Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 expression increased in HCC. (A) The mRNA expression of PUS1 between normal tissue and primary HCC tissue analyzed based on the UALCAN website. (B) The protein expression of PUS1 between normal tissue and primary HCC tissue analyzed based on the CPTAC database in UALCAN website. (C–H) The mRNA expression of PUS1 between normal tissue and primary HCC tissue analyzed based on E-MTAB-6695, E-MTAB-4171, GSE39791, GSE47197, GSE54236, and GSE25079. (I) The protein expression of PUS1 between normal liver cell lines (LO2, WRL68) and HCC cell lines (Huh7, HepG2, SUN449, and PLC/PRF/5). (J, K) The immunohistochemistry of PUS1 in liver tissue and HCC from THE HUMAN PROTEIN ATLAS. (L, M) . The expression of PUS1 between liver tissue and HCC from 45 patients. (*P < 0.05, ***P < 0.001).

Article Snippet: The immunohistochemistry (IHC) staining and subcellular localization analysis of PUS1 in HCC were obtained from THE HUMAN PROTEIN ATLAS ( https://www.proteinatlas.org/ ) TISSUE and PHATHOLOGY module.

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: High PUS1 expression was positively correlated with occurrence and malignant progression of HCC. (A) The expression of PUS1 stratified by normal, hepatitis, cirrhosis, and HCC based on the GSE54238. (B, C) The expression of PUS1 stratified by cirrhosis and HCC based on the GSE17548 and GSE56140. (D) The expression of PUS1 based on different stage in 45 HCC patients. (E) The expression of PUS1 in primary and metastasis tumor in 45 HCC patients. (F) The expression of PUS1 stratified by different grade in HCC based on the E-MTAB-8887. (G) The expression of PUS1 stratified by different stage in HCC based on the UALCAN website. (H) The expression of PUS1 stratified by different grade in HCC based on the UALCAN website. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Article Snippet: The immunohistochemistry (IHC) staining and subcellular localization analysis of PUS1 in HCC were obtained from THE HUMAN PROTEIN ATLAS ( https://www.proteinatlas.org/ ) TISSUE and PHATHOLOGY module.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: High PUS1 expression in HCC predicted poor prognosis. (A–D) OS, DSS, PFS, and RFS Kaplan–Meier curves comparing the high and low expression of PUS1. (80324 is the RNA-seq ID of PUS1.).

Article Snippet: The immunohistochemistry (IHC) staining and subcellular localization analysis of PUS1 in HCC were obtained from THE HUMAN PROTEIN ATLAS ( https://www.proteinatlas.org/ ) TISSUE and PHATHOLOGY module.

Techniques: Expressing, RNA Sequencing

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 silencing inhibited HCC cell proliferation and colony formation, and promoted cell apoptosis. (A) RT-PCR analysis of PUS1 expression in SNU449 cell and HepG2 (B) after transfection with control or PUS1 siRNAs. (C, D) Cell proliferation of SNU449 and HepG2 cells transfected with NC or PUS1 siRNA analyzed by MTT assay. (E, F) Knockdown of PUS1 dramatically inhibited the cell colony formation ability. (G, H) Apoptosis assay by flow cytometric analysis revealed PUS1–siRNA promoted apoptosis in SNU449 and HepG2 cells. (I) Knockdown of PUS1 hardly inhibited the cell colony formation ability of LO2 and WRL68 cells. (J, K) Knockdown of PUS1 hardly inhibited the cell proliferation of LO2 and WRL68 cells. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, P>0.05).

Article Snippet: The immunohistochemistry (IHC) staining and subcellular localization analysis of PUS1 in HCC were obtained from THE HUMAN PROTEIN ATLAS ( https://www.proteinatlas.org/ ) TISSUE and PHATHOLOGY module.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, MTT Assay, Knockdown, Apoptosis Assay

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 promoted tumorigenesis and progression of HCC closely related to the mTOR and MYC pathways. (A) GSEA analysis between high and low PUS1 expression patients using hallmark gene sets based on TCGA–HCC database. (B–E) GSEA analysis revealed that MYC pathways, DNA repair, and MTORC1 pathways were significantly enriched in patients with high PUS1 expression. (F–H) The intersection analysis of the PUS1–dependent Ψ modification genes and MYC or MTORC1 pathway genes were applied. (I, J) RT–PCR analysis of PUS1 potential downstream targets in HepG2 cell after transfection with control or PUS1 siRNA. (**P<0.01, ***P<0.001).

Article Snippet: The immunohistochemistry (IHC) staining and subcellular localization analysis of PUS1 in HCC were obtained from THE HUMAN PROTEIN ATLAS ( https://www.proteinatlas.org/ ) TISSUE and PHATHOLOGY module.

Techniques: Expressing, Modification, Reverse Transcription Polymerase Chain Reaction, Transfection, Control

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 promotes cell proliferation depending on the mTOR and MYC pathways. (A, B) RT–PCR analysis of the mRNA expression of MYC, pre-MYC, mTOR and pre-mTOR in HCC cells after transfection with control or PUS1 siRNA. (C, D) WB testing of PUS1, MYC, mTOR, p-mTOR and P-S6 expression in HCC cells after transfection with control or PUS1 siRNA. (E-H) Cell proliferation of SNU449 and HepG2 cells transfected with PUS1 siRNA treatment with or without inhibitors of MYC (10058-F4, 10μM) or mTOR (rapamycin, 200nM) were analyzed by MTT assay. (**P<0.01, ***P<0.001, ****P<0.0001; ns, P>0.05).

Article Snippet: The immunohistochemistry (IHC) staining and subcellular localization analysis of PUS1 in HCC were obtained from THE HUMAN PROTEIN ATLAS ( https://www.proteinatlas.org/ ) TISSUE and PHATHOLOGY module.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, MTT Assay

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: A schematic view of PUS1 promoted tumorigenesis and progression of HCC dependent on the mTOR and MYC pathways.PUS1 expression was increased in HCC. However, knocking down PUS1 in HCC cells, the expression of MYC, p-mTOR and mTOR pathway‐related protein P-S6 were decreased. These results suggested that the role of PUS1 promoted tumorigenesis and progression of HCC is dependent on the mTOR and MYC pathways.

Article Snippet: The immunohistochemistry (IHC) staining and subcellular localization analysis of PUS1 in HCC were obtained from THE HUMAN PROTEIN ATLAS ( https://www.proteinatlas.org/ ) TISSUE and PHATHOLOGY module.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 expression increased in HCC. (A) The mRNA expression of PUS1 between normal tissue and primary HCC tissue analyzed based on the UALCAN website. (B) The protein expression of PUS1 between normal tissue and primary HCC tissue analyzed based on the CPTAC database in UALCAN website. (C–H) The mRNA expression of PUS1 between normal tissue and primary HCC tissue analyzed based on E-MTAB-6695, E-MTAB-4171, GSE39791, GSE47197, GSE54236, and GSE25079. (I) The protein expression of PUS1 between normal liver cell lines (LO2, WRL68) and HCC cell lines (Huh7, HepG2, SUN449, and PLC/PRF/5). (J, K) The immunohistochemistry of PUS1 in liver tissue and HCC from THE HUMAN PROTEIN ATLAS. (L, M) . The expression of PUS1 between liver tissue and HCC from 45 patients. (*P < 0.05, ***P < 0.001).

Article Snippet: Our result indicated that PUS1 expressed significantly higher in cancer tissues than adjacent liver tissues of HCC patients, in line with the results of the patient data analysis of THE HUMAN PROTEIN ATLAS database.

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: High PUS1 expression was positively correlated with occurrence and malignant progression of HCC. (A) The expression of PUS1 stratified by normal, hepatitis, cirrhosis, and HCC based on the GSE54238. (B, C) The expression of PUS1 stratified by cirrhosis and HCC based on the GSE17548 and GSE56140. (D) The expression of PUS1 based on different stage in 45 HCC patients. (E) The expression of PUS1 in primary and metastasis tumor in 45 HCC patients. (F) The expression of PUS1 stratified by different grade in HCC based on the E-MTAB-8887. (G) The expression of PUS1 stratified by different stage in HCC based on the UALCAN website. (H) The expression of PUS1 stratified by different grade in HCC based on the UALCAN website. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Article Snippet: Our result indicated that PUS1 expressed significantly higher in cancer tissues than adjacent liver tissues of HCC patients, in line with the results of the patient data analysis of THE HUMAN PROTEIN ATLAS database.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: High PUS1 expression in HCC predicted poor prognosis. (A–D) OS, DSS, PFS, and RFS Kaplan–Meier curves comparing the high and low expression of PUS1. (80324 is the RNA-seq ID of PUS1.).

Article Snippet: Our result indicated that PUS1 expressed significantly higher in cancer tissues than adjacent liver tissues of HCC patients, in line with the results of the patient data analysis of THE HUMAN PROTEIN ATLAS database.

Techniques: Expressing, RNA Sequencing

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 silencing inhibited HCC cell proliferation and colony formation, and promoted cell apoptosis. (A) RT-PCR analysis of PUS1 expression in SNU449 cell and HepG2 (B) after transfection with control or PUS1 siRNAs. (C, D) Cell proliferation of SNU449 and HepG2 cells transfected with NC or PUS1 siRNA analyzed by MTT assay. (E, F) Knockdown of PUS1 dramatically inhibited the cell colony formation ability. (G, H) Apoptosis assay by flow cytometric analysis revealed PUS1–siRNA promoted apoptosis in SNU449 and HepG2 cells. (I) Knockdown of PUS1 hardly inhibited the cell colony formation ability of LO2 and WRL68 cells. (J, K) Knockdown of PUS1 hardly inhibited the cell proliferation of LO2 and WRL68 cells. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, P>0.05).

Article Snippet: Our result indicated that PUS1 expressed significantly higher in cancer tissues than adjacent liver tissues of HCC patients, in line with the results of the patient data analysis of THE HUMAN PROTEIN ATLAS database.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, MTT Assay, Knockdown, Apoptosis Assay

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 promoted tumorigenesis and progression of HCC closely related to the mTOR and MYC pathways. (A) GSEA analysis between high and low PUS1 expression patients using hallmark gene sets based on TCGA–HCC database. (B–E) GSEA analysis revealed that MYC pathways, DNA repair, and MTORC1 pathways were significantly enriched in patients with high PUS1 expression. (F–H) The intersection analysis of the PUS1–dependent Ψ modification genes and MYC or MTORC1 pathway genes were applied. (I, J) RT–PCR analysis of PUS1 potential downstream targets in HepG2 cell after transfection with control or PUS1 siRNA. (**P<0.01, ***P<0.001).

Article Snippet: Our result indicated that PUS1 expressed significantly higher in cancer tissues than adjacent liver tissues of HCC patients, in line with the results of the patient data analysis of THE HUMAN PROTEIN ATLAS database.

Techniques: Expressing, Modification, Reverse Transcription Polymerase Chain Reaction, Transfection, Control

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: PUS1 promotes cell proliferation depending on the mTOR and MYC pathways. (A, B) RT–PCR analysis of the mRNA expression of MYC, pre-MYC, mTOR and pre-mTOR in HCC cells after transfection with control or PUS1 siRNA. (C, D) WB testing of PUS1, MYC, mTOR, p-mTOR and P-S6 expression in HCC cells after transfection with control or PUS1 siRNA. (E-H) Cell proliferation of SNU449 and HepG2 cells transfected with PUS1 siRNA treatment with or without inhibitors of MYC (10058-F4, 10μM) or mTOR (rapamycin, 200nM) were analyzed by MTT assay. (**P<0.01, ***P<0.001, ****P<0.0001; ns, P>0.05).

Article Snippet: Our result indicated that PUS1 expressed significantly higher in cancer tissues than adjacent liver tissues of HCC patients, in line with the results of the patient data analysis of THE HUMAN PROTEIN ATLAS database.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, MTT Assay

Journal: Frontiers in Oncology

Article Title: Pseudouridine synthase 1 promotes progression of hepatocellular carcinoma via mTOR and MYC signaling pathways

doi: 10.3389/fonc.2025.1576651

Figure Lengend Snippet: A schematic view of PUS1 promoted tumorigenesis and progression of HCC dependent on the mTOR and MYC pathways.PUS1 expression was increased in HCC. However, knocking down PUS1 in HCC cells, the expression of MYC, p-mTOR and mTOR pathway‐related protein P-S6 were decreased. These results suggested that the role of PUS1 promoted tumorigenesis and progression of HCC is dependent on the mTOR and MYC pathways.

Article Snippet: Our result indicated that PUS1 expressed significantly higher in cancer tissues than adjacent liver tissues of HCC patients, in line with the results of the patient data analysis of THE HUMAN PROTEIN ATLAS database.

Techniques: Expressing

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 1. PUS1 expression levels in various tumor types and carcinogenic tissues. (A) PUS1 expression in unpaired normal and tumor samples from TCGA. (B) PUS1 expression in paired normal and tumor samples from TCGA. (C) PUS1 RNA expression in normal and tumor tissues of KIRC (n=613) from TCGA. (D) PUS1 RNA expression in KIRC samples (n=613) from TCGA. (E) ROC analysis for predicting elevated PUS1 expression. PUS1 expression was also assessed in GEO datasets GSE53757 (F, n=72), GSE66272 (G, n=54), and GSE15641 (H, n=92). (I) PUS1 mRNA levels in cancer cell lines from CCLE. (All data were shown as the mean ± SD; significance by Spearman’s rank correlation: *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Expressing, RNA Expression

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 2. Correlation of PUS1 expression with cancer prognosis and clinical characteristics. (A–C) Kaplan–Meier curves show OS, PFI, and DSS in low and high PUS1 expression groups (n=532, TCGA cohort). (D–F) ROC curves evaluate PUS1’s predictive value for OS, PFI, and DSS at 1-, 3-, and 5-year intervals. (G) Correlation analysis links PUS1 expression with clinical factors such as age, sex, tumor grade, stage, and TNM classification (n=532). (H) Heatmap shows PUS1 expression associations with clinical parameters. Univariate (I) and multivariate (J) Cox regression analyses assess PUS1’s prognostic significance regarding tumor stage, grade, age, sex, and PUS1 score. (K) Nomograms predict KIRC OS at 1-, 3-, and 5-year intervals, with calibration curves (L) assessing accuracy. (All data were shown as the mean ± SD. Statistical tests include Wilcoxon rank-sum (A–C), unpaired two-tailed t-test for PUS1-clinical factor comparisons (G), and chi-squared test for heatmap data (H). Cox regression determined p-values for univariate (I) and multivariate (J) analyses, * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Expressing, Two Tailed Test

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 3. Enrichment analysis of the differentially expressed PUS1 genes within the TCGA–KIRC dataset. (A) Volcano mapping indicating differential expression of the PUS1 gene. (B) Comparison of low and high expression groups using GSEA. (C) Differentially expressed PUS1 genes in KIRC were analyzed using KEGG. (D) Differentially expressed PUS1 genes in KIRC were identified using GO pathway analysis.

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Quantitative Proteomics, Comparison, Expressing

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 4. Correlation of PUS1 expression with TME cell infiltration. (A) In ccRCC, variation in immune cell infiltration levels was observed. (B) PUS1 expression and immune cell ratio of tumor-infiltrating cells were displayed using a lollipop plot. (C) Correlation analysis of TME scores with PUS1 expression in ccRCC. (D, E) Relationship between OS in KIRC and StromalScore and ImmuneScore. (F) Scores of immune-related functions between low and high PUS1 expression groups. (All data were shown as the mean ± SD, P-values were calculated using Spearman’s rank correlation; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Expressing

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 5. PUS1 Knockdown and Overexpression Affect Migration in ccRCC Cells. (A-D) The efficiency of PUS1 gene knockdown and overexpression was validated in 769-P (A, C) and A498 (B, D) cells using qRT-PCR and Western blot analysis. (n=3) (E-H) The correlation between PUS1 gene knockdown or overexpression and the regulation of cell migration was analyzed in 769-P (E, G) and A498 (F, H) cells. (n=3) Bar = 50μm. (I-L) The correlation between PUS1 gene knockdown or overexpression and the regulation of scratch wound healing was examined in 769-P (I, K) and A498 (J, L) cells. (n=3) Bar = 100μm. (All data were shown as the mean ± SD, P-values were calculated using an unpaired two-tailed t-test; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Knockdown, Over Expression, Migration, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 6. PUS1 facilitates the migration of ccRCC cells through a mechanism reliant on mRNA pseudouridylation. (A-D) The levels of pseudouridylated mRNA in stable cell lines overexpressing or knocking down PUS1 in 769-P (A, B) and A498 (C, D) cells were detected by dot blotting, with methylene blue (MB) staining serving as a loading control. (n=3) (E) Western blot analysis was employed to confirm the stable overexpression of PUS1WT and PUS1Mut in 769-P cells. (F) Dot blot analysis was conducted to assess the pseudouridylated mRNA levels in 769-P cells overexpressing PUS1WT and PUS1Mut, with MB staining serving as the loading control. (n=3) (G) The correlation between the overexpression of PUS1WT or PUS1Mut and the regulation of cell migration in 769-P cells was analyzed. (n=3) Bar = 50μm. (H) The relationship between the overexpression of PUS1WT or PUS1Mut and the regulation of scratch wound healing in 769-P cells was investigated. (n=3) Bar = 100μm. (All data were shown as the mean ± SD, P-values were calculated using an unpaired two-tailed t-test; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Migration, Stable Transfection, Staining, Control, Western Blot, Over Expression, Dot Blot, Two Tailed Test

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 7. USF1 Directly Modulates PUS1 Transcription by Binding to Specific Promoter Elements in ccRCC Cells. (A-D) We evaluated PUS1 protein abundance in control, USF1 knockdown, and overexpressing 769-P (A, B) and A498 (C, D) cells using Western blotting. (E) USF1 knockdown or PUS1 overexpression aimed to demonstrate the reversal effect of oePUS1 in 769-P cells. (F) We explored how USF1 overexpression or PUS1 knockdown reverses the effects of shPUS1 in 769-P cells. (G, I) We validated the reversal effects of oePUS1 on migration (G) (Bar = 50μm) and scratch healing (I) (Bar = 100μm) in 769-P cells via USF1 knockdown or PUS1 overexpression. (n=3) (H, J) We investigated how USF1 overexpression or PUS1 knockdown reverses migration (H) (Bar = 50μm) and scratch (J) (Bar = 100μm) inhibition induced by shPUS1 in 769-P cells. (n=3) (K) This study identifies URE motifs on the PUS1 promoter and their sequences. (L) JASPAR predictions indicate three putative UREs on the PUS1 promoter that play significant roles in gene regulation. (M) We performed ChIP assays on PUS1 promoter UREs in 769-P cells to analyze binding. (N) We co-transfected pGL3-luciferase constructs with wild-type or mutated URE#3 PUS1 promoter into 769-P cells(n=3), with or without oeUSF1 and siUSF1, to assess activity. Luciferase assays evaluated promoter activity and expression levels under varying treatment conditions. (All data were shown as the mean ± SD, P-values were calculated using an unpaired two-tailed t-test; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Binding Assay, Quantitative Proteomics, Control, Knockdown, Western Blot, Over Expression, Migration, Inhibition, Transfection, Luciferase, Construct, Activity Assay, Expressing, Two Tailed Test

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 8. PUS1 Enhances ccRCC Cells Migration by Stabilizing SMOX mRNA through Pseudouridylation. (A, B) The abundance of SMOX protein in 769-P (A) and A498 (B) cells was evaluated using Western blot analysis in both control groups and those with PUS1 knockdown or overexpression. (C, D) The efficiency of SMOX gene knockdown and overexpression in 769-P (C) and A498 (D) cells was validated by Western blot analysis. (E, F) The correlation between SMOX gene knockdown or overexpression and cell migration regulation was analyzed in 769-P (E) and A498 (F) cells. (n=3) Bar = 50μm. (G, H) The impact of PUS1 gene knockdown or overexpression on scratch wound healing regulation was investigated in 769-P (G) and A498 (H) cells. (n=3) Bar = 100μm. (I) Selected PUS1 and SMOX mRNA in 769-P cells were validated by RIP. (n=3) (J) The changes in relative expression levels over time in different experimental groups after Actinomycin D treatment in 769-P cells. (n=3) (All data were shown as the mean ± SD, P-values were calculated using an unpaired two-tailed t-test; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Migration, Western Blot, Control, Knockdown, Over Expression, Expressing, Two Tailed Test

Journal: Cellular signalling

Article Title: PUS1 facilitates cell migration in clear cell renal cell carcinoma through the promotion of mRNA pseudouridylation and the stabilization of the SMOX gene.

doi: 10.1016/j.cellsig.2025.111700

Figure Lengend Snippet: Fig. 9. The Interaction between SMOX and PUS1 in Cell Migration and Wound Healing in 769-P Cells. (A) Knockdown of SMOX or overexpression of PUS1 was performed to demonstrate that shSMOX can reverse the effects caused by the overexpression of PUS1 in 769-P cells. (B) Conversely, overexpression of SMOX or knockdown of PUS1 was conducted to show that oeSMOX can reverse the biological effects resulting from the knockdown of shPUS1 in 769-P cells. (n=3) (C, E) Additionally, the knockdown of SMOX or overexpression of PUS1 was utilized to illustrate that shSMOX can reverse the enhanced cell migration capacity (C) (Bar = 50μm) and accelerated wound healing (E) (Bar = 100μm) induced by oePUS1 in 769-P cells. (D, F) Furthermore, overexpression of SMOX or knockdown of PUS1 was employed to indicate that oeSMOX can reverse the reduction in cell migration capacity (D) (Bar = 50μm) and the delay in wound healing (F) (Bar = 100μm) caused by shPUS1 in 769-P cells. (n=3) (All data were shown as the mean ± SD, P-values were calculated using an unpaired two-tailed t-test; * p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: The IgG control group was supplemented with 2 μg of IgG, while the experimental group was treated with the PUS1 antibody (sc-390,043, Santa Cruz Biotechnology, USA) and incubated overnight at 4◦C.

Techniques: Migration, Knockdown, Over Expression, Two Tailed Test

Journal: Nature Methods

Article Title: Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

doi: 10.1038/s41592-024-02439-8

Figure Lengend Snippet: a , Integrated view of the PUS-dependent Ψ profiles of HeLa cy-tRNAs and mt-tRNAs. Ψ 55 in HeLa cy-tRNAs is partially dependent on TRUB1, which is labeled by the dashed line. b , Comparison of the modification levels of Ψ 55 in HeLa cy-tRNAs and mt-tRNAs upon TRUB1 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA Ψ 55 , n = 180; mt-tRNA Ψ 55 , n = 6). c , Scatterplot illustrating all TRUB1-dependant Ψ sites across the HeLa transcriptome. d , Sequence motifs of TRUB1-dependent Ψ sites in mt-tRNAs and poly-A-tailed RNA. e , Comparison of the modification levels of Ψ sites at selected positions of HeLa cy-tRNAs upon PUS7 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA: Ψ 13 , n = 43; Ψ 20B , n = 12; Ψ 35 , n = 7; Ψ 36 , n = 4; Ψ 50 , n = 3). f , Comparison of the modification levels of Ψ 50 in mt-tRNA Met between wild-type (WT) and PUS7-KO cell lines. g , Sequence motifs of PUS7-dependent Ψ sites in tRNAs and poly-A-tailed RNA. h , Comparison of the modification levels of Ψ sites at selected positions of HeLa cy-tRNAs and mt-tRNAs upon PUS1 depletion. Box plots visualize all Ψ sites at each position; boxes represent the 25th to 75th percentiles with a line at the median; whiskers correspond to 1.5 times the interquartile range (cy-tRNA: Ψ 27/28 , n = 130; mt-tRNA: Ψ 27/28 , n = 21; Ψ 66/67/68 , n = 4). i , Scatterplot illustrating all PUS1-dependant Ψ sites in HeLa mt-mRNAs. j , Sequence motifs of PUS1-dependent Ψ sites in cy-tRNAs and mt-tRNAs.

Article Snippet: Finally, clones were expanded and picked for western blot validation with TRUB1 antibody (Proteintech, 12520-1-AP; 1:1,000 dilution), PUS7 antibody (Abcam, ab226257; 1:10,000 dilution) and PUS1 antibody (Proteintech, 11512-1-AP; 1:1,000 dilution).

Techniques: Labeling, Comparison, Modification, Sequencing