Journal: Oncotarget

Article Title: Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ β-arrestin-1 dependent manner in NSCLC

doi:

Figure Lengend Snippet: Microarray was performed in ARRB1 depleted and nicotine stimulated A549 cells Nicotine induced and ARRB1 dependent genes from the microarray data were analyzed. We identified differentially regulated genes that were regulated by nicotine in a β-arrestin-1 dependent fashion and top 10 up/down regulated genes from the list were used for prognosis prediction. Assessment of the expression of these genes for smoking revealed that SCF (highlighted in red) strongly differentiated smokers from non-smokers implying an important role of this gene in lung carcinogenesis induced by smoking

Article Snippet: A human lung cancer tissue microarray slide (Catalog number IMH-358; Imgenex, San Diego, CA) was immunostained for SCF.

Techniques: Microarray, Expressing, Control

Journal: Translational Oncology

Article Title: Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis

doi: 10.1016/j.tranon.2019.09.001

Figure Lengend Snippet: Increased vasorin expression in human lung cancer and HBECs exposed to tobacco carcinogens . (A) Tissue arrays were stained for vasorin with immunohistochemistry. The normal airway and alveolar epithelial cells are weakly positive while the tumor cells are strongly positive. (B) Results of tissue arrays with comparison of the staining between vasorin and normal lung epithelial cells. (C) Vasorin was examined in nontransformed (HBEC-1 and -2) and lung cancer cell lines. Relative vasorin fold increase (HBEC-2 was set to 1) was shown. (D) BEAS-2B and HBEC-2 cells were treated with CSE for 24 hours. (E) HBEC-2B (BPDE-transformed) cells were compared with HBEC-2 cells for vasorin expression. Protein was detected by western blot in C to E with β-actin as loading control.

Article Snippet: Immunohistochemistry staining using VECTASTAIN ABC Kit and DAB (3,3′-diaminobenzidine) Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA) and result assessment of human lung cancer tissue array (Imgenex; Novus Biologicals, Centennial, CO) has been described previously [ ].

Techniques: Expressing, Staining, Immunohistochemistry, Comparison, Transformation Assay, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 gene and protein expression is downregulated in patients with lung cancer from The Cancer Gene Atlas, Gene Expression Omnibus and Human Protein Atlas databases, as well as in CS-exposed cells. (A) Differential analysis of YTHDC2 mRNA expression in lung cancer tissues based on the Gene Expression Profiling Interactive Analysis tool. * P<0.05 vs. normal tissues. Differential analysis of YTHDC2 mRNA expression in lung cancer tissues from (B) GSE32665 and (C) GSE19188 datasets. (D) Representative IHC images showed that YTHDC2 staining was found in the cell cytoplasm in lung cancer and normal lung tissues. High expression of YTHDC2 could be found in adjacent normal tissues, while its expression was decreased in the majority of lung cancer tissues. (E) Differential analysis of YTHDC2 staining positive ratio quantitated by IHC Profiler in lung cancer tissue arrays. YTHDC2 staining positive ratio in lung cancer tissues with different (F) maximum diameter, (G) pathological stage and (H) invasion depth. (I) Relative mRNA expression level of YTHDC2 in CS-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. Western blot analysis (J) and quantitative results (K) of YTHDC2 protein expression in CS-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. S10, S20 and S30 represent BEAS-2B cells exposed to CS for 10, 20 and 30 passages, respectively. **P<0.01 vs. normal BEAS-2B cells. IHC, immunohistochemistry; YTHDC2, YTH domain containing 2; CS, cigarette smoke.

Article Snippet: Human lung cancer tissue arrays (cat. no. LAC-1402 and LAC-1403) were purchased from Wuhan Servicebio Technology Co., Ltd.

Techniques: Expressing, Gene Expression, Staining, Western Blot, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 mRNA expression was regulated by gene amplification. Distribution of patients with (A) LUAD and (D) LUSC with different YTHDC2 amplification status. YTHDC2 mRNA expression in (B) LUAD and (E) LUSC tissues with different YTHDC2 amplification status. Different letters (a, b, c and d) represent statistically significant group differences. Pearson's correlation analysis revealed a significant positive correlation between YTHDC2 mRNA expression and copy numbers in (C) LUAD and (F) LUSC. The line represents linear regression of data (LUAD: y=1.065x+9.177, R 2 =0.385; LUSC: y=0.965x+9.318, R 2 =0.198). (G) The Oncomine datasets for the corresponding YTHDC2 copy numbers in lung cancer were obtained with a threshold P=0.001 and ≥2 fold-change. The data in the graphic show significant downregulation (blue column) of YTHDC2 copy numbers in lung cancer versus normal tissue. The intensity of the blue color represents the respective levels of YTHDC2 copy number. (H) Copy number variation in LUAD samples with different smoking histories. Different letters (a, b, c and d) represent statistically significant group differences. (I) Copy number variation of YTHDC2 in BEAS-2B cells and cigarette smoke-exposed cells (grey block), as well as in two lung cancer cell lines (black block). The dotted line (copy number = 2) represents the copy number of the reference gene RNase P. * P<0.05, ** P<0.01 vs. BEAS-2B cells. YTHDC2, YTH domain containing 2; LUSC, lung squamous cell carcinoma; LUAD , lung adenocarcinoma.

Article Snippet: Human lung cancer tissue arrays (cat. no. LAC-1402 and LAC-1403) were purchased from Wuhan Servicebio Technology Co., Ltd.

Techniques: Expressing, Amplification, Blocking Assay

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) Scatter plot shows an upregulation of the PP2A-A subunit in the tumor samples (p=0.0144). A Mann-Whitney U test was used for comparison between the normal and SCLC samples. (B) IHC for PP2A was conducted on TMA tissue sections and images were captured at 4x or 20x using a 3D-Histech PANNORAMIC SCAN whole slide scanner (3D-Histech, Budapest, Hungary). PP2A subunit A positively immunostained the cytoplasm and nucleus of normal lung and tumor tissue but was highly upregulated in tumor tissue. TMAs were scored in normal (n=24) and tumor (n=79) cores on a scale from 0 (no staining/no protein expression) to 3+ (strong staining/high protein expression). (C) Summary bar graph of the average PP2A subunit staining. IHC staining intensity of normal and tumor cores. There was a statistically significant difference between normal and tumor tissue (***, p<0.001). Student’s t test was used for comparison between the normal and SCLC samples. (D) In order to compare the expression of PP2A subunits A and C, cell lysates from seven SCLC cell lines and HBEC 3KT (non-malignant cell line) were subjected to western blotting (n=3 biological replicates). (E) PP2A activity was determined using a serine/threonine phosphatase activity assay (Millipore) after 24 h exposure to cantharidin (10 µM) and LB100 (5 µM) (n=3 biological replicates). ***, p<0.001, results were analyzed by ANOVA with Tukey post-test. (F) The inset showed reduction of PP2A subunit Aα in H524 cells as well as inhibition of cell proliferation due to PP2A subunit Aα knockdown (n=3 biological replicates). p<0.05, Student’s t test was used for comparison between the groups. LB100 alone or in combination with carboplatin inhibited proliferation and colony formation in SCLC cells. The Cell Counting Kit-8 assay detected cell H524 and H69 cell viability. (n=3 biological replicates). (G, H) Cells were treated with LB100, carboplatin and etoposide, as a single treatment or in combination, at constant ratio. The combination index (CI) was calculated using Chou-Talalay method to find synergism between LB100 with carboplatin and etoposide (CompuSyn software: www.combosyn.com). **, p<0.01, ANOVA with Tukey post-test was used for comparison between LB100, LB100/carboplatin and LB100/etoposide. Colony formation assays were used to count the ability of H524 (I) and H69 (J) cells to form colonies. Drug concentrations are listed for two assays with H524 and H69 respectively: LB100 (2.5 µM; 20 µM), carboplatin (4 µM; 20 µM), etoposide (3 µM; 30 µM), LB100/carboplatin (2.5&4 µM; 20&20 µM) and LB100/etoposide (2.5&3 µM; 20&30 µM). Representative images of colonies at 4x are shown under the graph (n=2). *p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Results were analyzed by ANOVA with Tukey post-test.

Article Snippet: Tissue Microarray Small cell lung cancer TMAs were from US Biomax Inc. (Rockville, MD; LC818).

Techniques: MANN-WHITNEY, Comparison, Staining, Expressing, Immunohistochemistry, Western Blot, Activity Assay, Phosphatase Assay, Inhibition, Knockdown, Cell Counting, Software

Journal: Molecular cancer therapeutics

Article Title: Protein phosphatase 2A as a therapeutic target in small cell lung cancer

doi: 10.1158/1535-7163.MCT-21-0013

Figure Lengend Snippet: (A) Significant changes were observed for signal transduction and metabolic pathways. (B) MicroArray analysis showed that overnight treatment with 20 µM treatment with LB100 inhibited utilization of carbon substrate sources. Table includes 10 carbon sources affected by LB100 (n=3). (C) LB100 significantly inhibited two carbon substrates utilization by H69 cells. P < 0.001 (***) for control (untreated cells) vs. LB100. Results were analyzed by ANOVA with Tukey post-test. (D) Amplex Red Glucose/Oxidase assay kit was used to measure glucose level in cell culture media. Glucose level was significantly higher in cell culture medium from cells treated with LB100 (20 µM). Glucose concentration detected in initial medium and counted as 100%. Subtracting final medium level of glucose from initial glucose medium concentration yielded % glucose in the medium with cells. Level of glucose dropped in control with cells and in LB100 treated groups (p < 0.0001 (****). Results were analyzed by ANOVA with Tukey post-test, (n=3 biological replicates). Effect of LB100 on MET phosphorylation. (E) H524 and H69 cells were treated overnight with LB100 (H524 – 5 µM and H69 – 20 µM) following by stimulation with 100 ng/ml HGF in 10 min. Cells were collected and lysed for WB analysis with pMET and total MET antibody. Pan-actin was used as loading control (n=3 biological replicates). (F) H524 cell lysates (control, LB100, carboplatin and combination (LB100/carboplatin) were analyzed by western blots to check phosphorylation status of MET at Ser985 and Tyr1234/1235. Actin was used as a loading control (n=3 biological replicates).

Article Snippet: Tissue Microarray Small cell lung cancer TMAs were from US Biomax Inc. (Rockville, MD; LC818).

Techniques: Transduction, Microarray, Control, Glucose Oxidase Assay, Cell Culture, Concentration Assay, Western Blot