCFBs (Fig. 3A).

Fig. 3.

Conditionally immortalized cardiac fibroblasts (iCFBs) retain primary cardiac fibroblast (pCFB) identity. (A) RT-qPCR analysis of fibroblast-associated marker genes (COL1A1, DDR2, POSTN, and VIM) and cardiac-specific genes (GJA1 and TBX20) in pCFBs and iCFBs cultured in the presence or absence of dox. (B) Representative immunofluorescence images showing the expression of COL-1 (green) and CX43 (red) in iCFBs, pCFBs, and FFBs. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 µm. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; COL1A1, collagen type I alpha 1 chain; DDR2, discoidin domain receptor tyrosine kinase 2; POSTN, periostin; VIM, vimentin; GJA1, gap junction protein alpha 1; TBX20, T-box transcription factor 20; COL-1, Collagen type I; CX43, connexin 43; pCFB, primary cardiac fibroblast; iCFB, conditionally immortalized cardiac fibroblast; iCFB+dox, iCFB cultured in the presence of dox; FFB, foreskin fibroblast; DNA, deoxyribonucleic acid. Data were presented as mean $\pm$ standard deviation (SD; n = 3). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc test. *p 0.05, **p 0.01, ***p 0.001; #not significant.

Consistent with the transcriptional data, immunofluorescence staining verified comparable COL-1 and CX43 protein expression in pCFBs and dox-withdrawn iCFBs, confirming restoration of the pCFB molecular profile in iCFBs (Fig. 3B). FFBs, as non-cardiac fibroblast controls, expressed COL-1, but not CX43, further supporting the cardiac-specific identity of dox-withdrawn iCFBs (Fig. 3B). Taken together, these results indicate that, in the absence of dox-induced proliferative pressure, the molecular identity of iCFBs closely resemble that of pCFBs. Accordingly, iCFBs cultured for 4 d without dox were used for all subsequent functional experiments.

A series of functional assays was performed to compare the biological behaviors of iCFBs and pCFBs. TGF-$\beta$1, a central mediator of cardiac fibrosis, is well known to induce myofibroblast transdifferentiation and to stimulate the production of collagen and $\alpha$-smooth muscle actin ($\alpha$-SMA). To evaluate myofibroblast differentiation, iCFBs were cultured in dox-free medium for 4 days to suppress LT expression; both iCFBs and pCFBs were then stimulated with TGF-$\beta$1 for 2 days.

Immunofluorescence analysis showed that TGF-$\beta$1 treatment markedly increased COL-1 expression in both cell types (Fig. 4A). Furthermore, $\alpha$-SMA staining was localized to prominent stress fiber-like filaments that traversed the cytoplasm and aligned along the longitudinal axis of the cell. This characteristic fibrillar localization is a hallmark of mature contractile myofibroblasts, confirming the successful phenotypic conversion of iCFBs (Fig. 4A). Consistent with these observations, RT-qPCR analysis demonstrated that the mRNA levels of COL1A1 and ACTA2 (encoding $\alpha$-SMA) were significantly elevated in differentiated iCFBs (iCFB-differentiated myofibroblasts, iCMFBs) and pCFBs (primary cardiac myofibroblasts, pCMFBs) compared to their undifferentiated controls (Fig. 4B).

Fig. 4.

Conditionally immortalized cardiac fibroblasts (iCFBs) retain key biological functions of primary cardiac fibroblasts (pCFBs). (A) Representative immunofluorescence images showing the expression of COL-1 (green) and $\alpha$-SMA (red) in pCFBs, pCMFBs, iCFBs, and iCMFBs. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 µm. (B) RT-qPCR analysis of COL1A1 and ACTA2 expression levels in pCFBs, pCMFBs, iCFBs, and iCMFBs. (C) Quantification of collagen gel contraction assays for each group. The remaining gel surface area was normalized to that of the pCFB control group. Gel area is inversely proportional to contractile activity. COL1A1, collagen type I alpha 1 chain; COL-1, collagen type I; ACTA2, actin alpha 2; $\alpha$-SMA, $\alpha$-smooth muscle actin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; pCFB, primary cardiac fibroblast; pCMFB, primary cardiac myofibroblast; iCFB, conditionally immortalized cardiac fibroblast; iCMFB, differentiated iCFBs; TGF-$\beta$1, transforming growth factor-$\beta$1; DNA, deoxyribonucleic acid. Data were presented as mean $\pm$ standard deviation (SD; n = 3). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc test. *p 0.05, **p 0.01, ***p 0.001; #not significant.

Enhanced contractile activity is a hallmark of myofibroblast differentiation. In line with this, collagen gel contraction assays revealed that TGF-$\beta$1 stimulation induced significant gel contractions in iCFBs. The extent of contraction was comparable between iCFBs and pCFBs, with no statistically significant differences observed (Fig. 4C). Taken together, these functional analyses demonstrated that iCFBs retain a myofibroblast differentiation capacity that is highly similar to that of pCFBs.

To assess the scalability of iCFBs as in vitro models, we examined their phenotypic stability and functional capacity during long-term subculture. Marker gene expression and TGF-$\beta$1-induced transdifferentiation were compared between iCFBs on PD30, iCFBs on PD60, and pCFBs. RT-qPCR analysis revealed highly consistent expression levels of the signature genes COL1A1, POSTN, GJA1, and TBX20 across all three groups, indicating that iCFBs preserved their core molecular identity, even after continuous passaging to PD60 (Fig. 5A).

Fig. 5.

Conditionally immortalized cardiac fibroblasts (iCFBs) preserve phenotypic and functional properties during long-term expansion. (A) RT-qPCR analysis of COL1A1, POSTN, GJA1, and TBX20 expression levels in pCFBs and iCFBs at PD30 and PD60. (B) Representative immunofluorescence images showing COL-1 (green) and CX43 (red) expression in pCFBs and iCFBs at PD30 and PD60. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 µm. (C) Representative immunofluorescence images showing COL-1 (green) and $\alpha$-SMA (red) expression in pCMFB, iCMFB-PD30, and iCMFB-PD60. (D) RT-qPCR analysis of COL1A1 and ACTA2 expression levels in pCMFB, iCMFB-PD30, and iCMFB-PD60. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 µm. COL-1, collagen type I; CX43, connexin 43; $\alpha$-SMA, $\alpha$-smooth muscle actin; COL1A1, collagen type I alpha 1 chain; POSTN, periostin; GJA1, gap junction protein alpha 1; TBX20, T-box transcription factor 20; ACTA2, actin alpha 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; pCFB, primary cardiac fibroblast; iCFB, conditionally immortalized cardiac fibroblast; iCMFB, iCFB-differentiated myofibroblasts; pCMFB, primary cardiac myofibroblast; PD, population doubling; DNA, deoxyribonucleic acid. Data were presented as mean $\pm$ standard deviation (SD; n = 3). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc test. #not significant.

Immunofluorescence staining further demonstrated that iCFBs at both PD30 and PD60 displayed cellular morphology and expression patterns of COL-1 and CX43 that were highly similar to those observed in pCFBs, with no spontaneous differentiation (Fig. 5B).

After TGF-$\beta$1 treatment, all three groups (pCFB, iCFB-PD30, iCFB-PD60) successfully underwent myofibroblast differentiation with enhanced COL-1 deposition and $\alpha$-SMA⁺ stress fiber formation (Fig. 5C). RT-qPCR analysis further demonstrated that the expression levels of the fibrotic marker genes COL1A1 and ACTA2 in iCMFB-PD30 and iCMFB-PD60 cells were not significantly different from those in pCMFBs, suggesting that iCFBs maintained a robust and stable differentiation response despite long-term passaging (Fig. 5D).

Taken together, these results demonstrate that iCFBs maintain their core molecular identity and myofibroblast transdifferentiation potential after prolonged in vitro expansion.

To evaluate the suitability of iCFB model for drug screening, we assessed its responsiveness to multiple pro-fibrotic stimuli. After 4 days of culture in dox-free medium, iCFBs were treated with TGF-$\beta$1, Ang II, or PA to mimic the multifactorial pathways that contribute to cardiac fibrosis. Using ACTA2 and COL1A1 expression as readouts of fibroblast activation, we observed that all three stimuli significantly increased the levels of these fibrotic markers in iCFBs compared to that in the control conditions (Fig. 6A), indicating a robust induction of pro-fibrotic phenotypes in vitro.

Fig. 6.

Conditionally immortalized cardiac fibroblasts (iCFBs) serve as a platform for pro- and anti-fibrotic drug screening. (A) RT-qPCR analysis of myofibroblast marker gene expression (COL1A1 and ACTA2) in iCFBs treated with pro-fibrotic stimuli (TGF-$\beta$1, Ang II, and PA) or TGF-$\beta$1 in combination with anti-fibrotic agents (NS8593 or PFD). Data were presented as mean $\pm$ standard deviation (SD; n = 3). (B) Representative immunofluorescence images showed COL-1 (green) and $\alpha$-SMA (red) expression in iCFBs treated with TGF-$\beta$1 alone or in combination with NS8593. The nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 µm. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; COL1A1, collagen type I alpha 1 chain; ACTA2, actin alpha 2; COL-1, collagen type I; $\alpha$-SMA, $\alpha$-smooth muscle actin; ctrl, control; TGF-$\beta$1, transforming growth factor beta 1; Ang II, angiotensin II; PA, palmitic acid; NS8593, TRPM7 channel inhibitor; PFD, pirfenidone; DNA, deoxyribonucleic acid. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc test. *p 0.05, **p 0.01; #not significant.

Having established the broad pro-fibrotic responsiveness of the model, we examined its utility for evaluating anti-fibrotic compounds. Specifically, we tested whether the transient receptor potential melastatin 7 (TRPM7) channel antagonist NS8593 and the clinically approved anti-fibrotic drug PFD could suppress TGF-$\beta$1-induced myofibroblast transdifferentiation. Both NS8593 and PFD effectively attenuated the TGF-$\beta$1-induced COL1A1 and ACTA2 expression upregulation in iCFBs (Fig. 6A).

Consistent with these molecular findings, immunofluorescence analysis showed that TGF-$\beta$1-induced fibrotic morphological changes in iCFBs were significantly attenuated by NS8593 co-treatment (Fig. 6B). Collectively, these results indicated that the iCFB model is a reliable and sensitive in vitro platform for screening both pro-fibrotic stimuli and candidate anti-fibrotic therapeutics.