mouse llc cells  (Procell Inc)

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Identification of 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK)-binding small-molecule compounds by high-throughput screening (HTS). (A) Small molecule compounds are printed onto homemade phenyl-isocyanate-functionalized glass slides and immobilized through amino, hydroxyl, and thiol groups as well as other interactions. In oblique-incidence reflectivity difference (OI-RD) scanning of small-molecule microarrays (SMMs), successful binding of the target protein at a specific location with small-molecule compounds increases the molecular layer thickness, which OI-RD detects as white spots. The binding of proteins to small molecule compounds is usually coupled mediated by nucleophilic groups (hydroxyl, amino, and thiol). Interactions such as hydrogen bonding will be further formed thereby mediating protein interactions with small molecule compounds. No spot is observed if binding does not occur. Created with www.BioRender.com . (B) Categories of the compound library. Approximately 4389 compounds, including 1678 Food and Drug Administration (FDA)-approved drugs, 1456 natural products, and 1255 known inhibitors, were printed on the chip. (C) A representative image showing the binding of the recombinant AMPK protein to compounds printed on an HTS chip. (D) A real-time OI-RD assay depicting the association‒dissociation curves of surface-immobilized Ribavirin binding to the recombinant AMPK protein. (E) A surface plasmon resonance (SPR) experiment was used to evaluate the interaction kinetics and binding affinity between Ribavirin and AMPK. (F, G) Quantitative real-time polymerase chain reaction (qRT-PCR) measurement of Ampk transcripts in mouse Lewis lung carcinoma (LLC) cells (F) or human non-small cell lung cancer cells (A549) (G). The data are presented as the relative fold induction of Ampk/actin transcripts. Arrows indicate groups with significantly reduced Ampk transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Binding Assay, High Throughput Screening Assay, Drug discovery, Recombinant, SPR Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: The impact of Ribavirin on inflammatory factors is regulated downstream of the 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway. (A, B) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of interleukin-6 ( Il - 6 ) transcript levels in mouse Lewis lung carcinoma (LLC) cells (A) and human non-small cell lung cancer cells (A549) (B) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Il - 6/actin transcripts. Arrows indicate groups with significantly reduced Il - 6 transcripts. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001. (C, D) qRT-PCR analysis of Tnf α transcript levels in LLC (C) and A549 (D) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Tnf α/actin transcripts. Arrows indicate groups with significantly reduced Tnf α transcripts. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (E, F) qRT-PCR analysis of C-X-C motif chemokine ligand 10 ( Cxcl10 ) transcript levels in LLC (E) and A549 (F) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Cxcl10/actin transcripts. The arrows indicate groups with significantly reduced Cxcl10 transcript levels. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (G, H) qRT-PCR analysis of C–C motif chemokine ligand 5 ( Ccl5 ) transcript levels in LLC (G) and A549 (H) cells following 8 h of hypoxia treatment. The data are presented as the relative fold induction of Ccl5/actin transcripts. Arrows indicate groups with significantly reduced Ccl5 transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗ P < 0.05. mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Ribavirin suppresses lung cancer cell growth both in vitro and in vivo . (A, B) Viability of mouse Lewis lung carcinoma (LLC) cells (A) and human non-small cell lung cancer cells (A549) (B) cells treated with various concentrations of Ribavi rin. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. (C, D) Viability of LLC (C) and A549 (D) cells following treatment with 10 μM Ribavirin or the control for 1–5 days. The data shown are the means ± SEM from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (E, F) Colony formation of LLC (E) and A549 (F) cells after treatment with 10 μM Ribavirin or the control. The data shown are the means ± SEM from n = 3 biological replicates. Statistical significance was determined by two-way ANOVA, followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001. (G) Tumor anatomy of LLC subcutaneous tumor-bearing mice. (H) Tumor volume of LLC subcutaneous tumor-bearing mice. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 5 mice per group, with differences denoted by ∗ P < 0.05. (I) Tumor weights of LLC subcutaneous tumor-bearing mice. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 8 mice per group, with differences denoted by ∗∗ P < 0.01. PBS: phosphate buffered saline.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: In Vitro, In Vivo, Control, Saline

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: The interaction of Ribavirin with 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK). (A) Biotin was conjugated to the amide residue of Ribavirin to produce biotin-conjugated Ribavirin. (B) Immunoblotting (IB) of the streptavidin precipitates of the mixture of biotin or biotin-Ribavirin and the lysates of human embryonic kidney epithelial cells (HEK293T) cells transfected with Flag-AMPK. The data shown are representative of n = 3 biological replicates. (C–F) The molecular docking assay illustrates the interaction between Ribavirin and AMPK, identifying the key amino acids involved (C); the blue protein represents AMPK (D), the green small molecule represents Ribavirin (E), and the yellow dashed lines represent the hydrogen bonds formed (F). A binding energy below −5 suggests high reliability of the docking model. (G, H) Immunoblotting of phosphorylated AMPK (p-AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lysates of mouse Lewis lung carcinoma (LLC) cells (G) and human lung adenocarcinoma cells (PC9) (H) cells in the presence of Ribavirin (10 μg/mL). The data shown are representative of n = 3 biological replicates. (I, J) Immunoblotting of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (p-4EBP1) and GAPDH in the lysates of LLC (I) and PC9 (J) cells in the presence of Ribavirin (10 μg/mL). The data shown are representative of n = 3 biological replicates. (K, L) Quantitative real-time polymerase chain reaction (qRT-PCR) measurement of 4ebp1 (K) and Mtor (L) transcripts in LLC cells in the presence of phosphate buffered saline (PBS) or the indicated concentrations of Ribavirin (10 μm). The data are presented as the fold induction of 4ebp1/Gapdh transcripts and Mtor/Gapdh transcripts. The data shown are the means ± standard error of the mean (SEM) from n = 3 biological replicates. Statistical significance was determined by two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. ns: not significant; IP: immunoprecipitation; mRNA: messenger RNA.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Residue, Western Blot, Transfection, Docking Assay, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Saline, Immunoprecipitation

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Ribavirin has synergistic effects on 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) downregulation. (A) Three AMPK-targeting short hairpin RNAs (shRNAs) were screened to evaluate their knockdown efficiency and suppress AMPK expression in lung cancer cells. The data shown are representative of n = 3 biological replicates. (B) Tumor anatomy of mouse Lewis lung carcinoma (LLC) cells subcutaneous tumor-bearing mice in different treatment groups. (C) Tumor volume of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± standard error of the mean (SEM); n = 8 mice per group, with differences denoted by ∗ P < 0.05 and ∗∗ P < 0.01. (D) Tumor weights of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 8 mice per group, with differences denoted by ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (E) Schematic illustration of the mechanism by which Ribavirin regulates AMPK activation. AMPK is phosphorylated in response to an increased AMP/adenosine triphosphate (ATP) or adenosine diphosphate (ADP)/ATP ratio, influencing cell growth and proliferation by inactivating mechanistic target of rapamycin complex 1 (mTORC1). Ribavirin exerts its regulatory effect by inhibiting AMPK phosphorylation. Created with www.BioRender.com . shControl: non-targeting control short hairpin RNA; shAMPKα: AMPKα-targeting short hairpin RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PBS: phosphate buffered saline; mTOR: mechanistic target of rapamycin; Raptor: regulatory-associated protein of mTOR; Deptor: DEP domain-containing mTOR-interacting protein; PRAS40: proline-rich Akt substrate of 40 kDa; mLST8: mammalian lethal with SEC13 protein 8; 4EBP1:eukaryotic translation initiation factor 4E-binding protein 1; P: phosphorylation.

Article Snippet: Human embryonic kidney epithelial cells (HEK293T; ATCC CRL-11268), human non-small cell lung cancer cells (A549; ECACC 86012804), human lung adenocarcinoma cells (PC-9; ECACC 90071810), and mouse Lewis lung carcinoma (LLC) cells (ATCC CRL-1642) (FuHeng, Shanghai, China) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA).

Techniques: Knockdown, Expressing, Activation Assay, Phospho-proteomics, Control, shRNA, Saline, Binding Assay

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) Schematic diagram of experimental design. Wild-type LLC lung cancer cells were subcutaneously injected into C57BL/6 mice, and the tumors were dissected after 14 days. ( B ) YAP/TAZ expression showed heterogeneity in mouse lung tumor tissues. Lewis lung carcinoma (LLC) cells were subcutaneously injected into C57BL/6 mice, and the resulting tumors were harvested 14 days after transplantation. Paraffin-embedded tumor tissues were subjected to immunohistochemical analysis using YAP/TAZ antibodies. Representative image from four tumors are shown. Black arrowheads indicate YAP/TAZ high cancer cells and white arrowheads indicate YAP/TAZ low cells. Scale bar, 100 µm. ( C ) LLC cells lacking YAP/TAZ protect their surrounding cells from ferroptosis. Wild-type (WT) LLC cells expressing EGFP (green) were co-cultured with YAP/TAZ double-knockout (dKO) LLC cells expressing tdTomato (red) in the presence of RSL3 (400 nM) for 10 h. Quantitative analysis of cell numbers is shown on the right. Data are presented as the means ± SD of five randomly selected images. The experiment was repeated three times, and data from one representative experiment are shown. Scale bars, 100 µm. **** (a) P = 0.000000000000105; **** (b) P = 0.000000001145261 (one-way ANOVA test followed by Tukey’s multiple comparison test). .

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Injection, Expressing, Transplantation Assay, Immunohistochemical staining, Cell Culture, Double Knockout, Comparison

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) DNA mutation in YAP/TAZ double-knockout (dKO) LLC cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. gDNA, genomic DNA. ( B ) Immunoblotting (IB) of cell extracts from wild-type (WT) and YAP/TAZ dKO LLC cells with antibodies against the indicated proteins. ( C ) Reverse transcription (RT) and real-time PCR analysis of the YAP/TAZ target gene Ankrd1 in WT and YAP/TAZ dKO LLC cells. Data are means ± SD of three biologically independent samples from a representative experiment. **** P = 0.000001244429960 (unpaired t test).

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Mutagenesis, Double Knockout, CRISPR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) Actinomycin D-induced cell death was rescued by the apoptosis inhibitor Z-VAD-FMK. Wild-type (WT) LLC cells were pretreated (or not) with Z-VAD-FMK (50 µM) for 1 h, followed by stimulation with Actinomycin D (500 nM) for 24 h. Dead cells were stained with propidium iodide (PI), and the percentage of the PI-negative live cell population was calculated using the flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000000065; **** (b) P = 0.000000000000065 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( B ) TCZ-induced cell death was rescued by the necroptosis inhibitor Necrostatin-1. WT LLC cells were pretreated (or not) with Necrostatin-1 for 1 h, followed by stimulation with TCZ [T, TNFα (30 ng/mL); C, cycloheximide (30 µg/mL); Z, Z-VAD-FMK (20 µM)] for 24 h. The percentage of live cell population was calculated using PI and flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000012723845; **** (b) P = 0.000000720360306 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( C ) RSL3-induced cell death was rescued by the ferroptosis inhibitor Ferrostatin-1. WT LLC cells were pretreated (or not) with Ferrostatin-1 (5 µM) for 30 min, followed by stimulation with RSL3 (400 nM) for 10 h. The percentage of live cell population was calculated using PI and flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000048729652; **** (b) P = 0.000000056548852 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( D ) YAP/TAZ loss sensitizes LLC cells to apoptosis. WT and YAP/TAZ dKO LLC cells were treated with Actinomycin D (500 nM) for 24 h, followed by cell viability assay using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** P = 0.000030539048461 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( E ) YAP/TAZ loss sensitizes LLC cells to necroptosis. WT and YAP/TAZ dKO LLC cells were treated with TCZ [T, TNFα (30 ng/mL); C, cycloheximide (30 µg/mL); Z, Z-VAD-FMK (20 µM)] for 24 h, followed by cell viability assay using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** P = 0.000000000000054 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( F ) YAP/TAZ loss confers resistance to ferroptosis in LLC cells. WT and YAP/TAZ dKO LLC cells were treated with RSL3 (400 nM) for 10 h, followed by cell viability assay using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** P = 0.000000000000051 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( G ) Cell viability analysis was performed on WT and YAP/TAZ dKO LLC cells treated with indicated concentrations of RSL3 for 10 h, followed by cell viability assay using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. ( H ) Cell death induced by ferroptosis-inducing reagents or cysteine and cystine starvation (CCS) was rescued by the ferroptosis inhibitor Ferrostatin-1. WT LLC cells were pretreated (or not) with Ferrostatin-1 (5 µM) for 30 min, followed by stimulation with Erastin (5 µM) for 10 h, L-Buthionine-(S,R)-Sulfoximine (L-BSO; 10 mM) for 24 h, or Sulfasalazine (SAS; 1 mM) for 24 h. For CCS, cells were cultured in the cysteine and cystine-starved medium for 24 h. The percentage of live cell population was calculated using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000005959; **** (b) P = 0.000000096903963; **** (c) P = 0.000000191080058; **** (d) P = 0.000000000000065 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( I ) YAP/TAZ loss confers resistance to ferroptosis in LLC cells. WT and YAP/TAZ dKO LLC cells were treated with Erastin (5 µM) for 10 h, L-BSO (10 mM) for 24 h, or SAS (1 mM) for 24 h. For CCS, cells were cultured in the cysteine and cystine-starved medium for 24 h. The percentage of live cell population was calculated using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000000769; **** (b) P = 0.000000000883037; **** (c) P = 0.000000000024749; **** (d) P = 0.000000000000051 (one-way ANOVA test followed by Tukey’s multiple comparison test).

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Staining, Flow Cytometry, Comparison, Viability Assay, Cell Culture

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) YAP/TAZ depletion suppresses lipid peroxidation in LLC cells. WT and YAP/TAZ dKO LLC cells were treated with RSL3 (400 nM) for 4 h, followed by lipid peroxidation measurement using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000000051; **** (b) P = 0.000000000000051 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( B ) Immunoblot (IB) analysis of cell extracts from WT and YAP/TAZ dKO LLC cells infected (or not infected) with HA-YAP expression vectors. ( C ) RT and real-time PCR analysis of the YAP/TAZ target Ankrd1 gene in WT and YAP/TAZ dKO LLC cells infected (or not infected) with HA-YAP expression vectors. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000023517185; **** (b) P = 0.000027011157926 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( D ) Re-expression of YAP sensitizes YAP/TAZ-deficient LLC cells to ferroptosis. WT and YAP/TAZ dKO LLC cells infected (or not) with expression vectors for HA-YAP were treated with RSL3 (400 nM) for 10 h, followed by cell viability assay using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** P = 0.000000004002113 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( E ) Left: Representative image of Phos-tag immunoblotting (IB) analysis of cell extracts from A549, LK-2, PC-9 and H1975 human lung cancer cells using antibodies against YAP. Right: The relative activity of YAP in each cell line was assessed based on the ratio of non-phosphorylated YAP (black arrowhead) to fully phosphorylated YAP (white arrowhead). Data are means ± SD of three biologically independent samples from a representative experiment. * P = 0.044457726502518 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( F ) Immunoblotting (IB) of cell extracts from WT and YAP/TAZ dKO LK-2 cells with antibodies against the indicated proteins. ( G ) WT and YAP/TAZ dKO LK-2 cells were treated with Erastin (5 µM) for 10 h, followed by cell viability assay using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** P = 0.000002538628468 (one-way ANOVA test followed by Tukey’s multiple comparison test).

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Flow Cytometry, Comparison, Western Blot, Infection, Expressing, Real-time Polymerase Chain Reaction, Viability Assay, Activity Assay

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) RT and real-time PCR analysis of the ferroptosis-related genes in WT and YAP/TAZ dKO LLC cells. Data are means ± SD of three biologically independent samples from a representative experiment. ( B ) Intracellular glutathione (GSH) levels were measured using LC-MS/MS. Data are means ± SD of three biologically independent samples from a representative experiment. ns indicates no significant difference, ns, not significant ( P = 0.658668395593572) (unpaired t test). ( C ) RT and real-time PCR analysis of the YAP/TAZ target Ctgf gene in WT LLC cells cultured with or without serum for 32 h (left) or at low (L) or high (H) cell densities for 52 h (right). Data are means ± SD of three biologically independent samples from a representative experiment. * P = 0.029089468282210; ***P = 0.000272320651963 (unpaired t test). ( D ) The correlation between GCH1 and YAP1 mRNA levels across TCGA normal datasets was analyzed using the GEPIA (Gene Expression Profiling Interactive Analysis) tool with Pearson correlation. ( E ) The correlation between GCH1 mRNA levels and the YAP/TAZ transcriptional target signature across TCGA normal datasets was analyzed using the GEPIA tool with Pearson correlation.

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Real-time Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Cell Culture, Gene Expression

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) Differences in the transcriptomes of WT and YAP/TAZ dKO LLC cells. Differentially expressed genes (DEGs; fold change >2 and adjusted P value < 0.01) in YAP/TAZ dKO LLC cells compared to WT cells are displayed in a volcano plot (blue, downregulated; red, upregulated). The ferroptosis-related genes are highlighted in green. Data represent the mean of three biologically independent samples from a representative experiment. Statistical analysis of differential gene expression was performed using DESeq2. ( B ) GCH1 accumulates in YAP/TAZ-deficient LLC cells. Immunoblotting (IB) of cell extracts from WT and YAP/TAZ dKO LLC cells with antibodies against indicated proteins was shown. ( C ) Loss of YAP/TAZ induces Gch1 transcription. Total RNA extracted from WT or YAP/TAZ dKO LLC cells were subjected to reverse transcription (RT) and real-time PCR analysis. Data are means ± SD of three biologically independent samples from a representative experiment. *** P = 0.000253798986094 (unpaired t test). ( D ) Gch1 mRNA abundance is increased in YAP/TAZ-deficient lung tumor tissues. Equal numbers of WT or YAP/TAZ dKO LLC cells were subcutaneously injected into C57BL/6 mice. Fourteen days after transplantation, tumors were harvested and then subjected to RNAscope analysis of Gch1 mRNA. Quantitative analysis of signal Gch1 intensity is shown on the right. Data are means ± SD of five randomly selected images. Scale bars, 100 µm. *** P = 0.000361619614793 (unpaired t test). ( E ) YAP re-expression prevents GCH1 accumulation in YAP/TAZ-deficient LLC cells. WT and YAP/TAZ dKO LLC cells infected (or not) with expression vectors for HA-YAP were subjected to immunoblot (IB) analysis of the indicated proteins. ( F ) Re-expression of YAP suppresses Gch1 transcription in YAP/TAZ dKO LLC cells. Total RNA extracted from the indicated cells were subjected to RT and real-time PCR analysis. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000309035446; **** (b) P = 0.000000796405245 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( G ) YAP/TAZ inhibition conditions induce Gch1 transcription. WT LLC cells were cultured with or without serum for 32 h (left) or at low (L) or high (H) cell density for 52 h (right), and then subjected to RT and real-time PCR analysis. Data are means ± SD of three biologically independent samples from a representative experiment. * P = 0.021959987914851; **** P = 0.000029784548704 (unpaired t test). ( H ) YAP/TAZ loss affects chromatin accessibility of Gch1 . WT and YAP/TAZ dKO LLC cells were subjected to assay for transposase-accessible chromatin with sequencing (ATAC-seq). Differential peaks indicative of the open chromatin status are highlighted in yellow. .

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Gene Expression, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Injection, Transplantation Assay, RNAscope, Expressing, Infection, Comparison, Inhibition, Cell Culture, Sequencing

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) Schematic representation of the tetrahydrobiopterin biosynthesis pathway. GCH1 GTP cyclohydrolase 1, DHFR dihydrofolate reductase, BH4 tetrahydrobiopterin, BH2 dihydrobiopterin. ( B ) YAP/TAZ loss increases BH4 production. Peaks with the corresponding multiple reaction monitoring (MRM) indicate intracellular BH4 levels (left, red arrows). Quantitative analysis of the BH4 levels is shown on the right-hand side. Data are means ± SD of three biologically independent samples from a representative experiment. *** P = 0.000144582491651 (unpaired t test). ( C ) Generating YAP/TAZ/GCH1 triple-knockout (tKO) cells. Immunoblot (IB) analysis of cell extracts from WT, YAP/TAZ dKO, and YAP/TAZ/GCH1 tKO LLC cells with antibodies to the indicated proteins was shown. ( D ) GCH1 is required for BH4 accumulation in YAP/TAZ dKO cells. The levels of BH4 in WT, YAP/TAZ dKO, and YAP/TAZ/GCH1 tKO LLC cells were measured by LC-MS/MS. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000001766; **** (b) P = 0.000000000006759 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( E ) GCH1 is required for ferroptosis resistance in YAP/TAZ-deficient cells, and BH4 supplementation confers such resistance. WT, YAP/TAZ dKO and YAP/TAZ/GCH1 tKO LLC cells were pretreated (or not) with BH4 (40 µM) for 30 min, followed by stimulation with RSL3 (400 nM) for 10 h. Dead cells were stained with propidium iodide (PI), and the percentage of the PI-negative live cell population was calculated using the flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000084054; **** (b) P = 0.000000005054431; **** (c) P = 0.000000006325909 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( F ) YAP/TAZ-deficient cells lacking GCH1 expression fail to protect surrounding cells from ferroptosis. WT LLC cells expressing EGFP (green) were co-cultured with YAP/TAZ/GCH1 tKO LLC cells expressing tdTomato (red) in the presence of RSL3 (400 nM) for 10 h. Quantitative analysis of cell numbers is shown on the right. Data are means ± SD of five randomly selected images. The experiment was repeated three times, and data from one representative experiment are shown. The relative number of WT LLC cells co-cultured with YAP/TAZ dKO LLC cells in Fig. is shown in a lighter color for reference. Scale bars, 100 µm. **** (a) P = 0.000000000597045; **** (b) P = 0.000000000000105; **** (c) P = 0.000000000000219 (one-way ANOVA test followed by Tukey’s multiple comparison test). .

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Targeted Proteomics, Triple Knockout, Western Blot, Liquid Chromatography with Mass Spectroscopy, Comparison, Staining, Flow Cytometry, Expressing, Cell Culture

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) Intracellular dihydrobiopterin (BH2) levels in WT and YAP/TAZ dKO LLC cells. Peaks with the corresponding multiple reaction monitoring (MRM) indicate intracellular BH2 (left, red arrows). Quantitative analysis of BH2 levels is shown on the right. Data are means ± SD of three biologically independent samples from a representative experiment. **** P = 0.000001790642720 (unpaired t test). ( B ) BH2 levels in the culture media from WT and YAP/TAZ dKO LLC cells. Peaks with the corresponding MRM indicate extracellular BH2 (red arrows). Quantitative analysis of extracellular BH2 levels is shown on the right. Data are means ± SD of three biologically independent samples from a representative experiment. *** P = 0.000211344236570 (unpaired t test). ( C ) DNA mutation in YAP/TAZ/GCH1 tKO LLC cells using the CRISPR/Cas9 system. gDNA, genomic DNA. ( D ) BH2 levels in WT, YAP/TAZ dKO, and YAP/TAZ/GCH1 tKO LLC cells. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000357087917378; **** (b) P = 0.000357087917378 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( E ) WT, YAP/TAZ dKO, and YAP/TAZ/GCH1 triple-knockout (tKO) LLC cells were pretreated (or not) with BH4 (40 µM) for 30 min, followed by stimulation with RSL3 (400 nM) for 4 h. The percentage of cells positive for lipid peroxidation was calculated using flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000005554; **** (b) P = 0.000000155138171; **** (c) P = 0.000000230915225 (one-way ANOVA test followed by Tukey’s multiple comparison test).

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Targeted Proteomics, Mutagenesis, CRISPR, Comparison, Triple Knockout, Flow Cytometry

Journal: EMBO Reports

Article Title: Hippo pathway controls biopterin metabolism to shield adjacent cells from ferroptosis in lung cancer

doi: 10.1038/s44319-025-00515-4

Figure Lengend Snippet: ( A ) Pharmacological inhibition of GCH1 using 2,4-diamino-6-hydroxypyrimidine (DAHP) sensitizes YAP/TAZ-deficient cells to ferroptosis. YAP/TAZ double-knockout (dKO) LLC cells were pretreated (or not) with Ferrostatin-1 (Fer-1; 5 µM) or BH4 (40 µM) for 30 min, and then stimulated with RSL3 (400 nM) and/or DAHP (10 mM) for 24 h. The percentage of live cell population was calculated using PI and flow cytometer. Data are means ± SD of three biologically independent samples from a representative experiment. **** (a) P = 0.000000000000026; **** (b) P = 0.000000000000026; **** (c) P = 0.000000000000026 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( B ) Schematic diagram of experimental design. Wild-type LLC lung cancer cells were subcutaneously injected into C57BL/6 mice, and the drug administration was started at day 5. Tumor-bearing mice were treated with intratumoral injections of vehicle (10% DMSO, 45% PEG300, and 45% PBS), DAHP (100 mg/kg body weight in the vehicle), or RSL3 (50 mg/kg body weight in the vehicle) three times a week for the whole duration of the experiment, until a mouse was sacrificed. ( C ) Administering the GCH1 inhibitor DAHP sensitizes LLC tumors to RSL3. LLC cells were transplanted into C57BL/6 mice, and tumor growth over time following indicated treatments was monitored. Data are presented as the means ± SEM; n = 12 tumors for each group. ns, not significant ( P = 0.975133563807377 for Vehicle v.s. DAHP, P = 0.873296292458696 for Vehicle v.s. RSL3); **** P = 0.000000000479864 (two-way ANOVA test). ( D ) The RSL3 and DAHP combination suppressed LLC tumor growth. C57BL/6 mice were injected with LLC cells, and tumor weight was determined after 2 weeks of drug treatment. Data are presented as the means ± SD; n = 12 tumors for each group. ns, not significant ( P = 0.689283972138939 for Vehicle v.s. DAHP, P = 0.617118340941646 for Vehicle v.s. RSL3); **** P = 0.000007129017268 (one-way ANOVA test followed by Tukey’s multiple comparison test). ( E ) Combination RSL3 with DAHP therapy improves the survival of mice bearing LLC tumors. Kaplan–Meier tumor-free survival curves for tumor-bearing mice treated with the indicated drugs were shown. n = 10 mice per group. *** P = 0.000996737657140 (log-rank test). .

Article Snippet: Mouse LLC cells , ATCC , CRL-1642.

Techniques: Inhibition, Double Knockout, Flow Cytometry, Comparison, Injection