Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: High PD-L1 expression is associated with poorer survival outcomes (A and B) Kaplan-Meier curve comparing progression-free survival (PFS) (A) and overall survival (OS) (B) with first-line osimertinib in patients with EGFR -mutated advanced NSCLC having high (tumor proportion score [TPS] ≥50%) or low (TPS <50%) PD-L1 TPS. (C and D) Outcomes of patients stratified by PD-L1 expression: PD-L1 negative (A, TPS <1%), PD-L1 low (B, TPS 1%–49%), and PD-L1 high (C, TPS ≥50%). Tick marks represent censored patients. Risk table below indicates the number of patient at risk at each time point. Hazard ratios and p values were calculated using the log rank test. (E and F) Univariate and multivariate Cox regression analyses identifying risk factors for PFS (E) and OS (F) with first-line osimertinib of patients with EGFR -mutated advanced NSCLC.

Article Snippet: Primary antibodies against PD-L1 (#13684), STAT3 (#9139), p-STAT3 (#9145), EGFR (#4267), p-EGFR (#3777), and β-Tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: Upregulation of IFNG and IL-6/JAK/STAT3 signaling pathways in patients with high PD-L1 expression (A) Volcano plot showing differentially expressed genes between high and low PD-L1 expression groups. (B) Reactome pathway enrichment analysis of the differentially expressed genes in tumors with high PD-L1 expression compared to those with low expression. (C) Gene set enrichment analysis highlighting key pathways enriched in tumors with high PD-L1 expression compared to those with low expression. (D) Deconvolution analysis of immune cell infiltration between PD-L1 expression groups. Group 1: PD-L1 TPS <50%; group 2: PD-L1 TPS ≥50% (data are presented as mean ± SD; ∗ p < 0.05; ns, not statistically significant).

Article Snippet: Primary antibodies against PD-L1 (#13684), STAT3 (#9139), p-STAT3 (#9145), EGFR (#4267), p-EGFR (#3777), and β-Tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Protein-Protein interactions, Expressing

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: Increased proportion of CD56 bright NK cells in patients with high PD-L1 expression (A and B) Flow cytometric analysis of CD56 + and CD16 + cells within CD3 − populations in patients with high PD-L1 expression (A) and low PD-L1 expression (B). (C) Bar graphs showing the ratio of CD56 dim NK cell (CD56 + CD16 + ) subsets to CD56 bright NK cell (CD56 + CD16 − ) subsets across all patients (data are presented as mean ± SD; ∗ p < 0.05).

Article Snippet: Primary antibodies against PD-L1 (#13684), STAT3 (#9139), p-STAT3 (#9145), EGFR (#4267), p-EGFR (#3777), and β-Tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: STAT3 expression is elevated in tumor tissues from patients with high PD-L1 expression (A) Representative immunohistochemical images showing PD-L1 and STAT3 expression in high vs. low PD-L1 tumors. Scale bars: 100 and 25 μm for full and zoom images. (B) Boxplots comparing the percentage of STAT3-positive cells in high vs. low PD-L1 expression groups (data are presented as mean ± SD; ∗∗∗ p < 0.001).

Article Snippet: Primary antibodies against PD-L1 (#13684), STAT3 (#9139), p-STAT3 (#9145), EGFR (#4267), p-EGFR (#3777), and β-Tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Immunohistochemical staining

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: IFN-γ induces PD-L1 expression in EGFR -mutant NSCLC cell lines via STAT3 activation (A) Quantitative real-time PCR analysis of IFNG and PD-L1 gene expression in various lung cancer cell lines. (B–D) Western blot analysis demonstrating PD-L1 expression across cell lines (B), STAT3 phosphorylation following treatment with STAT3 inhibitor C188-9 in PC-9 (left) and HCC827 (right) cells (C), and PD-L1 expression and STAT3 phosphorylation after IFN-γ treatment for 30 min or 18 h in PC-9 (left) and HCC827 (right) cells. Ctrl denotes vehicle control. (E) Flow cytometry analysis of cell surface PD-L1 expression following IFN-γ stimulation (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (F and G) Western blot analysis of PD-L1 expression in PC-9 (F) and HCC827 (G) cells after STAT3 knockdown, STAT3 overexpression, or C188-9 treatment. (H) Flow cytometry analysis of cell surface PD-L1 expression following C188-9 treatment (∗∗∗ p < 0.001). (I and J) Immunofluorescence analysis of STAT3 localization in HCC827 (I) and PC-9 (J) cells that overexpress STAT3 (STAT3-OE) or treated with IFN-γ (IFNG). NC indicates negative control. Nuclei were stained with DAPI. Red boxes in the merge images highlight areas shown at higher magnification in the fourth column. Scale bars: 10 and 2 μm for full and zoom images (data are presented as mean ± SD).

Article Snippet: Primary antibodies against PD-L1 (#13684), STAT3 (#9139), p-STAT3 (#9145), EGFR (#4267), p-EGFR (#3777), and β-Tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Mutagenesis, Activation Assay, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Phospho-proteomics, Control, Flow Cytometry, Knockdown, Over Expression, Immunofluorescence, Negative Control, Staining

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: Schematic model of PD-L1 regulation in EGFR -mutated NSCLC cells EGFR -mutated NSCLC tumors with low PD-L1 expression harbor a higher proportion of CD56 dim natural killer (NK) cells, whereas tumors with high PD-L1 expression exhibit increased CD56 bright NK cells, which secrete IFN-γ, leading to STAT3 activation and upregulation of PD-L1 expression.

Article Snippet: Primary antibodies against PD-L1 (#13684), STAT3 (#9139), p-STAT3 (#9145), EGFR (#4267), p-EGFR (#3777), and β-Tubulin (#2146) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Activation Assay

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: E3 ubiquitin ligase RNF125 is downregulated in LC. (A) GSE datasets showing the expression profile of LUAD and non-cancer samples were downloaded and analyzed. DEGs in these three datasets were overlapped with the gene list of human E3 ubiquitin ligases downloaded from the UbiNet 2.0 database. A total of 10 overlapping genes were identified and RNF125 was the only novel gene in LC. (B) The heatmap shows the expression of RNF125 in the three datasets. (C) Representative images of IHC staining for RNF125 in 21 pairs of LUAD tissues and adjacent non-cancerous tissues are shown. Scale bar, 200 or 50 µm. The statistical analysis result of the IHC score is shown in the right panel. **P<0.01. (D) The representative image of immunohistochemical staining of RNF125 in LUAD tissues was downloaded from HPA. RNF125, ring finger protein 125; LUAD, lung adenocarcinoma; LC, lung cancer; DEG, differentially expressed gene; HPA, Human Protein Atlas.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Ubiquitin Proteomics, Expressing, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: Clinical significance of RNF125 expression in human LC. (A) Pan-cancer analysis of RNF125 expression was performed using the TNMplot platform. (B) RNF125 expression analysis based on the metastatic status of LC was performed using the TNMplot platform. (C) The association between RNF125 expression and LUAD tumor stage was performed using the TISIDB web portal. Spearman correlation analysis was performed using data from all tumor stages. (D) Overall survival analysis based on RNF125 expression levels was performed using the Gene Expression Omnibus datasets via the PROGgeneV2 database. RNF125, ring finger protein 125; LUAD, lung adenocarcinoma; LC, lung cancer.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Expressing, Gene Expression

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: RNF125 inhibits the proliferation and colony formation of LUAD cells. (A) RT-qPCR shows RNF125 expression in six LC cell lines (NCI-H1975, NCI-H2228, NCI-H1395, HCC-827, CALU-3 and NCI-H1437). RT-qPCR and western blotting demonstrate plasmid-mediated RNF125 (B) knockdown or (C) overexpression in NCI-H1395 and HCC-827 cells. MTT assays show the effect of RNF125 (D) knockdown or (E) overexpression on the proliferation of NCI-H1395 and HCC-827 cells. Colony formation analysis shows the effect of RNF125 knockdown or overexpression on colony formation of (F) NCI-H1395 and (G) HCC-827 cells. Data are presented as mean ± SD. **P<0.01 vs. shNC or vector. LUAD, lung adenocarcinoma; RT-qPCR, reverse transcription-quantitative PCR; RNF125, ring finger protein 125; sh, short hairpin RNA; NC, negative control; OE, overexpression; OD, optical density.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Knockdown, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Negative Control

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: RNF125 inhibits migration and invasion of NCI-H1395 and HCC-827 cells. Transwell assays in the absence of Matrigel show the effect of RNF125 on the migration of (A) NCI-H1395 and (B) HCC-827 cells. Representative images (top) and the mean number of migrated cells (bottom) are shown. Transwell assays in the presence of Matrigel show the effect of RNF125 on the invasion of (C) NCI-H1395 and (D) HCC-827 cells. Representative images (top) and the mean number of invaded cells (bottom) are shown. Scale bar, 100 µm. Data are presented as mean ± SD. **P<0.01 vs. shNC or vector. RNF125, ring finger protein 125; sh, short hairpin RNA; NC, negative control; OE, overexpression.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Migration, Plasmid Preparation, shRNA, Negative Control, Over Expression

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: RNF125 increases the sensitivity of NCI-H1395 and HCC-827 cells to cisplatin. MTT assays show the effect of RNF125 on the sensitivity of (A) NCI-H1395 and (B) HCC-827 cells to cisplatin at the indicated time points. Quantitative analysis demonstrates the effect of RNF125 (C) knockdown or (D) overexpression on caspase-3 activity in NCI-H1395 and HCC-827 cells in the presence of cisplatin. The concentration of p NA indicates the activity of caspase-3. Western blotting determines the levels of cleaved PARP in (E) RNF125-silenced or (F) RNF125-overexpressing NCI-H1395 and HCC-827 cells in the presence of cisplatin. Data are presented as mean ± SD. *P<0.05, **P<0.01 vs. shNC or vector. RNF125, ring finger protein 125; PARP, poly(ADP-ribose) polymerase; p NA, p -nitroaniline; sh, short hairpin RNA; NC, negative control; OE, overexpression.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Knockdown, Over Expression, Activity Assay, Concentration Assay, Western Blot, Plasmid Preparation, shRNA, Negative Control

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: Knockdown of RNF125 enhances immune evasion in LUAD. (A) The PPI network of RNF125 interactors was constructed using the GeneMANIA database. (B) Gene Ontology Biological Process and Reactome pathway enrichment analysis of RNF125 interactors was performed using the DAVID database. (C) Co-IP assays show the interactions between RNF125 and PD-L1 in NCI-H1395 and HCC-827 cells. (D) Western blotting determines that knockdown of RNF125 reduces PD-L1 protein expression levels in NCI-H1395 and HCC-827 cells. (E) Correlation analysis between RNF125 expression and immune cell infiltration in LUAD was performed using the TIMER2.0 database. (F) Co-culture of T cells and RNF125-silenced cancer cells decreased IL-2 secretion from T cells. **P<0.01 vs. shNC. (G) Co-culture of NK cells and RNF125-silenced cancer cells attenuated NK cell-mediated lysis of NCI-H1395 and HCC-827 cells. **P<0.01 vs. sh1-RNF125; †† P<0.01 vs. sh2-RNF125. Data are presented as mean ± SD. PPI, protein-protein interaction; RNF125, ring finger protein 125; PD-L1, programmed cell death 1 ligand 1; LUAD, lung adenocarcinoma; sh, short hairpin RNA; NC, negative control; NK, natural killer; TPM, transcripts per million.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Knockdown, Construct, Co-Immunoprecipitation Assay, Western Blot, Expressing, Co-Culture Assay, Lysis, shRNA, Negative Control

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: MBNL1 is an upstream modulator of RNF125 in LUAD. (A) Potential RNF125-binding RBPs were predicted using two RBP databases (RBPDB and RBPmap) and overlapped with RBPs involved in lung cancer progression. (B) Correlation analysis between RNF125 and the indicated RBPs in LUAD was performed using the TIMER2.0 portal. RT-qPCR and western blotting show MBNL1 and RNF125 expression in NCI-H1395 cells 48 h after transfection of (C) MBNL1-specific siRNAs or (D) plasmids containing MBNL1 coding sequences (MBNL1 OE). **P<0.01 vs. siNC or empty vector. (E) RIP-PCR and agarose gel electrophoresis showed that RNF125 transcripts were detected in the MBNL1 antibody RIP products from NCI-H1395 cells. After treatment with actinomycin D, RT-qPCR analysis showed the % of remaining RNF125 mRNA relative to 0 h at the indicated time points in (F) MBNL1-silenced (**P<0.01 vs. si1-MBNL1; † P<0.05, †† P<0.01 vs. si2-MBNL1) or (G) MBNL1-overexpressing NCI-H1395 cells (**P<0.01 vs. empty vector). (H) MTT assays determine the viability of NCI-H1395 cells 48 h after co-transfection of MBNL1 OE and shRNF125 plasmids. (I) Representative images (right) of Transwell assays in the presence of Matrigel and the mean number of invaded cells (left) are shown. Scale bar, 100 µm. **P<0.01. Data are presented as mean ± SD. MBNL1, muscleblind-like 1; RNF125, ring finger protein 125; LUAD, lung adenocarcinoma; RBP, receptor binding protein; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; OE, overexpression; NC, negative control; sh, short hairpin RNA; TPM, transcripts per million.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Cotransfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Over Expression, Negative Control, shRNA

Journal: Oncology Reports

Article Title: RNA‑binding protein MBNL1 regulates tumor growth, chemosensitivity and antitumor immunity in lung adenocarcinoma by controlling the expression of tumor suppressor RNF125

doi: 10.3892/or.2025.8907

Figure Lengend Snippet: Schematic diagram of the function and molecular regulatory mechanism of RNF125 in LC. RNF125 is downregulated in lung cancer, and its low expression is associated with advanced-stage disease. RNF125 inhibits the LUAD cell growth and invasiveness and enhances the chemosensitivity of LUAD cells to cisplatin. RNF125 acts as an E3 ubiquitin ligase of PD-L1. Knockdown of RNF125 suppresses PD-L1 degradation, thereby impairing Tcell activation and antitumor cytokine secretion. Mechanistically, the RBP MBNL1 serves as an upstream regulator of RNF125 by stabilizing RNF125 mRNA. The figure contains elements from Servier Medical Art ( https://smart.servier.com/ ) under a Creative Commons 3.0 license. MBNL1, muscleblind-like 1; RNF125, ring finger protein 125; PD-L1, programmed cell death 1 ligand 1; LUAD, lung adenocarcinoma.

Article Snippet: The membranes were blocked with a Western blocking buffer (cat. no. SW3010; Beijing Solarbio Science & Technology Co., Ltd) at room temperature for 1 h, followed by incubation with primary antibodies against RNF125 (cat. no. DF4024; 1:1,000; Affinity Biosciences), cleaved poly ADP-ribose polymerase (PARP; cat. no. AF7023; 1:1,000; Affinity Biosciences), PD-L1 (cat. no. BF8035; 1:1,000; Affinity Biosciences), MBNL1 (cat. no. A8054; 1:1,000; ABclonal Biotech, Co., Ltd.) and β-actin (cat. no. 66009-1-Ig; 1:10,000; Proteintech Group, Inc.) at 4°C overnight.

Techniques: Expressing, Ubiquitin Proteomics, Knockdown, Activation Assay