Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The knockdown of FGL1 and RNA-seq results. (A) The knockdown of FGL1 confirmed by western blot. (B) The knockdown of FGL1 confirmed by immunofluorescent staining. (C) Volcano map of differential gene changes in PC9 cells after FGL1 knockdown. (D) Volcano map of differential gene changes in Jurkat T cells after FGL1 knockdown. (E) The enrichment of differential genes in PC9 cells, analyzed by GSEA. **, P<0.01; ***, P<0.001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: PC9 lung cancer cells and HCC827 lung cancer cells (human; iCell Bioscience Inc., Shanghai China) were cultured in RPMI 1640 (Gibco, Shanghai, China) with 10% fetal calf serum at 37 °C in 5% CO 2 .

Techniques: Knockdown, RNA Sequencing, Western Blot, Staining, Negative Control

Journal: Translational Lung Cancer Research

Article Title: The downregulation of fibrinogen-like protein 1 inhibits the proliferation of lung adenocarcinoma via regulating MYC -target genes

doi: 10.21037/tlcr-22-151

Figure Lengend Snippet: The experiments related to cell proliferation. (A,B) Effects of FGL1 knockdown on the cycle of PC9 cells and Jurkat T cells. (C) Real-time cell analyzer presents the real-time condition of cell proliferation. (D) Colony formation confirms the cell proliferation of PC9 cells and HCC827 cells, the magnifications in the figure are 40× and 200×, respectively, both of them were stained with crystal violet. **, P<0.01; ***, P<0.001; ****, P<0.0001. NC, negative control; KD, knockdown; LUAD, lung adenocarcinoma; FGL1, fibrinogen-like protein 1.

Article Snippet: PC9 lung cancer cells and HCC827 lung cancer cells (human; iCell Bioscience Inc., Shanghai China) were cultured in RPMI 1640 (Gibco, Shanghai, China) with 10% fetal calf serum at 37 °C in 5% CO 2 .

Techniques: Knockdown, Staining, Negative Control

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 expression is induced in EGFR‐mutant NSCLC cells upon EGFR‐TKI treatment. (A) Differential gene expression between EGFR‐TKI‐treated versus ‐untreated EGFR‐mutant NSCLC cells in GSE75308 (left) and GSE57156 (right) were analyzed. (B) EGFR‐mutant PC9 and H1975 cells, and EGFR‐wild‐type RERF‐LC‐Ad1 and H522 cells were treated with osimertinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Gray: isotype control; dotted line: untreated; line: treated. Data are representative of four independent experiments. (C) Numbers of CD24 molecules expressed on cells with or without EGFR‐TKI treatment in vitro. Data are presented as mean ± SEM from four independent experiments. Numbers of the receptors were calculated using the BD QuantiBrite kit as described in Materials and Methods. (D) CD24 gene expression in tumor cells with or without EGFR‐TKI treatment in vitro were analyzed by qRT‐PCR. Data are presented as mean ± SEM of technical replicates from at least two independent experiments. (E) EGFR‐mutant PC9 and EGFR‐wild‐type H522 cells were treated either with osimertinib, gefitinib, or afatinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Data are representative of two independent experiments. * q < 0.01.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Mutagenesis, Gene Expression, In Vitro, Flow Cytometry, Control, Quantitative RT-PCR

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 induction in EGFR‐mutant NSCLC cells is a target of antibody‐dependent cellular phagocytosis. (A) The fraction of immune cell types in the tumor microenvironment of NSCLC from the TCGA‐LUAD dataset (B) Arbitrary gene expression levels of SIGLEC‐10 and MRC1 across macrophages in the tumor microenvironment of NSCLC (C) Siglec‐10 expression was analyzed by flow cytometry. Gray: isotype control, line: Siglec‐10. Human monocyte‐derived macrophages were differentiated according to the protocols shown in Supplementary Table . Data are a representative of at least two independent experiments from three healthy donors. (D) M‐CSF differentiated macrophages and EGFR‐TKI‐treated PC9 cells were incubated for 9 h with anti‐CD24 antibodies or isotype control antibodies. Cytochalasin D was used to inhibit macrophage phagocytosis as described in Materials and Methods. Data are presented as the mean ± SEM of technical replicates from at least two independent experiments. *** p < 0.001.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Gene Expression, Expressing, Flow Cytometry, Control, Derivative Assay, Incubation

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: EGFR‐TKIs accelerate the release of the cell‐free DNA activating type I IFN response in a STING‐dependent manner. (A) EGFR‐mutant tumor cells were treated with either osimertinib (EGFR‐TKI) or paclitaxel. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (B) cfDNA concentrations in the supernatant were measured as described in Materials and Methods. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (C) THP‐1‐Dual and THP‐1‐Dual STING‐knockout (KO) cells were cultured either with LPS (500 ng/ml), IFNa2a (10,000 U/ml), tumor‐derived cfDNA (PC9 and H1975 cfDNA)/lipofectamine, or lipofectamine alone. IRF (left) and NF‐κB (right) activities were analyzed as described in Materials and Methods. (D) PMA‐differentiated THP‐1 monocytes were pretreated either with lipofectamine, poly(dA:dT) (100 ng/ml), tumor‐derived cfDNA (PC9) (100 ng/ml)/lipofectamine, or IFNa2a (20,000 U/ml) followed by IFN‐γ stimulation (100 ng/ml). (E) Expression levels of CXCL9 and CXCL10 were analyzed using flow cytometry. * p < 0.05, ** p < 0.01, **** p < 0.0001, * q < 0.01. ns, not significant.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Knock-Out, Cell Culture, Derivative Assay, Expressing, Flow Cytometry

Journal:

Article Title: Tumorigenic Role of Orphan Nuclear Receptor NR0B1 in Lung Adenocarcinoma

doi: 10.2353/ajpath.2009.090010

Figure Lengend Snippet: High NR0B1 expression in SP of A549. A: Dot blot analysis of A549 cells stained with Hoechst 33342 dye in the absence (left) or presence (right) of verapamil. SP and MP cells were boxed. B: Quantitative real-time RT-PCR was performed with mRNA obtained from SP and MP of A549. The amount of NR0B1 mRNA was normalized for the amount of GAPDH mRNA. The values represent the mean ± SE of three experiments. *P < 0.01 by the Student’s t-test.

Article Snippet: The human lung adenocarcinoma cell lines A549 and PC-14 were purchased from the American Type Culture Collection (Rockville, MD) and the Riken Bioresource Center (Tsukuba, Japan), respectively.

Techniques: Expressing, Dot Blot, Staining, Quantitative RT-PCR

Journal:

Article Title: Tumorigenic Role of Orphan Nuclear Receptor NR0B1 in Lung Adenocarcinoma

doi: 10.2353/ajpath.2009.090010

Figure Lengend Snippet: Expression of apoptosis- and invasion-related genes in NR0B1-knocked down A549 cells. A: Semiquantitative RT-PCR. B: Real-time quantitative RT-PCR to examine the expression level of Bcl-2 and MMP-2. The value is shown as the mean ± SE. *P < 0.01 by the Student’s t-test.

Article Snippet: The human lung adenocarcinoma cell lines A549 and PC-14 were purchased from the American Type Culture Collection (Rockville, MD) and the Riken Bioresource Center (Tsukuba, Japan), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cancers

Article Title: Nicotine Induces Resistance to Erlotinib Therapy in Non-Small-Cell Lung Cancer Cells Treated with Serum from Human Patients

doi: 10.3390/cancers11030282

Figure Lengend Snippet: Treatment of ( a ) PC9 and ( b ) HCC827 cells with serum from a smoker reduces sensitivity to erlotinib therapy. Treatment of cells for 72 h with 1 μM erlotinib and serum from smoker No. 4 (serum cotinine level: 488.4 ng/mL) resulted in a significant reduction of sensitivity to erlotinib compared with serum from a non-smoker control (serum cotinine level: 0.6 ng/mL) in both cell lines (** p < 0.001). Cell survival was assessed by using a cell-counting kit (CCK)-F. Results are means ± SEM of four independent experiments.

Article Snippet: We purchased PC9 cells from the RIKEN BioResource Center (Ibaraki, Japan), and obtained the HCC827 cells from Kyushu University (Fukuoka, Japan).

Techniques: Control, Cell Counting

Journal: Cancers

Article Title: Nicotine Induces Resistance to Erlotinib Therapy in Non-Small-Cell Lung Cancer Cells Treated with Serum from Human Patients

doi: 10.3390/cancers11030282

Figure Lengend Snippet: Comparisons of ( a ) PC9 and ( b ) HCC827 cell lines cultured for 72 h with various concentrations of erlotinib (0, 0.1, and 1 μM), and serum from the non-smoker and smoker No. 4. Serum from the smokers demonstrated significant resistance to erlotinib treatment at all concentrations in both cell lines, compared with serum from the non-smoker (at 1 μM erlotinib in the PC9 cell, p = 0.0018; for all other comparisons, p < 0.001). Cell survival was assessed using a cell counting kit (CCK)-F. Results are means ± SEM of four independent experiments. ( c ) Immunoblot analysis of PC9 cells incubated with erlotinib (1 μM), and serum from the non-smoker or smoker No. 4 for 1 h. The combination of erlotinib with serum from the smoker elevated the protein levels of the phosphorylated AKT (Ser 473) considerably. AKT phosphorylation was inhibited by erlotinib and serum from the non-smoker. Erlotinib inhibited the phosphorylation of EGFR and ERK, independent of serum addition. The control is untreated cells.

Article Snippet: We purchased PC9 cells from the RIKEN BioResource Center (Ibaraki, Japan), and obtained the HCC827 cells from Kyushu University (Fukuoka, Japan).

Techniques: Cell Culture, Cell Counting, Western Blot, Incubation, Phospho-proteomics, Control

Journal: Cancers

Article Title: Nicotine Induces Resistance to Erlotinib Therapy in Non-Small-Cell Lung Cancer Cells Treated with Serum from Human Patients

doi: 10.3390/cancers11030282

Figure Lengend Snippet: Comparison between smokers No. 1 and 4 with the lowest and highest serum cotinine levels (33.0 and 488.4 ng/mL), respectively. Serum with the highest levels showed stronger resistance to erlotinib therapy over 72 h. ( a ) PC9 cells treated with 0.1 and 1 μM erlotinib, p = 0.8077 and 0.4242, respectively. ( b ) HCC827 cells treated with 0.1 and 1 μM erlotinib, ** p < 0.001. Cell survival was assessed using a cell counting kit (CCK)-F. The Results are means ± SEM of four independent experiments.

Article Snippet: We purchased PC9 cells from the RIKEN BioResource Center (Ibaraki, Japan), and obtained the HCC827 cells from Kyushu University (Fukuoka, Japan).

Techniques: Comparison, Cell Counting

Journal: Cancers

Article Title: Nicotine Induces Resistance to Erlotinib Therapy in Non-Small-Cell Lung Cancer Cells Treated with Serum from Human Patients

doi: 10.3390/cancers11030282

Figure Lengend Snippet: Comparisons of PC9 cell lines cultured for 72 h with various concentrations of ( a ) afatinib and ( b ) osimertinib (0, 0.1, and 1 μM) and serum from the non-smoker and smoker No. 4. Serum from the smoker demonstrated significant resistance to afatinib and osimertinib treatment, compared with serum from non-smoker ( p < 0.001). Cell survival was assessed using a cell counting kit (CCK)-F. Results are means ± SEM of five independent experiments.

Article Snippet: We purchased PC9 cells from the RIKEN BioResource Center (Ibaraki, Japan), and obtained the HCC827 cells from Kyushu University (Fukuoka, Japan).

Techniques: Cell Culture, Cell Counting

Journal: Cancers

Article Title: Nicotine Induces Resistance to Erlotinib Therapy in Non-Small-Cell Lung Cancer Cells Treated with Serum from Human Patients

doi: 10.3390/cancers11030282

Figure Lengend Snippet: Treatment of PC9 cells with serum from smoker-induced erlotinib resistance following treatment with serum from an erlotinib user. Analysis of PC9 cells treated with serum from a non-smoker or smoker No. 4 for 24 h, followed by serum from erlotinib users (1:10 or 1:2 dilutions, or undiluted) for 120 h. Serum from smokers significantly reduced erlotinib sensitivity (** p < 0.001). Cell survival was assessed using a cell counting kit (CCK)-F. Results are means ± SEM of four independent experiments.

Article Snippet: We purchased PC9 cells from the RIKEN BioResource Center (Ibaraki, Japan), and obtained the HCC827 cells from Kyushu University (Fukuoka, Japan).

Techniques: Cell Counting