Journal: The American Journal of Pathology

Article Title: Laminin-γ2–NR6A1 Fusion Protein Promotes Metastatic Potential in Non–Small-Cell Lung Carcinoma Cells without Epidermal Growth Factor Receptor Mutation

doi: 10.1016/j.ajpath.2025.03.006

Figure Lengend Snippet: Screening for Lm-γ2 and Lm-γ2F in serum-free conditioned media (SF-CM) from non–small-cell lung carcinoma (NSCLC) cells. A: Schematic representation of Lm-γ2 and Lm-γ2F proteins, including the epitopes recognized by the anti–Lm-γ2 monoclonal antibodies (mAbs) D4B5 and 23B1. B: Western blot of Lm-γ2 and its related chains. Concentrated conditioned media (100×) from NSCLC and SKOV-3 cells were subjected to Western blot analysis using mAbs 23B1 and D4B5 (NSCLC: 10 μL; SKOV-3: 2 μL). CBB, Coomassie Brilliant Blue R-250.

Article Snippet: The NSCLC cell lines PC-9, NCI-H1650, NCI-H1975, EKVX, RERF-LC-KJ, and VMRC-LCD were obtained from the Japanese Collection of Research Bioresource Cell Bank (National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan).

Techniques: Bioprocessing, Western Blot

Journal: The American Journal of Pathology

Article Title: Laminin-γ2–NR6A1 Fusion Protein Promotes Metastatic Potential in Non–Small-Cell Lung Carcinoma Cells without Epidermal Growth Factor Receptor Mutation

doi: 10.1016/j.ajpath.2025.03.006

Figure Lengend Snippet: Lm-γ2F expression promotes cell proliferation and survival through epidermal growth factor receptor (EGFR) signaling pathway. A: Western blot analysis of EGFR protein in non–small-cell lung carcinoma (NSCLC) cells. B: Western blot analysis of enforced Lm-γ2F expression in NSCLC cells using anti–Lm-γ2 (D4B5), anti–Lm-γ2F (23B1), and anti-V5 monoclonal antibodies. EKVX and RERF-LC-KJ cells expressed wild-type EGFR, whereas VMRC-LCD cells did not. C – E: Effect of enforced Lm-γ2F expression on cell proliferation in NSCLC cells (EKVX, RERF-LC-KJ, and VMRC-LCD) under two-dimensional (2-D) culture conditions with 10% fetal calf serum (FCS). Cell counts were performed using Trypan blue staining after 3, 5, and 7 days of incubation. F and G: Effect of gefitinib on NSCLC cell proliferation. Control and Lm-γ2F–expressing cells were cultured in 6-well plates with or without gefitinib under 2-D culture conditions with 10% FCS (EKVX: 500 nmol/L; RERF-LC-KJ: 250 nmol/L). Cell counts were performed using Trypan blue staining after 5 days of incubation. H and I: Effect of enforced Lm-γ2F expression on cell proliferation in NSCLC cells (EKVX and RERF-LC-KJ) under three-dimensional collagen gel culture conditions with 10% and 20% FCS, respectively. J and K: Effect of enforced Lm-γ2F expression on spheroid growth in NSCLC cells (EKVX and RERF-LC-KJ) under poly-HEMA–coated plate conditions with 10% FCS. Spheroid formation ability was quantified using ImageJ software version 1.54g. All experiments were performed in duplicate, and three independent experiments were conducted. Bar graphs represent data of at least three replicates. Statistical analysis was performed using one-way analysis of variance with Dunnett multiple comparisons test or Tukey multiple comparisons test. Data are given as means ± SD ( C – K ). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001; † P < 0.05, ††† P < 0.001. Scale bars = 100 μm ( J and K ). NS, not significant.

Article Snippet: The NSCLC cell lines PC-9, NCI-H1650, NCI-H1975, EKVX, RERF-LC-KJ, and VMRC-LCD were obtained from the Japanese Collection of Research Bioresource Cell Bank (National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan).

Techniques: Expressing, Western Blot, Bioprocessing, Staining, Incubation, Control, Cell Culture, Software

Journal: The American Journal of Pathology

Article Title: Laminin-γ2–NR6A1 Fusion Protein Promotes Metastatic Potential in Non–Small-Cell Lung Carcinoma Cells without Epidermal Growth Factor Receptor Mutation

doi: 10.1016/j.ajpath.2025.03.006

Figure Lengend Snippet: Summary of Lm-γ2F–induced malignant progression in non–small-cell lung carcinoma (NSCLC) without epidermal growth factor receptor (EGFR) mutations. A: Ectopic expression of Lm-γ2F activates EGFR and its downstream signaling pathways, including AKT and extracellular signal-regulated kinase (ERK), in NSCLC cells lacking EGFR mutations. B: This activation promotes cell proliferation, motility, survival, invasion, and metastasis. EGFR tyrosine kinase inhibitors are suggested to inhibit the acquisition of malignant traits in patients with NSCLC without EGFR mutations expressing Lm-γ2F.

Article Snippet: The NSCLC cell lines PC-9, NCI-H1650, NCI-H1975, EKVX, RERF-LC-KJ, and VMRC-LCD were obtained from the Japanese Collection of Research Bioresource Cell Bank (National Institute of Biomedical Innovation, Health and Nutrition, Osaka, Japan).

Techniques: Expressing, Protein-Protein interactions, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: Immunohistochemical staining for ARID1A expression in EGFR-mutant LUAD tissues (50×and 200×). (A) ARID1A low expression. (B) ARID1A high expression.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Immunohistochemical staining, Staining, Expressing, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: SWI/SNF subunit mutations in lung adenocarcinoma (LUAD). (A) The frequencies of EGFR and SWI/SNF subunit mutations in LUAD. (B) The frequency of SWI/SNF subunit mutations in EGFR-mutant LUAD. (C) The types of SWI/SNF subunit mutations in EGFR-mutant LUAD.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: The role of SWI/SNF subunit mutations in the prognosis of EGFR-mutant LUAD. (A-F) ARID1A, ARID1B, ARID2, ARID5B, SMARCA4, and SMARCB1.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: ARID1A mutation confers a poor prognosis for patients with EGFR-mutant LUAD. (A) The frequencies of genes exhibiting coexisting mutations with EGFR. Survival analysis for patients with the EGFR mutation, ARID1A/EGFR comutation and (B) TP53/EGFR, (C) KRAS/EGFR, (D) CDKN2A/EGFR, (E) PIK3CA/EGFR, (F) RB1/EGFR, and (G) PTEN/EGFR comutations.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: The role of ARID1A expression in the prognosis of EGFR-mutant LUAD patients receiving EGFR-TKIs as a first-line treatment after postoperative progression. (A) Comparison of PFS between patients with low ARID1A expression and patients with high ARID1A expression. (B) Forest plots for the univariate and multivariate analyses of PFS.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: The role of ARID1A in the prognosis of EGFR-mutant LUAD patients receiving postoperative adjuvant EGFR-TKI treatments. (A) Comparison of DFS between patients with low ARID1A expression and patients with high ARID1A expression. (B) Forest plots for univariate and multivariate analyses of DFS. (C) Nomogram for the prediction of 1-, 2- and 3-year survival. (D) Calibration curves of the nomogram.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis, Adjuvant, Comparison, Expressing

Journal: Cell Death & Disease

Article Title: SPIN1 accelerates tumorigenesis and confers radioresistance in non-small cell lung cancer by orchestrating the FOXO3a/FOXM1 axis

doi: 10.1038/s41419-024-07225-0

Figure Lengend Snippet: A The relative mRNA expression of SPIN1 in lung adenocarcinoma tissues compared with that in normal lung tissues obtained from the Oncomine database. B SPIN1 protein levels in fresh lung cancer tissues and paired normal tissues ( n = 8) were analysed via western blotting. C The expression of SPIN1 in six NSCLC cell lines and normal bronchial epithelioid cells was detected by western blotting. D Representative images of SPIN1 immunohistochemistry in NSCLC tissues and adjacent nontumorous tissues (Left, scale bar: 100 μm; Right, scale bar: 50 μm). E Quantification of SPIN1 protein expression levels in NSCLC tissues and normal adjacent tissues. F IHC staining scores of the IHC images of NSCLC and adjacent nontumorous tissues. G Kaplan-Meier curves of patients with high and low SPIN1 expression in NSCLC ( n = 120, log-rank test, p < 0.05). n.s., no significant difference, * p < 0.05, ** p < 0.01. The cell experiments were conducted more than 3 times independently, and the data are presented as the means ± standard deviations (SDs).

Article Snippet: Human NSCLC cell lines (95-D, A549, HCC827, H358, H1299 and PC-9), as well as normal human bronchial epithelioid cells (Beas-2B), were purchased from Procell Life Science and Technology (Wuhan, China) and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium.

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: SPIN1 accelerates tumorigenesis and confers radioresistance in non-small cell lung cancer by orchestrating the FOXO3a/FOXM1 axis

doi: 10.1038/s41419-024-07225-0

Figure Lengend Snippet: A Western blotting assays were conducted to validate the transfection efficiency of SPIN1 siRNA in A549 (left) and HCC827 (right) cells. B CCK-8 assays were used to evaluate the growth ability of A549 and HCC827 cells transfected with SPIN1 siRNAs. C A colony formation assay was performed in SPIN1-depleted NSCLC cells. Scratch wound healing ( D ) and transwell ( E ) assays were conducted to evaluate the migratory and invasive abilities of NSCLC cells upon SPIN1 depletion. F Representative images of xenograft tumours in two groups (scramble groups and shSPIN1 groups). G Growth curves of xenograft tumours derived from A549 cells expressing scramble or SPIN1 shRNA are presented. H Histograms of tumour weights from the above experiments. ** p < 0.01. At least three replicate experiments were performed, and the final results are presented as the means ± SDs.

Article Snippet: Human NSCLC cell lines (95-D, A549, HCC827, H358, H1299 and PC-9), as well as normal human bronchial epithelioid cells (Beas-2B), were purchased from Procell Life Science and Technology (Wuhan, China) and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium.

Techniques: Western Blot, Transfection, CCK-8 Assay, Colony Assay, Derivative Assay, Expressing, shRNA

Journal: Cell Death & Disease

Article Title: SPIN1 accelerates tumorigenesis and confers radioresistance in non-small cell lung cancer by orchestrating the FOXO3a/FOXM1 axis

doi: 10.1038/s41419-024-07225-0

Figure Lengend Snippet: Representative images ( A ) and quantification analysis ( B ) of flow cytometry data depicting the cell cycle distribution of NSCLC cells (A549 and HCC827) transfected with NC and SPIN1 siRNAs 6 h after IR (6 Gy). C , D Representative images of neutral comet assays performed 4 h after IR exposure of SPIN1-depleted or control cells. Scale bar: 25 μm. E , F Immunofluorescence staining was performed to detect γ-H2AX foci formation in A549 and HCC827 cells transfected with SPIN1 siRNAs or negative control siRNAs. Scale bar: 10 μm. ** p < 0.01. All the experiments were performed three times independently, and the results are presented as the means ± SDs.

Article Snippet: Human NSCLC cell lines (95-D, A549, HCC827, H358, H1299 and PC-9), as well as normal human bronchial epithelioid cells (Beas-2B), were purchased from Procell Life Science and Technology (Wuhan, China) and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium.

Techniques: Flow Cytometry, Transfection, Control, Immunofluorescence, Staining, Negative Control

Journal: Cell Death & Disease

Article Title: SPIN1 accelerates tumorigenesis and confers radioresistance in non-small cell lung cancer by orchestrating the FOXO3a/FOXM1 axis

doi: 10.1038/s41419-024-07225-0

Figure Lengend Snippet: A Cells transfected with SPIN1 siRNAs and HA-FOXM1 plasmids were harvested, and the expression of relevant molecules was detected via western blotting. B The proliferation and viability of the indicated NSCLC cells were analysed via CCK-8 assays. Scale bar: 100 μm. C The results of the colony formation assay performed with the indicated cells. D A clonogenic survival assay was used to detect the sensitivity of the indicated cells (A549 and HCC827) transfected with SPIN1 siRNAs or/and FOXM1 plasmids. E The results of neutral comet assay performed in the three groups. Scale bar: 25 μm. F The number and number of Rad51 foci detected by immunofluorescence staining. Scale bar: 10 μm. n.s., no significant difference, * p < 0.05, ** p < 0.01. All experiments were performed independently at least three times, and the results are presented as the means ± SDs.

Article Snippet: Human NSCLC cell lines (95-D, A549, HCC827, H358, H1299 and PC-9), as well as normal human bronchial epithelioid cells (Beas-2B), were purchased from Procell Life Science and Technology (Wuhan, China) and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium.

Techniques: Transfection, Expressing, Western Blot, CCK-8 Assay, Colony Assay, Clonogenic Cell Survival Assay, Neutral Comet Assay, Immunofluorescence, Staining

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: High expression of ERO1A is associated with poor prognosis in all-NSCLC( A ), when considering squamous ( B ) and adenocarcinoma histology ( C ) and when accounting for EGFR mutation status ( D , E ). ERO1A quartiles are based on expression in all-NSCLC patients and were consistently applied across all subtypes of NSCLC.

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: Expressing, Mutagenesis

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: ERO1A quartile cutoffs were determined based on the expression of ERO1A in EGFRmt-LUAD. A Patients with tumors expressing high ERO1A levels have increased survival. B Molecular alterations associated with ERO1A expression prevalent in either cohort at ≥2% and statistically significantly different are shown in the table. C Molecular alterations with a prevalence difference of at least 5% between the cohorts were incorporated as co-variates in a complete-case multivariate analysis. High ERO1A expression and TP53 mutations were independently associated with poor OS in EGFRmt-LUAD ( n = 960).

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: High expression of ERO1A was associated with an enrichment of a number of Hallmark Pathways as revealed by GSEA. The second most significant pathway was associated with regulating protein secretion ( A ). The association of ERO1A expression was established with the expression of genes involved in Hallmark protein secretion pathway. Genes were sorted based on their Spearman correlation with ERO1A and are listed to the right side of the heatmap ( B ). Subset of genes involved in vesicle trafficking from ER to Golgi as well as regulation of exosomes were investigated and observed to be significantly enriched in ERO1A -Q4 tumors ( C ). Correlation between ERO1A and levels of other proteins downloaded from CPTAC, a publicly available lung adenocarcinoma tumor proteomics dataset, were analyzed using LinkedOmics ( http://linkedomics.org/data_download/CPTAC-LUAD/ ) ( D ). 7 out of 28 targets with highest correlation to ERO1A levels are extracellular matrix proteins ( E ).

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: Shown is a representative image demonstrating CRISPR deletion of ERO1A in PC-9 and HCC006 cell line. ERO1B expression levels do not compensate for loss of ERO1A. Representative western blots demonstrate that ERO1A KO does not affect total levels of EGFR or p-EGFR in two EGFR MUT -NSCLC cell lines ( A ). ERO1A KO cells form smaller number of colonies in soft agar and on tissue culture plates. Representative fields of view from one independent experiment are shown. *** p = 0.0004 by one-way ANOVA, * p = 0.0111 by repeated measures ANOVA, n = 3–4 independent experiments performed in triplicates ( B , C ). ERO1A KO clones reveal a decrease in total tumor sphere formation in both PC-9 and HCC4006 cell lines. Representative fields of view from one independent experiment are shown. Shown is the means of 3-4 independent experiments performed in triplicates. Numbers are mean ± SEM. **** p < 0.0001, ** p = 0.0072, * p < 0.05; Dunnett’s multiple comparisons test following one-way ANOVA ( D – E ).

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: CRISPR, Expressing, Western Blot, Clone Assay

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: Levels of LAMC2 and LOXL2 are dramatically reduced in conditioned medium collected from ERO1A KO cells. Representative western blots on whole cell lysates and conditioned media in PC-9 cell line are shown ( A ). Concentration of LAMC2 secreted into conditioned media by ERO1A KO cell lines is reduced compared to control cells. ELISA was performed in triplicate and the average ± S.D. of n = 3 combined results are shown. **** p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA ( B – C ). Representative images of immunofluorescence staining for LOXL2 and LAMC2 in HCC4006 spheroids, scale bar - 50 µm ( D – E ). Quantification of LOXL2 and LAMC2 immunofluorescent staining in control and ERO1A KO HCC4006 cells cultured as tumor spheres. * p = 0.0209 by two-tailed unpaired t -test, n = 5, experiment was repeated 3 times. ns – not significant ( F ).

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: Western Blot, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Cell Culture, Two Tailed Test

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: Soft agar colony formation assay using regular non-conditioned medium (Normal), medium conditioned by control cells (Conditioned) and conditioned medium from control cells after heat denaturation (Heat denatured), **** p < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 3 ( A ). Conditioned medium generated by ERO1A KO cells does not rescue colony formation abilities of ERO1A KO cells ( B ). Representative immunoblot showing level of LAMC2 and LOXL2 knockdown in PC-9 cells. LAMC2 or LOXL2 knockdown dramatically decrease levels of secreted LAMC2 and LOXL2, accordingly ( C ). Conditioned medium generated by sh LOXL2 and sh LAMC2 PC-9 cells partially rescue colony formation abilities of ERO1A KO cells in soft agar assay. GFP—empty vector control. Data are means ± SD. **** p < 0.0001, Tukey’s multiple comparisons test following two-way ANOVA. ns – not significant ( D ).

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: Soft Agar Assay, Control, Generated, Western Blot, Knockdown, Plasmid Preparation

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: Picture of tumors formed and H & E staining from the lungs of ERO1A replete and KO mice. H & E was performed on 5 random lungs from each group selected by a random number generator and tumor margins quantified using ImageJ ( A , B ). Each data point represents the average on three sections from each mouse. * p = 0.0110, unpaired t -test ( C ). Kaplan–Meier curve showing that ERO1A KO increased overall survival compared to the control group. p = 0.0064, log-rank (Mantel-Cox) test ( D ).

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: Staining, Control

Journal: NPJ Precision Oncology

Article Title: ERO1A levels are a prognostic indicator in EGFR mutated non small cell lung cancer

doi: 10.1038/s41698-024-00736-1

Figure Lengend Snippet: Antibodies used for Western blot experiments

Article Snippet: 2 million of either PC-9 control or PC-9 ERO1A knockout cell line were injected via tail vein into SCID-Beige mice Charles River Laboratories.

Techniques: Western Blot