Journal: Cell Death & Disease

Article Title: Akt kinase LANCL2 functions as a key driver in EGFR -mutant lung adenocarcinoma tumorigenesis

doi: 10.1038/s41419-021-03439-8

Figure Lengend Snippet: a LANCL2 mRNA expression levels in PC9, HCC827, and A549 cells. b Immunoblotting analysis of RPS4X, LANCL2, and EIF3C protein expression in PC9 cells following vehicle control (Ctrl) or 10 nM gefitinib + 20 nM pemetrexed (Gef + PTX) combination therapy. c mRNA and d protein expression levels of LANCL2 in shLANCL2- or shCtrl-infected PC9 cells. e CCK-8 cell proliferation assay (raw OD450 data (left) and relative fold-change in OD450 (right)) and f flow cytometry analysis of apoptosis using Annexin V staining in shLANCL2- and shCtrl-infected PC9 cells. g Caspase-3/7 activity in shLANCL2- and shCtrl-infected PC9 cells. * p < 0.05; ** p < 0.01; and *** p < 0.001 vs. shCtrl. Data presented as means ± SDs. All in vitro experiments: three biological replicates × three technical replicates.

Article Snippet: The gefitinib-sensitive human EGFR -mutant LUAD cell lines PC9 (EGFR p.Glu746_Ala750del) and HCC827 (EGFR p.Glu746_Ala750del) , as well as the non- EGFR -mutant LUAD cell line A549 were purchased from American Type Culture Collection.

Techniques: Expressing, Western Blot, Control, Infection, CCK-8 Assay, Proliferation Assay, Flow Cytometry, Staining, Activity Assay, In Vitro

Journal: Cell Death & Disease

Article Title: Akt kinase LANCL2 functions as a key driver in EGFR -mutant lung adenocarcinoma tumorigenesis

doi: 10.1038/s41419-021-03439-8

Figure Lengend Snippet: a Volcano plot analysis of gene chip array results revealed 496 upregulated and 839 downregulated DEGs after LANCL2 knockdown (DEG criteria: p < 0.05; absolute fold-change >2.0). b Heatmap of DEGs after LANCL2 knockdown. Each row refers to one DEG. Each column represents one sample. The legend depicts the color code for Row Z-score fold-change values. c IPA interaction network analysis of DEGs detecting putative regulatory interactors of LANCL2. Note the highlighted putative interactions between LANCL2-EGFR, LANCL2-APP, and LANCL2-Akt. d IPA upstream regulator analysis of DEGs evidencing putative transcriptional regulators of LANCL2. e IPA downstream effects analysis of DEGs identifying putative dysregulated biological functions.

Article Snippet: The gefitinib-sensitive human EGFR -mutant LUAD cell lines PC9 (EGFR p.Glu746_Ala750del) and HCC827 (EGFR p.Glu746_Ala750del) , as well as the non- EGFR -mutant LUAD cell line A549 were purchased from American Type Culture Collection.

Techniques: Knockdown

Journal: Open Medicine

Article Title: STK11 (LKB1) mutation suppresses ferroptosis in lung adenocarcinoma by facilitating monounsaturated fatty acid synthesis

doi: 10.1515/med-2023-0845

Figure Lengend Snippet: qRT-PCR primers

Article Snippet: STK11 wild-type LUAD cell lines (Calu-3, H1299) and STK11-mutant LUAD cell lines (A549, H23, H2030) were got from American Type Culture Collection (ATCC, USA).

Techniques: Sequencing

Journal: Open Medicine

Article Title: STK11 (LKB1) mutation suppresses ferroptosis in lung adenocarcinoma by facilitating monounsaturated fatty acid synthesis

doi: 10.1515/med-2023-0845

Figure Lengend Snippet: STK11 mutation causes aberrant gene expression and increased expression of ferroptosis inhibition-related proteins. (a) Correlation between STK11 and key ferroptosis inhibition genes; (b) qRT-PCR analysis of STK11 expression in different cell lines; and (c) WB analysis of STK11, SLC7A11, and GPX4 expression levels in cells; * indicates P < 0.05.

Article Snippet: STK11 wild-type LUAD cell lines (Calu-3, H1299) and STK11-mutant LUAD cell lines (A549, H23, H2030) were got from American Type Culture Collection (ATCC, USA).

Techniques: Mutagenesis, Gene Expression, Expressing, Inhibition, Quantitative RT-PCR

Journal: Open Medicine

Article Title: STK11 (LKB1) mutation suppresses ferroptosis in lung adenocarcinoma by facilitating monounsaturated fatty acid synthesis

doi: 10.1515/med-2023-0845

Figure Lengend Snippet: STK11 mutation suppresses ferroptosis in LUAD. (a) TEM observation of mitochondrial morphological changes; (b) CCK-8 analysis of cell viability; (c) flow cytometry analysis of cell apoptosis; (d) GSH assay kit analysis of GSH levels in cells; (e) MDA assay kit analysis of lipid peroxidation levels; (f) ROS assay kit analysis of ROS levels in cells; and (g) Fe 2+ assay kit analysis of Fe 2+ levels in cells; * indicates P < 0.05.

Article Snippet: STK11 wild-type LUAD cell lines (Calu-3, H1299) and STK11-mutant LUAD cell lines (A549, H23, H2030) were got from American Type Culture Collection (ATCC, USA).

Techniques: Mutagenesis, CCK-8 Assay, Flow Cytometry, GSH Assay, Multiple Displacement Amplification, ROS Assay

Journal: Open Medicine

Article Title: STK11 (LKB1) mutation suppresses ferroptosis in lung adenocarcinoma by facilitating monounsaturated fatty acid synthesis

doi: 10.1515/med-2023-0845

Figure Lengend Snippet: STK11 mutation affects MUFA synthesis to suppress cell ferroptosis. (a) Correlation between STK11 and SCD1; (b) oil red O staining to measure LD content; (c) MUFAs assay kit to quantify MUFA levels; (d) WB analysis of MUFA synthesis-related enzyme protein expression; (e) Flow cytometry analysis of cell apoptosis; (f) GSH assay kit to measure GSH levels in cells; (g) MDA assay kit to measure MDA levels; (h) ROS assay kit to measure ROS levels in cells; and (i) Fe 2+ assay kit to measure Fe 2+ levels in cells; * indicates P < 0.05.

Article Snippet: STK11 wild-type LUAD cell lines (Calu-3, H1299) and STK11-mutant LUAD cell lines (A549, H23, H2030) were got from American Type Culture Collection (ATCC, USA).

Techniques: Mutagenesis, Staining, Expressing, Flow Cytometry, GSH Assay, Multiple Displacement Amplification, ROS Assay

Journal: Molecular cancer therapeutics

Article Title: Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy

doi: 10.1158/1535-7163.MCT-20-0024

Figure Lengend Snippet: YAP1 promotes STAT3 phosphorylation via IL-6 upregulation. A, Immunoblotting shows YAP1, STAT3 and pSTAT3 expressions in YAP1-deficient (H1299 and H1437) and YAP1-overexpressing (A549) LUAD cells. YAP1 expression was positively associated with pSTAT3 expression. B, qRT-PCR data show IL-6 mRNA expression to be positively associated with YAP1 expression in H1299, H1437 and A549 cell lines. C, Secretion of IL-6 into media was significantly reduced in YAP1-sh cells, whereas higher levels were observed in YAP1-LV cells, as measured by ELISA in cell culture supernatant. Immunoblotting of pSTAT3 and β-actin in the respective cell lines are shown below the graph. D, Addition of recombinant human IL-6 (0.1, 1 or 10 ng/ml) to H1299 and H1437 YAP1-sh cells increased pSTAT3 levels dose-dependently. E, Blocking IL-6 activity with IL-6 neutralizing antibody (1 μg/ml) inhibited pSTAT3 in a time-dependent manner without influencing total STAT3 expression in A549 YAP1-LV cells. F, ChIP-PCR assay with YAP1 antibody was conducted using H1299, H1437 and A549 cells. DNA isolation followed by PCR showed that YAP1 protein directly binds to the IL-6 gene promoter region in three LUAD cells analyzed (white arrows). “Input” indicates DNA lysate before ChIP, which was diluted to 2% for PCR reactions. Histone H3 antibody and normal rabbit IgG served as positive and negative control, respectively. G, Schematic shows YAP1 binding to IL-6 promoter and upregulating its transcription to induce STAT3 phosphorylation. Error bars: mean ± SEM. *P<0.05, **P<0.001.

Article Snippet: To assess correlations among mRNA levels, we collected pathologically confirmed LUAD samples from 59 patients who underwent resections at the Johns Hopkins Hospital (Baltimore, MD, USA), the Johns Hopkins Bayview Medical Center (Baltimore, MD, USA), and the Medical College of Wisconsin Froedtert Memorial Hospital (Milwaukee, WI, USA) ( 20 ).

Techniques: Phospho-proteomics, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Blocking Assay, Activity Assay, DNA Extraction, Negative Control, Binding Assay