Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Increased TAG Synthesis in Lung CSCs via Upregulation of DGAT1/2 Expression. (A) Gene ontology (GO) analysis of RNA-sequencing data to identify spheroid-enriched genes between adherent and spheroid culture of A549 and H1993 cells. (B) Genes identified in TAG biosynthesis process term. (C) Western blot showing protein expression related to TAG biosynthesis process in adherent and spheroid culture. (D) Measurement of TAG levels in spheroid A549 cells treated with vehicle, DGAT1i (DGAT1 inhibitor, PF-04620110, 10 μM), DGAT2i (DGAT2 inhibitor, PF-06424439, 10 μM), or DGAT1i + DGAT2i for 24 h. (E) BODIPY 493/503 staining (green) for lipid droplets in spheroid A549 cells treated with DGAT1i + DGAT2i for 24h. Cell nuclei were counterstained with DAPI (blue). (F) Cell viability of spheroid A549 cells incubated without or with H 2 O 2 (250 μM) and/or indicated DGAT inhibitors (10 μM) for 24 h (G, H, and I) TAG, MDA, and 4-HNE levels in spheroid A549 cells incubated without treatment (CTR), treated with H 2 O 2 (250 μM), treated with DGATi (DGAT1 inhibitor at 10 μM + DGAT2 inhibitor at 10 μM), or in combination with H 2 O 2 and DGATi for 24 h. Data are presented as mean ± SD (one-way ANOVA followed by Tukey's post-hoc test). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: DGAT1 inhibitor (PF-04620110) and DGAT2 inhibitor (PF-06424439) were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Expressing, RNA Sequencing, Western Blot, Staining, Incubation

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Inhibition of Tumor Growth and Clinical Implications of DGAT1 and DGAT2 Suppression in Lung Cancer. (A) Cell viability was assessed after irradiation with 2 Gy, 4 Gy, or 6 Gy for 24 h in adherent and spheroid A549 cells. (B) Surviving fractions were calculated in adherent and spheroid A549 cells following irradiation with doses of 2 Gy, 4 Gy, or 6 Gy over a period of 7 days. (C) Cancer cells were subcutaneously injected. When the tumor size reached approximately 50 mm 3 , the mice were subjected to gamma irradiation (3 × 6 Gy) on days 0, 3, and 6, and tumor volumes were measured on the specified days. (D and E) Cell viability and surviving fractions in spheroid A549 cells with DGAT1 and/or DGAT2 inhibitors after irradiation with 6 Gy. (F) Spheroid A549 cells were subcutaneously injected. When the tumor size reached approximately 50 mm 3 , the mice underwent gamma irradiation (3 × 6 Gy) and were orally administered either a vehicle, a DGAT1 inhibitor (10 mg/kg body weight), or a DGAT2 inhibitor (10 mg/kg body weight) on day 0, day 3, and day 6. (G) Sphere formation in spheroid A549 cells with DGAT1 and/or DGAT2 knockdown. (H) The capability of tumor initiation was assessed through the subcutaneous injection of spheroid A549 cells with DGAT1 and/or DGAT2 knockdown. (I) Immunohistochemical staining of DGAT1 and DGAT2 in patients with lung cancer. Case 1 represents a patient with low expression of DGAT1 and DGAT2. Case 2 represents a patient with high expression of DGAT1 and DGAT2. (J) The survival curves of lung cancer patients with or without DGAT1 and DGAT2 expression (n = 59). Significance is calculated using the Kaplan-Meier method and comparisons are performed using the log-rank test. (K) Survival analysis of the 6-gene signature related to TAG synthesis in lung cancer. Data are presented as mean ± SD (student's two-tailed unpaired t -test is employed in A and B; two-way ANOVA followed with Tukey's multiple comparison test is employed in C; one-way ANOVA followed by Tukey's post-hoc test is employed in D, E, F, and G). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: DGAT1 inhibitor (PF-04620110) and DGAT2 inhibitor (PF-06424439) were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Irradiation, Injection, Knockdown, Immunohistochemical staining, Staining, Expressing, Two Tailed Test, Comparison

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Regulation of Lipid Metabolism by Cancer Stem Cells via the YAP1/TEAD Pathway. (A) Top pathways identified by KEGG analysis from RNA-sequencing data between adherent and spheroid culture of A549 and H1993 cells. (B) Representative immunofluorescence images showing the subcellular location of pYAP1 (Y357) and TEAD1 in adherent and spheroid A549 cells. Cell nuclei were counterstained with DAPI. (C) Western blot analysis of cytoplasmic (Cyt) and nuclear (Nuc) fractions revealing pYAP1 (Y357) and YAP1 protein expression in the adherent and spheroid culture of A549 cell cultures. (D) Co-immunoprecipitation of the nuclear fraction to detect the interaction between pYAP1 (Y357) and TEAD1. (E) Representative images of proximity ligation assay (PLA) illustrating protein-protein interactions between pYAP1 (Y357) and TEAD1. (F) ChIP-qPCR analysis using TEAD1 antibody to confirm protein-crosslinked genomic DNA fragments. (G) Western blot showing protein expression of ACSL1, ACSL4, DGAT1, DGAT2, LPIN2 and PNPLA3 in spheroid culture treated without or with 10 μM verteporfin (VP) for 24 h. (H and I) Assessment of TAG levels and cell viability in spheroid A549 cells treated without (CTR) or with 10 μM VP. (J) Western blot showing ACSL1, ACSL4, DGAT1, DGAT2, LPIN2 and PNPLA3 protein expression in spheroid A549 cells with YAP1 or TEAD1 knockdown. (K) Reduced TAG levels in spheroid A549 cells with YAP1 or TEAD1 knockdown. (L) Cell viability of spheroid A549 cells treated with or without YAP1 or TEAD1 knockdown, followed by treatment with H 2 O 2 (250 μM) for 24 h. Data are presented as mean ± SD (student's two-tailed unpaired t -test is employed in F and G; one-way ANOVA followed by Tukey's post-hoc test is employed in K and L). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: DGAT1 inhibitor (PF-04620110) and DGAT2 inhibitor (PF-06424439) were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: RNA Sequencing, Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Proximity Ligation Assay, Protein-Protein interactions, ChIP-qPCR, Knockdown, Two Tailed Test