Fig 1: Characterization of heterogeneity between SRGN-high malignant cells and SRGN-low malignant cells. (A) Subcluster of malignant cells with SRGN expression. (B) UMAP is colored by malignant cells from the SRGN-high subcluster or SRGN-low subcluster. (C) Heatmap shows the metabolic flux in the SRGN-high subcluster and SRGN-low subcluster. Volcano plots display the metabolism difference of glycolysis and TCA cycle(D), BCAA(E), O-linked glycan synthesis(F), and pyrimidine synthesis(G) between the two subclusters. (H) Dot plot shows the hallmark pathway condition in the two subclusters. Violin plots compare the metastasis score(I), proliferation score(J), ECM modeling score(K), and collagen formation score(L) for two subclusters. (M) SRGN-high vs. SRGN-low tumor size. (N) SRGN sera level comparison among different HCC statuses. (O) Clinical stages between SRGN-high and SRGN-low patients. (P) SRGN expression-AFP correlation.
Fig 2: Validation of SRGN expression is related to the HCC cell's stemness characteristic. (A, B) Knockdown of SRGN in MHCC-97H cells verified by qPCR and immunoblotting. (C) The number of suspended spheres was reduced after SRGN knockdown in MHCC-97H cells. (D) Quantification of tumor sphere size and number. (E, F) Side population cell assay and abundance comparison. (G) Immunoblotting of cancer stem cell-related markers following SRGN knockdown in MHCC-97H cells (72 h post-transfection). (H) Relative SRGN mRNA levels in the main and side populations of MHCC-97H cells were determined by qPCR (normalized to GAPDH).
Fig 3: Potential medication screening for targeting SRGN protein. (A) Workflow of screening. (B) SRGN protein structure. (C) Docking pocket 1 of SRGN protein. (D) Docking pocket 2 of SRGN protein. The docking pockets and poses, and binding residues of sorafenib (E), daurisoline (F), regorafenib (G), and baicalin (H) with SRGN protein.
Fig 4: CRISPLD2 is a novel YAP-TEAD1 target gene regulated by SRGN. (A) Luciferase activity of the CRISPLD2 promoter in YAP-overexpressing cells. (B) Luciferase activity of the CRISPLD2 promoter with VP intervention in YAP-overexpressing cells. (C) YAP-TEAD1 binding site 1 motif in the CRISPLD2 promoter. (D) Schematic of CRISPLD2 promoter binding sites within the TEAD1 motif. (E) ChIP analysis of YAP DNA in cells stably transfected with vector or SRGN, quantified by agarose gel electrophoresis and qPCR. (F) Five primer pairs were used to evaluate decreased YAP DNA levels after SRGN suppression. (G) Fold changes in promoter DNA copy numbers. (H) Conserved CRISPLD2 motif sequences across species.
Fig 5: CRISPLD2 functions as a downstream effector of the SRGN/YAP axis. (A) SRGN and CRISPLD2 expression were overlapped among three transcriptomes groups. (B) Positive correlation between SRGN and CRISPLD2 expression in HCC cohorts at the bulk-seq level. (C) SRGN dose-dependently upregulated CRISPLD2 mRNA levels. (D) Immunoblot quantification confirms SRGN-induced CRISPLD2 protein elevation. (E) Immunoblotting shows CRISPLD2 expression in HCC cells. (F) CRISPLD2 knockdown reduced Transwell migration in SRGN-activated cells. (G) Migratory cell quantification validated CRISPLD2-dependent motility. (H) Peptide 17 suppressed CRISPLD2 expression in SRGN-overexpressing cells. (I) Immunoblot validation of CRISPLD2 protein downregulation by peptide 17 in SRGN-overexpressing cells. (J) Mechanistic schema of peptide 17 targeting SRGN/YAP/CRISPLD2 signaling node. (K) Luciferase reporter assay demonstrated CRISPLD2 promoter activity. (L) CRISPLD2 overexpression reversed SRGN knockdown-induced migration defect. (M) Quantitative migration analysis of SRGN-knockdown and CRISPLD2-overexpression. (N) SRGN silencing reduced CRISPLD2 transcript levels. (O) VP attenuated SRGN-mediated migration in dose-dependent manner. (P) Suppression of CRISPLD2 reduced SRGN-driven migration.
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