Journal: Molecular medicine (Cambridge, Mass.)

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma.

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: Fig. 2 NUAK1 inhibits CD8+ T cell infiltration and activity in HCC. A, B Representative flow cytometry plots showing CD3+, CD8+ and CD8+GZMB+ T cells from Vector, NUAK1-OE, Sh-NC, sh-NUAK1-1#, sh-NUAK1-2# tumors (A) and quantification (B). *p < 0.05, **p < 0.01 compared with vector group, two tailed unpaired t test per group, n = 3; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with sh-NC group; one-way ANOVA, n = 4. C, D Immunofluorescence staining and quantitative statistics of NUAK1 (pink), CD8α (green) and GZMB (red) of tumoral NUAK1 and CD8+ from tumor-grafted mice constructed Vector, NUAK1-OE, sh-NC, sh-NUAK1-1#, sh-NUAK1-2# H22 cells. Scale bars, 50 μm. *p < 0.05, **p < 0.01 compared with vector group, two tailed unpaired t test per group; #p < 0.05, ##p < 0.01 compared with sh-NC group; one-way ANOVA, n = 3

Article Snippet: Rabbit anti-NUAK1 antibody (4458s, 1:1000 for Western blot), rabbit anti-p-GSK3β antibody (Ser9) (5558T, 1:500 for Western blot; 1:200 for immunohistochemistry), rabbit anti-Lamin B1 (13435 S, 1:500 for western blot) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Flow Cytometry, Plasmid Preparation, Two Tailed Test, Immunofluorescence, Staining, Construct

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma.

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: Fig. 3 NUAK1-mediated tumor immune escape is associated with PD-L1 expression A The expression levels of CD274 mRNA were evaluated in HCC with high (n = 364) and low (n = 364) NUAK1 mRNA expression from the TCGA database. The X axis represents the group with high or low expression of NUAK1, and the Y axis represents the expression level of CD274 mRNA using TPM (Transcripts Per Kilobase of exon model per Million mapped reads). B Correlation analysis of NUAK1 mRNA and of PD-L1 mRNA level in HCC (n = 370) based on the RNA-seq datasets derived from the TCGA database. The X axis represents the expression level of NUAK1 mRNA, and the Y-axis represents the expression level of CD274 mRNA. C Immunohistochemical staining of NUAK1 and PD-L1 in 20 human HCC specimens. Left: Representative images of two tumors. Right: Pearman correlation test between tumoral NUAK1 and PD-L1 IHC scores, note that the scores of some samples overlap. D Representative images of livers from normal and DEN-treated rats. E, F The mRNA (E) and protein (F) expression of NUAK1 and PD-L1 in the livers of normal rats and DEN-tread rats, n = 4. **p < 0.01, ***p < 0.001, two tailed unpaired t test. G IHC staining of tumoral NUAK1 and PD-L1 from tumor-grafted mice constructed with or without NUAK1-OE H22 cells, n = 8. ***p < 0.001, two tailed unpaired t test. H IHC staining of tumoral NUAK1 and PD-L1 from tumor-grafted mice constructed with or without NUAK1-knockdown H22 cells, n = 8. ***p < 0.001, one-way ANOVA

Article Snippet: Rabbit anti-NUAK1 antibody (4458s, 1:1000 for Western blot), rabbit anti-p-GSK3β antibody (Ser9) (5558T, 1:500 for Western blot; 1:200 for immunohistochemistry), rabbit anti-Lamin B1 (13435 S, 1:500 for western blot) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, RNA Sequencing, Derivative Assay, Immunohistochemical staining, Staining, Two Tailed Test, Immunohistochemistry, Construct, Knockdown

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma.

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: Fig. 4 NUAK1 promotes PD-L1 expression in HCC cells. A, B The effects of NUAK1 overexpression on the levels of CD274 mRNA (A) and PD-L1 protein (B) in HCC cells, n = 5. **p < 0.01, ***p < 0.001, one-way ANOVA. C, D Effects of NUAK1 knockdown on CD274 mRNA (C) and PD-L1 protein (D) expression in Huh-7 and HepG2 cells, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with sh-NC group; one-way ANOVA

Article Snippet: Rabbit anti-NUAK1 antibody (4458s, 1:1000 for Western blot), rabbit anti-p-GSK3β antibody (Ser9) (5558T, 1:500 for Western blot; 1:200 for immunohistochemistry), rabbit anti-Lamin B1 (13435 S, 1:500 for western blot) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Over Expression, Knockdown, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma.

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: Fig. 7 NUAK1 promotes PD-L1 expression in a GSK3β/β-catenin dependent manner in HCC. A Effects of GSK3β inhibitors SB216763 and LiCl on β-catenin total protein expression in HCC cells. n = 5, ***p < 0.001, one-way ANOVA. B, C Effects of β-catenin knockdown on NUAK1 overexpression-induced CD274 mRNA (B) and PD-L1 protein (C) expression in HCC cells. n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. D, E Effects of β-catenin overexpression on NUAK1 knockdown-induced CD274 mRNA (D) and PD-L1 protein (E) expression in Huh-7 and HepG2 cells. n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA

Article Snippet: Rabbit anti-NUAK1 antibody (4458s, 1:1000 for Western blot), rabbit anti-p-GSK3β antibody (Ser9) (5558T, 1:500 for Western blot; 1:200 for immunohistochemistry), rabbit anti-Lamin B1 (13435 S, 1:500 for western blot) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Knockdown, Over Expression

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma.

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: Fig. 8 Schematic representation of NUAK1-mediated PD-L1 expression in HCC. NUAK1 enhances the transcriptional expression of PD-L1 by activating the GSK3β/β-catenin signaling pathway, thereby suppressing cytotoxic T cell activity within the tumor microenvironment and facilitating immune evasion in hepatocellular carcinoma

Article Snippet: Rabbit anti-NUAK1 antibody (4458s, 1:1000 for Western blot), rabbit anti-p-GSK3β antibody (Ser9) (5558T, 1:500 for Western blot; 1:200 for immunohistochemistry), rabbit anti-Lamin B1 (13435 S, 1:500 for western blot) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Activity Assay

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: NUAK1 promotes tumor immune escape by inhibiting CD8 + T cell infiltration in HCC. A The expression of NUAK1 in normal ( n = 212) and tumor ( n = 222) tissues in HCC was evaluated based on the GEO database. The X-axis represents normal and HCC (Tumor), and the Y-axis represents the expression level of NUAK1 mRNA. B Kaplan–Meier overall survival curves of HCC ( n = 370, cutoff = 50%) patients with of high and low NUAK1 mRNA expression obtained from the GSE14520. The X-axis represents survival time (months), and the Y-axis represents the patient’s probability of survival. C Correlation analysis of NUAK1 mRNA level and the abundance of CD8 + T cells in HCC ( n = 370) based on the RNA-seq datasets derived from the TCGA database. The X axis represents the expression level of NUAK1 mRNA, and the Y axis represents the abundance of CD8 + T cells. D Immunohistochemical staining of NUAK1 and CD8 + in 20 human HCC specimens. Left: Representative images of two tumors. Scale bar, 50 μm. Right: Pearman correlation test between tumoral NUAK1 and CD8 + IHC scores. Note that the scores of some samples overlap. E Schematic diagram of experimental protocol for survival. F Kaplan-Meier survival curves of vector and NUAK1-overexpression (NUAK1-OE) and sh-vector (sh-NC) and two sh-NUAK1 (1 # and 2 # )-transfected H22 xenografts in BALB/c mice, n = 8 mice per group. G Schematic diagram of experimental protocol for tumor analysis. H Representative images of tumors, quantification of tumor volume and tumor weight collected from vector, NUAK1-OE and sh-NC, two sh-NUAK1 groups, n = 8. ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA. I IHC staining of tumoral NUAK1 and CD8 + from tumor-grafted mice constructed vector or NUAK1-OE H22 cells, n = 8. ** p < 0.01, two tailed unpaired t test per group. J IHC staining of tumoral NUAK1 and CD8 + from tumor-grafted mice constructed sh-NC and two sh-NUAK1 (1 # and 2 # ) H22 cells, n = 8. *** p < 0.001, one-way ANOVA

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Expressing, RNA Sequencing, Derivative Assay, Immunohistochemical staining, Staining, Plasmid Preparation, Over Expression, Transfection, Immunohistochemistry, Construct, Two Tailed Test

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: NUAK1 inhibits CD8 + T cell infiltration and activity in HCC. A , B Representative flow cytometry plots showing CD3 + , CD8 + and CD8 + GZMB + T cells from Vector, NUAK1-OE, Sh-NC, sh-NUAK1-1 # , sh-NUAK1-2 # tumors ( A ) and quantification ( B ). * p < 0.05, ** p < 0.01 compared with vector group, two tailed unpaired t test per group, n = 3; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with sh-NC group; one-way ANOVA, n = 4. C , D Immunofluorescence staining and quantitative statistics of NUAK1 (pink), CD8α (green) and GZMB (red) of tumoral NUAK1 and CD8 + from tumor-grafted mice constructed Vector, NUAK1-OE, sh-NC, sh-NUAK1-1 # , sh-NUAK1-2 # H22 cells. Scale bars, 50 μm. * p < 0.05, ** p < 0.01 compared with vector group, two tailed unpaired t test per group; # p < 0.05, ## p < 0.01 compared with sh-NC group; one-way ANOVA, n = 3

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Activity Assay, Flow Cytometry, Plasmid Preparation, Two Tailed Test, Immunofluorescence, Staining, Construct

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: NUAK1-mediated tumor immune escape is associated with PD-L1 expression A The expression levels of CD274 mRNA were evaluated in HCC with high ( n = 364) and low ( n = 364) NUAK1 mRNA expression from the TCGA database. The X axis represents the group with high or low expression of NUAK1, and the Y axis represents the expression level of CD274 mRNA using TPM (Transcripts Per Kilobase of exon model per Million mapped reads). B Correlation analysis of NUAK1 mRNA and of PD-L1 mRNA level in HCC ( n = 370) based on the RNA-seq datasets derived from the TCGA database. The X axis represents the expression level of NUAK1 mRNA, and the Y-axis represents the expression level of CD274 mRNA. C Immunohistochemical staining of NUAK1 and PD-L1 in 20 human HCC specimens. Left: Representative images of two tumors. Right: Pearman correlation test between tumoral NUAK1 and PD-L1 IHC scores, note that the scores of some samples overlap. D Representative images of livers from normal and DEN-treated rats. E , F The mRNA ( E ) and protein ( F ) expression of NUAK1 and PD-L1 in the livers of normal rats and DEN-tread rats, n = 4. ** p < 0.01, *** p < 0.001, two tailed unpaired t test. G IHC staining of tumoral NUAK1 and PD-L1 from tumor-grafted mice constructed with or without NUAK1-OE H22 cells, n = 8. *** p < 0.001, two tailed unpaired t test. H IHC staining of tumoral NUAK1 and PD-L1 from tumor-grafted mice constructed with or without NUAK1-knockdown H22 cells, n = 8. *** p < 0.001, one-way ANOVA

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Expressing, RNA Sequencing, Derivative Assay, Immunohistochemical staining, Staining, Two Tailed Test, Immunohistochemistry, Construct, Knockdown

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: NUAK1 promotes PD-L1 expression in HCC cells. A , B The effects of NUAK1 overexpression on the levels of CD274 mRNA (A) and PD-L1 protein (B) in HCC cells, n = 5. ** p < 0.01, *** p < 0.001, one-way ANOVA. C , D Effects of NUAK1 knockdown on CD274 mRNA (C) and PD-L1 protein (D) expression in Huh-7 and HepG2 cells, n = 5. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with sh-NC group; one-way ANOVA

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Expressing, Over Expression, Knockdown, Control

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: NUAK1 induces phosphorylation of GSK3β at the Ser9 residue, and inhibition of GSK3β promotes PD-L1 expression in HCC. A Effects of NUAK1 overexpression on the phosphorylation level of GSK3β at Ser 9 in Huh-7 and HepG2 cells, n = 5. ** p < 0.01, *** p < 0.001, one-way ANOVA. B Effects of NUAK1 knockdown on the phosphorylation level of GSK3β at Ser 9 in Huh-7 and HepG2 cells, n = 5. ** p < 0.01, *** p < 0.001 compared with control group; ## p < 0.01, ### p < 0.001 compared with sh-NC group; one-way ANOVA. C IHC staining of tumoral NUAK1 and p-GSK3β from tumor-grafted mice constructed with or without NUAK1-overexpressing H22 cells. Left: Representative images. Scale bar, 50 μm. Right: quantification analysis. n = 8, *** p < 0.001, two tailed unpaired t test. D IHC staining of tumoral NUAK1 and p-GSK3β from tumor-grafted mice constructed with or without NUAK1-knockdown H22 cells. n = 8, *** p < 0.001, one-way ANOVA. E Immunohistochemical staining of NUAK1 and p-GSK3β in 20 human HCC specimens. Left: Representative photos of two tumors. Right: Pearman correlation test between tumoral NUAK1 and p-GSK3β. Note that the scores of some samples overlap. F The protein expression of p-GSK3β in the livers of normal rats and DEN-tread rats ( n = 4). G , H Effects of GSK3β inhibitors SB216763 and lithium chloride (LiCl) on CD274 mRNA ( G ) and PD-L1 protein ( H ) expression in Huh-7 and HepG2 cells. n = 5, *** p < 0.001, one way ANOVA

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Phospho-proteomics, Residue, Inhibition, Expressing, Over Expression, Knockdown, Control, Immunohistochemistry, Construct, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: NUAK1 promotes β-catenin expression and nuclear accumulation in HCC A Effects of NUAK1 overexpression on the expression of β-catenin total protein in HCC cells. n = 5, ** p < 0.01, *** p < 0.001, one-way ANOVA. B Effects of NUAK1 knockdown on the β-catenin total protein expression in HCC cells. n = 5, *** p < 0.001 compared with Ctrl group; ## p < 0.01, ### p < 0.001 compared with sh-NC group; one-way ANOVA. C IHC staining of tumoral NUAK1 and β-catenin from tumor-grafted mice constructed with or without NUAK1-overexpressing H22 cells. n = 8, * p < 0.05, two tailed unpaired t test. D IHC staining of tumoral NUAK1 and β-catenin from tumor-grafted mice constructed with or without NUAK1-knockdown H22 cells. Data were expressed by mean ± SEM ( n = 8). * p < 0.05, two-tailed unpaired t test. E Pearman correlation test between tumoral NUAK1 and p-GSK3β in 20 human HCC specimens. Note that the scores of some samples overlap. F The protein expression of β-catenin in the livers of normal rats and DEN-tread rats ( n = 4). G Effects of NUAK1 overexpression on the β-catenin nuclear protein expression in Huh-7 and HepG2 cells. Lamin B1 was used as internal control. n = 5, *** p < 0.001, two-tailed unpaired t test. H Effects of NUAK1 knockdown on the β-catenin nuclear protein expression in Huh-7 and HepG2 cells. Lamin B1 was used as internal control. Data were expressed by mean ± SEM ( n = 5). *** p < 0.001; one-way ANOVA. I Immunofluorescence staining of β-catenin in Huh-7 cells stably transfected with vector, NUAK1-overexpression (NUAK1-OE), sh-Vector (sh-NC), NUAK1-knockdown # 1 (sh-NUAK1 # 1) and NUAK1-knockdown # 2 (sh-NUAK1 # 2) groups

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Expressing, Over Expression, Knockdown, Immunohistochemistry, Construct, Two Tailed Test, Control, Immunofluorescence, Staining, Stable Transfection, Transfection, Plasmid Preparation

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: NUAK1 promotes PD-L1 expression in a GSK3β/β-catenin dependent manner in HCC. A Effects of GSK3β inhibitors SB216763 and LiCl on β-catenin total protein expression in HCC cells. n = 5, *** p < 0.001, one-way ANOVA. B , C Effects of β-catenin knockdown on NUAK1 overexpression-induced CD274 mRNA ( B ) and PD-L1 protein ( C ) expression in HCC cells. n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA. D , E Effects of β-catenin overexpression on NUAK1 knockdown-induced CD274 mRNA ( D ) and PD-L1 protein ( E ) expression in Huh-7 and HepG2 cells. n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Expressing, Knockdown, Over Expression

Journal: Molecular Medicine

Article Title: NUAK1 acts as a novel regulator of PD-L1 via activating GSK-3β/β-catenin pathway in hepatocellular carcinoma

doi: 10.1186/s10020-025-01088-7

Figure Lengend Snippet: Schematic representation of NUAK1-mediated PD-L1 expression in HCC. NUAK1 enhances the transcriptional expression of PD-L1 by activating the GSK3β/β-catenin signaling pathway, thereby suppressing cytotoxic T cell activity within the tumor microenvironment and facilitating immune evasion in hepatocellular carcinoma

Article Snippet: Rabbit anti-β-catenin antibody (66379-1-Ig, 1:1000 for western blot) and rabbit anti-NUAK1 antibody (22723-1-AP, 1:200 for immunofluorescence or immunohistochemistry) were purchased from Protein Tech Group (Chicago, IL, USA).

Techniques: Expressing, Activity Assay