Fig 1: Proteomics workflow and analysis of recurrent and non-recurrent hepatocellular carcinoma (HCC) tumour explant samples obtained at the time of transplantation. A Schematic diagram of the proteomics workflow including the 11 HCC samples. B Proteomics analysis workflow to identify the most significant proteins between recurrent and non-recurrent HCC. C Volcano plot illustrating proteins differentially expressed (p < 0.05) in recurrent vs non-recurrent HCC tumors. Proteins in red are significantly increased in recurrent, while those in blue are significantly decreased in the recurrent cases. The three proteins bolded and marked with stars (ALDH1A1, LGALS3 and LGALS3BP) were subsequently selected for validation. Some protein names were removed for clarity, to minimize overlap. HCC, hepatocellular carcinoma; SCX, strong cationic exchange; LC, liquid chromatography; MS/MS, tandem mass spectrometry; ALDH1A1, retinal dehydrogenase 1, LGALS3, galectin-3; LGALS3BP, galectin-3-binding protein
Fig 2: LncRNA ROR upregulates ALDH1A1/TESC to promote TPC-1 cell proliferation, migration, and invasion while inhibiting cell apoptosis.A The co-expression relationship between TESC and ALDH1A1 from MEM analysis. ALDH1A1 has multiple probes, i.e., multiple co-expression relationships are shown in the figure. B Venn diagram of differentially expressed genes in thyroid cancer samples from the GSE3467, GSE33630, and GSE97001 datasets and co-expression genes predicted using MEM. C PPI network diagram of 10 intersection genes and their related genes constructed by GeneMANIA. The larger the circle for the gene is, the higher the core degree is. D ALDH1A1 expression in PTC tissue and adjacent tissue samples of patients with PTC as detected by immunohistochemistry (×400, n = 85). *p < 0.05 vs. adjacent tissues. E TESC expression after alteration of TESC in TPC-1 cells as determined by RT-qPCR, normalized to GAPDH. *p < 0.05 vs. oe-NC. #p < 0.05 vs. sh-NC. F ALDH1A expression after alteration of TESC in TPC-1 cells as determined by RT-qPCR, normalized to GAPDH. *p < 0.05 vs. oe-NC. #p < 0.05 vs. sh-NC. G ALDH1A protein expression after alteration of TESC in TPC-1 cells as determined by western blot analysis, normalized to GAPDH. *p < 0.05 vs. oe-NC. #p < 0.05 vs. sh-NC. H ALDH1A1 expression after ALDH1A1 alteration and TESC overexpression in TPC-1 cells as determined by RT-qPCR. I TPC-1 cell viability after ALDH1A1 alteration and TESC overexpression, as detected by MTT. J Colony formation of TPC-1 cells after ALDH1A1 alteration and TESC overexpression, as detected by colony formation assay. K TPC-1 cell migration and invasion after ALDH1A1 alteration and TESC overexpression as detected by Transwell assay (×200). L TPC-1 cell apoptosis after ALDH1A1 alteration and TESC overexpression as detected by flow cytometry. In panel H–L, *p < 0.05 vs. oe-NC, #p < 0.05 vs. sh-NC, &p < 0.05 vs. oe-TESC. M The expression of ALDH1A1 in TPC-1 cells transduced with oe-lncRNA ROR + sh-TESC-1 or sh-ALDH1A1-1, as determined by RT-qPCR, normalized to GAPDH. N TPC-1 cell viability after transduction with oe-lncRNA ROR + sh-TESC-1 or sh-ALDH1A1-1, as detected by MTT. O Colony formation of TPC-1 cells after transduction with oe-lncRNA ROR + sh-TESC-1 or sh-ALDH1A1-1, as detected by colony formation assay. P TPC-1 cell migration and invasion after transduction with oe-lncRNA ROR + sh-TESC-1 or sh-ALDH1A1-1 as detected by Transwell assay (×200). Q TPC-1 cell apoptosis after transduction with oe-lncRNA ROR + sh-TESC-1 or sh-ALDH1A1-1 as detected by flow cytometry. In panel M–Q, *p < 0.05 vs. oe-lncRNA ROR + sh-NC. Cell experiments were repeated three times independently.
Fig 3: Silencing of S100A4 induced differentiation of rCSCs following corneal alkali burn. S100A4 shRNA vector, overexpressed S100A4 vector, and NC vector were constructed and transfected into rCSCs after alkali burn modeling. RT-qPCR and Western blot assay showing mRNA (A) and protein (B) levels of CD34 (CD34), keratocan (KERA), COL5A1 (COL5A1), ALDH1A1 (ALDH1A1), and ALDH1A1 (ALDH3A1) in rCSCs in the corneal alkali burn model. Replicates = 3. Data are expressed as mean ± standard deviation. Two-way or one-way ANOVA and Tukey's multiple comparisons test were used for data analysis. *P < 0.05. **P < 0.01.
Fig 4: Kaplan–Meier and dot plots for: A ALDH1A1 gene and protein; B LGALS3 gene and protein; C LGALS3BP gene and protein in HCC patients. The levels of the gene/protein were grouped by interquartile ranges. For ALDH1A1 gene: low level ≤ 10.40; medium level [10.40–12.00]; high level ≥ 12.00. For ALDH1A1 protein: low level ≤ 28.59; medium level [28.59–30.82]; high level ≥ 30.82. For LGALS3 gene: low level ≤ 4.10; medium level [4.10–6.10]; high level ≥ 6.10. For LGALS3 protein: low level ≤ 25.88; medium level [25.88–27.80]; high level ≥ 27.80. For LGALS3BP gene: low level ≤ 9.10; medium level [9.10–10.80]; high level ≥ 10.80. For LGALS3BP protein: low level ≤ 22.69; medium level [22.69–26.09]; high level ≥ 26.09. LGALS3, galectin-3; LGALS3BP, galectin-3-binding protein
Fig 5: ALDH1A1 activates TUBB3 to facilitate TPC-1 cell proliferation, migration, and invasion while inhibiting cell apoptosis.A TUBB3 expression in PTC tissue and adjacent tissue samples of patients with PTC as detected by immunohistochemistry (×400, n = 85). *p < 0.05 vs. adjacent tissues. B TUBB3 expression after alteration of ALDH1A1 in TPC-1 cells as determined by RT-qPCR, normalized to GAPDH. C TUBB3 protein expression after alteration of ALDH1A1 in TPC-1 cells as determined by western blot analysis, normalized to GAPDH. D TUBB3 expression after alteration of TUBB3 in TPC-1 cells as determined by RT-qPCR. E The effects of TUBB3 on TPC-1 cell viability, as detected by MTT. F The effects of TUBB3 on TPC-1 cell colony formation, as detected by colony formation assay. G TPC-1 cell migration and invasion after ALDH1A1 overexpression and TUBB3 silencing, as detected by Transwell assay (×200). H TPC-1 cell apoptosis after ALDH1A1 overexpression and TUBB3 silencing, as detected by flow cytometry. In panel B–H, *p < 0.05 vs. oe-NC, #p < 0.05 vs. sh-NC, @p < 0.05 vs. oe-ALDH1A1. Cell experiments were repeated three times independently.
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