Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Involvement of BAG5 interactome in regulation of actin cytoskeleton, focal adhesion and RNA metabolism. (A) Coomassie blue staining of SDS-PAGE gel analyzed BAG5 immunocomplex from a panel of immortalized lung epithelial and NSCLC cell lines. (B) SDS-PAGE gel from lung epithelial cell line BEAS-2B and NSCLC cell line A549 were identified by mass spectrometry. Ten members of Hsp70/HSC70 family were identified to interact with BAG5 in A549 cell. (C) Venn diagram showed the different interacting proteins between BEAS-2B and A549 cell. (D, E) GO (D) and KEGG pathways (E) enrichment analysis of 130 unique BAG5-interacting proteins in A549 cell. (F, G) Cytoscape ClueGO visualizing the enriched pathways of TOP 100 (F) and TOP 5 (G) hub proteins unique in A549 cell. (H) Co-IP was performed to confirm the potential interaction of BAG5 with the top 5 of BAG5-interacting proteins influencing protein translation. (I) BAG5-interacting proteins were analyzed by western blot analysis.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Staining, SDS Page, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: BAG5 was highly expressed in tumor epithelial cells and correlated with NSCLC metastasis. (A, B) Expression levels of BAG5 in tumor versus normal tissues were analyzed using a combined TCGA and GTEx dataset. Comparisons include lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), as well as subgroups with lymph node (N) or distant metastasis (M). *** p < 0.001; The statistical difference of two groups was compared through the Wilcox test, significance difference of three groups was tested with Kruskal-Wallis test. (C) Kaplan-Meier survival curve was constructed for NSCLC patients from TCGA based on BAG5 expression levels (BAG5-High vs. BAG5-Low). Significance ( p ) was evaluated by Log-rank test. HR: hazard ratio. (D) Forest plot of hazard ratios (HR) for the association of BAG5 expression with overall survival (OS) across 27 NSCLC datasets from the OSluca online database. Detailed statistics, including HR, 95% CI, and p-values for each dataset, are provided in Supplemental Table OSluca. (E–G) The public single-cell RNA-seq dataset ( GSE119911 ) was reanalyzed. BAG5 expression levels across major cell types in NSCLC versus normal lung tissues are shown. In panel H, average expression levels of BAG5 are presented as a heatmap across annotated cell types. (H) Western blotting analysis was performed to examine BAG5 expression in a panel of normal and NSCLC cell lines, with GAPDH used as the loading control. Representative immunoblots are shown (upper), and densitometric quantification of relative BAG5 protein levels normalized to GAPDH is presented (lower). Statistical significance was evaluated using one-way ANOVA, ****p < 0.0001. (I) BAG5 protein level was investigated using Western blot in paired fresh NSCLC tumor (T) and paratumor (P) normal tissues, and representative images were provided. β-actin was used as a loading control. LUAD (J) Scatter plots showing relative expression of BAG5 in paired NSCLC tumor (T) and paratumor (P) normal tissues. ** p < 0.01.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Expressing, Construct, RNA Sequencing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: BAG5 knockout inhibited proliferation and invasion of NSCLC in vitro and in vivo . (A, B) Control and BAG5 knockout NSCLC cells were injected subcutaneously on the right flanks of nude mice (n = 5-6 mice per group). Tumors of each group were removed and photographed after sacrifice of animals. Tumor weight and volumes (mean ± SD) were analyzed. * p < 0.05. (C) Xenografts were sectioned and stained with BAG5 and Ki67. Scale bars, 25μm. (D, E) To evaluate spontaneous metastasis, BAG5 knockout and control A549 cells were i.v. injected to nude mice (n = 8 mice per group). Representative images ( D left), quantified values ( D right) and H&E staining (E) of metastatic nodules in lung. ** p < 0.01. Scale bars, 2mm and 250μm. (F) H&E and immunohistochemical staining images of NSCLC cancer tissues, PDX and derived PDO. Scale bars, 50 µm. (G) To evaluate heterogeneous expression of BGA5 in tumor, the constructed PDOs were separated with a filter with 100μm pore size and BAG5 expression was investigated using histochemical staining. (H) The PDO (PDO#1) with higher basal BAG5 expression were infected with gRNA guided BAG5 using CRISPR/Cas9 system for knockout. Western blot analysis confirming effective BAG5 knockout in PDO#1. β-actin was used as a loading control. (I) Control and BAG5 knockout PDOs were seeded and maintained for 1 week. Representative photographs (left) of colony formation and their quantitative analysis (right) were presented. Data represent the mean ± SD of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test; **p < 0.001. (J) Invasion of cells to the lower compartment and chemotaxis of the upper compartment organoids was evaluated by Matrigel-uncoated Transwell. Representative photographs (left) and their numbers (right). These data show mean ± SD of three independent experiments. * p < 0.05.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Knock-Out, In Vitro, In Vivo, Control, Injection, Staining, Immunohistochemical staining, Derivative Assay, Expressing, Construct, Pore Size, Infection, CRISPR, Western Blot, Two Tailed Test, Chemotaxis Assay

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Enrichment of hallmark genes involved in EMT and metabolic reprogramming in BAG5 + NSCLC tumor cells. (A) The t-SNE plot of BAG5 expression landscape in NSCLC tumor tissues. Heterogeneity in the expression of BAG5 was shown in tumor epithelial cells. (B) The relative proportion of BAG5 + cells from different cell types in NSCLC tissues. (C, D) Single sample GSVA (ssGSVA) analysis (BAG5 + vs. BAG5 - tumor epithelial cells) showing most enriched KEGG pathways (C) and Hallmark annotation (D) .

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Regulation of metabolic reprogramming by BAG5 in NSCLC. (A) Heat map of differentially expressed proteins including metabolic reprogramming, EMT and mitodynamics-related ones in CON and BAG5-KD A549 cells by quantitative proteomics. (B) Downregulation of GLUT3 by BAG5 knockout was confirmed by western blot analysis in NSCLC cells. (C) Glucose uptake by NSCLC cells was analyzed by 2-NBDG incorporation experiments. (D, E) OCR (D) and ECAR (E) were measured using seahorse instrument in control and BAG5 knockout A549 and PC9 cells. (F, G) Glucose consumption (F) and lactate production (G) was analyzed by spectrophotometric methods. Data represent mean ± SD; * p < 0.05.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Quantitative Proteomics, Knock-Out, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Regulation of mitochondrial dynamics by BAG5 in NSCLC. (A) co-expression of BAG5 and DRP1 transcripts was explored using the single cell transcriptome. (B) Regulation of NFN2 and DRP1 by BAG5 in NSCLC cells was investigated via western blot analysis. (C) Representative images of the mitochondrial morphological change by mitotracker staining in A549 cells with control or BAG5 knockout (left). The proportion of cells (n = 100 cells for each sample) with fragmented, intermediate and elongated mitochondria was quantifified (right).Scale bars, 1 μm or 2 μm. (D) MFI of mitotracker staining was measured using flow cytometry in control or BAG5 knockout NCSCLs. (E) Representative TEM images of A549 cells confirmed the mitochondrial morphological changes by BAG5 downregulation. (F) Cells with control or BAG5 knockout were incubated with 10 μM DCFH-DA for 30 min. The MFI of intracellular ROS level was measured with flow cytometry. Data represent mean ± SD; *p < 0.05; n.s. means no significance (Student’s t test). (G) A proposed model for function and mechanism of BAG5 in NSCLC. Based on the multi-omics data, BAG5, as an oncogene, was shown to affect the malignant phenotype of NSCLC cancer cells, and participate in multiple tumor pathways, including regulation of mitochondrial morphology, metabolic reprogramming, RNA metabolism, cytoskeleton, and EMT, which consequently promoting tumor growth and metastasis.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Expressing, Western Blot, Staining, Control, Knock-Out, Flow Cytometry, Incubation, Biomarker Discovery

Journal: Scientific Reports

Article Title: KIAA1429 promotes non-small-cell lung cancer cell proliferation through the TRERNA1/HOXA6 axis

doi: 10.1038/s41598-025-09441-w

Figure Lengend Snippet: KIAA1429 is highly expressed in NSCLC and promotes NSCLC cell proliferation. A - B : The expression of KIAA1429 in different cells was detected by RT-qPCR and Western blot. ** versus BEAS-2B, p < 0.01. C - D : The expression of KIAA1429 in different cells after transfection with siRNA was detected by RT-qPCR and Western blot. ** versus si-NC, p < 0.01. E : Cell proliferation was measured by CCK-8 assay. * versus si-NC, p < 0.05; ** versus si-NC, p < 0.01. F : Colony formation assay was performed to assess cell proliferation. ** versus si-NC, p < 0.01. G : EdU staining was used to measure the EdU incorporation rate. ** versus si-NC, p < 0.01. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Data comparisons among multiple groups in panels A-B were analyzed by one-way ANOVA, and data comparisons among multiple groups in panels C-G were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B) and human NSCLC cell lines (H1573, A549, SK-MES-1 and H520) were procured from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: KIAA1429 promotes non-small-cell lung cancer cell proliferation through the TRERNA1/HOXA6 axis

doi: 10.1038/s41598-025-09441-w

Figure Lengend Snippet: Overexpression of lncRNA TRERNA1 alleviates the inhibitory effect of KIAA1429 downregulation on NSCLC cell proliferation. A : RT-qPCR was performed to detect the expression of TRERNA1 in A549 cells transfected with pc-NC or pc-TRERNA1. ** versus pc-NC, p < 0.01. B : Cell proliferation was measured using the CCK-8 assay. C : Colony formation assay was used to assess cell proliferation. D : EdU staining was performed to measure the EdU incorporation rate. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Data comparisons between two groups in panel A were analyzed by t -test. Data comparisons among multiple groups in panels C-D were analyzed by one-way ANOVA, and data comparisons among multigroup in panel B were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. ** p < 0.01.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B) and human NSCLC cell lines (H1573, A549, SK-MES-1 and H520) were procured from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO 2 .

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Colony Assay, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: KIAA1429 promotes non-small-cell lung cancer cell proliferation through the TRERNA1/HOXA6 axis

doi: 10.1038/s41598-025-09441-w

Figure Lengend Snippet: Downregulation of HOXA6 alleviates the inhibitory effect of KIAA1429 downregulation on NSCLC cell proliferation. A : RT-qPCR was performed to detect the expression of HOXA6 in A549 cells transfected with si-NC or si-HOXA6. ** versus si-NC, p < 0.01. B : Western blot was used to detect the expression of HOXA6 in A549 cells. C : Cell proliferation was measured using the CCK-8 assay. D : Colony formation assay was used to assess cell proliferation. E : EdU staining was performed to measure the EdU incorporation rate. The experiments were independently repeated three times, and the data are presented as the mean ± standard deviation. Data comparisons among multiple groups in panels A-B and D-E were analyzed by one-way ANOVA, and data comparisons among multiple groups in panel C were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. ** p < 0.01.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B) and human NSCLC cell lines (H1573, A549, SK-MES-1 and H520) were procured from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO 2 .

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, CCK-8 Assay, Colony Assay, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: KIAA1429 promotes non-small-cell lung cancer cell proliferation through the TRERNA1/HOXA6 axis

doi: 10.1038/s41598-025-09441-w

Figure Lengend Snippet: Downregulation of KIAA1429 inhibits NSCLC cell proliferation in vivo. A : Tumor formation was induced by subcutaneous injection of A549 cells into nude mice. Tumor size was measured every 3 days, and the mice were euthanized on day 21, and tumor tissues were photographed. B : Tumor tissue weight and representative images. C : Immunohistochemistry was performed to detect the positive rates of Ki67 and PCNA in tumor tissues. D : m6A quantitative analysis was conducted to measure the m6A levels in the tissues. E - F : RT-qPCR and Western blot were used to detect the expression of KIAA1429, TRERNA1, and HOXA6 in tumor tissues. N = 6, and the experiments were independently repeated three times. The data are presented as the mean ± standard deviation. Data comparisons between two groups in panels B and D were analyzed by the t -test. Data comparisons among multiple groups in panels A, C, E, and F were analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. ** versus sh-NC, p < 0.01.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B) and human NSCLC cell lines (H1573, A549, SK-MES-1 and H520) were procured from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO 2 .

Techniques: In Vivo, Injection, Immunohistochemistry, Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation

Journal: Scientific Reports

Article Title: KIAA1429 promotes non-small-cell lung cancer cell proliferation through the TRERNA1/HOXA6 axis

doi: 10.1038/s41598-025-09441-w

Figure Lengend Snippet: Mechanism of KIAA1429-mediated m6A modification in promoting NSCLC cell proliferation. KIAA1429 targets m6A modification sites on the lncRNA TRERNA1, enhancing TRERNA1 RNA stability and upregulating the expression of TRERNA1, which in turn strengthens TRERNA1 recruitment of EZH2, leading to increased H3K27me3 modification in the HOXA6 promoter, thereby suppressing HOXA6 expression and promoting NSCLC cell proliferation.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B) and human NSCLC cell lines (H1573, A549, SK-MES-1 and H520) were procured from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO 2 .

Techniques: Modification, Expressing