Fig. 5: Effect of TUFT1 knockdown on TGF-β1-mediated gene transcription in HSCs and activation of TGF-β/SMAD signaling induces TUFT1 expression.

A Volcano plots showing differentially expressed genes between the two groups. |log2 fold change| >1, P < 0.05. B Venny diagram revealing 690 TUFT1-dependent TGF-β1-inducible transcriptional targets. |log2 fold change| >1, P < 0.05. C A heatmap revealing expression levels of 690 differentially expressed genes (DEGs) affected by TUFT1 knockdown. |log2 fold change| >1, P < 0.05. The minimum expression level (blue) to maximum expression level (red) is shown by a color bar. D Enrichment analysis revealed that alterations of the expression of 690 DEGs potentially affect biological processes, cellular components, and molecular functions of the cells. Analyzed by R package “clusterProfiler”. E Gene Set Enrichment Analysis (GSEA) identified 4 pathways with their gene expression levels affected by TUFT1 knockdown in TGFβ1-stimulated HSCs. NES scores and P values are shown in the plots. F A heatmap showing that the transcripts of a gene set related to HSC activation and extracellular matrix remodeling are impacted by TUFT1 knockdown. The minimum expression level (blue) to maximum expression level (red) is shown by a color bar. G RNA sequencing revealed that TGF-β1 stimulation increased the transcripts of TUFT1 in HSCs. ***P < 0.001 by t-test, n = 3. FPKM: fragments per kilobase per million mapped fragments. H Luciferase reporter assay showed that the promoter activity of TUFT1 was elevated upon TGF-β1 stimulation. Not significant (n.s., P > 0.05), ***P < 0.001 by t-test, n = 3. I ChIP-qPCR showed that SMAD2/3 protein bound to the promoter of TUFT1 in primary human HSCs under TGF-β1 stimulation. ***P < 0.001 by t-test, n = 3.