Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Morphology of cells after treatment with gefitinib in PC9 and PC9/N cells. Cells were seeded in six-well culture plates, and after 24 hrs, the cells were treated with gefitinib (0, 0.1, or 1 µM) for 48 hrs. Cells were photographed under a phase contrast inverted microscope. Cells were stained with Wright and Giemsa (original magnification 200×). These results are representative of technical triplicate.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Inverted Microscopy, Staining

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Cell viability assay for PC9 and PC9/N cells cultured in the presence of gefitinib. Cell growth inhibition in response to gefitinib was evaluated by using the MTT assay. Cells were treated with the indicated concentrations of gefitinib and cell viability was determined 48 hrs later. There are mean of independent triplicate experiments. The data are presented as means ± SEM from technical triplicate. PC9/N: nicotine (1 µM) expose for 3 months in PC9 cells. *, P<0.05; **, P<0.01 and ***, P<0.001 compared with PC9 + gefitinib 0 µM. ## , P<0.01 and ### , P<0.001 compared with PC9/N + gefitinib 0 µM. $ , P<0.05 between two cells with gefitinib 0.01 µM. Data are presented as mean ± standard error of the mean (SEM). SEM, standard error.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Viability Assay, Cell Culture, Inhibition, MTT Assay

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: The mRNA expression of cells after treatment with gefitinib in PC9 and PC9/N cells. Cells were treated with gefitinib (0, 0.1, or 1 µM) for 48 hrs, and the mRNA expressions of α1-nAchR, and PD-L1 were examined by quantitative reverse transcription (qRT)-PCR (A) and RT-PCR (B). These results are representative of technical triplicate. *, P<0.05 and ***, P<0.001 compared with PC9 cells. ### , P<0.001 compared with PC9 cells + gefitinib 0 µM and $$$ , P<0.001 compared with PC9/N + gefitinib 0 µM. nAchR, nicotinic acetylcholine receptors; PD-L1, programmed death ligand 1.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

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

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: The protein expression levels of cells after treatment with gefitinib in PC9 and PC9/N cells. The cells were cultured with gefitinib (0, 0.1, or 1 µM) for 48 hrs, and EGFR, mTOR, AKT, PD-L1 and α1-nAchR was detected by Western blot. β-actin was used as an internal control. Western blot was quantified by densitometry and ImageJ. These results are representative of technical triplicate. EGFR, epidermal growth factor receptor; PD-L1, programmed death ligand 1; nAchR, nicotinic acetylcholine receptors.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Immunofluorescence staining of PD-L1 and phosphorylation of EGFR in PC9 and PC9/N cells treated with 0.1 µM gefitinib. The localizations of PD-L1 and p-EGFR (green signal; Alexa488) in PC9 and PC9/N cells were shown by immunofluorescence counterstained with DAPI (blue signal) and analyzed by confocal microscopy (original magnification 400×). These results are representative of technical triplicate. DAPI, 4',6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Immunofluorescence, Staining, Phospho-proteomics, Confocal Microscopy

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Overall proportion of PD-L1 TPS expression according to pack-year in NSCLC patients harboring activating EGFR mutation. Heavy smokers ( ≥ 30 PY) tended to have higher expression ( ≥ 50% PD-L1 TPS) than never and light smokers, however, Fisher exact test showed no statistical significance (P value =0.628). PD-L1, programmed death ligand 1; TPS, tumor proportion score; NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor receptor; PY, pack-year.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Expressing, Mutagenesis

Journal: Technology in Cancer Research & Treatment

Article Title: KRT6A Promotes EMT and Cancer Stem Cell Transformation in Lung Adenocarcinoma

doi: 10.1177/1533033820921248

Figure Lengend Snippet: Keratin 6A knockdown undermine the proliferation and migration ability of lung cancer cells. A and B, KRT6A was knocked down by siRNA, siRNA significantly knocked down KRT6A mRNA expression and protein level in A549 and PC-9 cell lines (the Western blots were quantified by Image J). C and D, CCK8 assay shown si-KRT6A could significantly undermine the proliferation of A549 and PC-9 cells. E, Would healing assay shown that KRT6A knockdown significantly undermined the migration ability in lung cancer cells. F, By measuring the supernatant LDH level, the cell death rate was determined, no significant difference was found between si-KRT6A and NC groups, which indicate KRT6A knockdown did not influence the cell death rate between NC and KRT6A knockdown group. CCK8 indicates Cell Counting Kit-8; KRT6A, keratin 6A; mRNA, messenger RNA; NC, negative control; siRNA, small interfering RNA.

Article Snippet: The lung cancer cell line, A549 was obtained from the ATCC (American Type Culture Collection) and PC-9 was obtained from NanJing KeyGen Biotech Co, Ltd. A549 and PC-9 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Inc, Waltham, MA; cat. no. 12491-015) supplemented with 10% fetal bovine serum (FBS; cat. no. 10500064; Gibco; Thermo Fisher Scientific, Inc) and penicillin/streptomycin cultured in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Knockdown, Migration, Expressing, Western Blot, CCK-8 Assay, Cell Counting, Negative Control, Small Interfering RNA

Journal: Technology in Cancer Research & Treatment

Article Title: KRT6A Promotes EMT and Cancer Stem Cell Transformation in Lung Adenocarcinoma

doi: 10.1177/1533033820921248

Figure Lengend Snippet: Keratin 6A is involved in CSCs transformation. A and B, CXCR4 high /CD133 high cells were identified as CSCs, compared with si-KRT6A groups, NC groups have significantly expanded CSCs population (Q2). C and D, The colony formation assay shown that compared with si-KRT6A A549 and PC-9 cells the normal control cancer cells (NC groups) have greater biological advantage in forming colonies. CSC indicates cancer stem cell; KRT6A, keratin 6A; NC, negative control.

Article Snippet: The lung cancer cell line, A549 was obtained from the ATCC (American Type Culture Collection) and PC-9 was obtained from NanJing KeyGen Biotech Co, Ltd. A549 and PC-9 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Inc, Waltham, MA; cat. no. 12491-015) supplemented with 10% fetal bovine serum (FBS; cat. no. 10500064; Gibco; Thermo Fisher Scientific, Inc) and penicillin/streptomycin cultured in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Transformation Assay, Colony Assay, Control, Negative Control

Journal: Cell Death & Disease

Article Title: ZNF32 contributes to the induction of multidrug resistance by regulating TGF- β receptor 2 signaling in lung adenocarcinoma

doi: 10.1038/cddis.2016.328

Figure Lengend Snippet: ZNF32 is upregulated by Sp1 in response to drug induction. ( a ) Immunohistochemistry (IHC) showing ZNF32 expression in human lung adenocarcinoma (AC) tissues and adjacent normal (AN) lung tissues from 52 patients. ( b ) qRT-PCR and immunoblot detection of ZNF32 expression in the cisplatin (CIS)-resistant cell line A549/CIS and the gefitinib (GEF)-resistant cell line PC9/GEF compared with wild-type cells. ( c ) A549 and PC9 cells were treated with CIS (10 μ M) or GEF (10 μ M), and ZNF32 expression was detected. ( d ) HEK293 cells were transfected with ZNF32 promoter constructs, treated with 10 μ M CIS for 24 h, and then analyzed using a dual-luciferase reporter assay. ( e ) A549 cells were transfected with pCGN-Sp1, and ZNF32 expression was then detected by qRT-PCR and immunoblot. ( f ) Nuclear extracts from A549 cells were incubated in biotin-labeled oligonucleotides corresponding to the ZNF32 promoter region −1326/−1302 or −49/−15. The arrow shows the specific DNA-protein complex. NS, non-significant difference. Each column and bar represents the mean±S.D. of three independent experiments. The photograph shows a representative result from three independent experiments

Article Snippet: The human lung adenocarcinoma cell lines A549, PC9 (EGFR mutant) and the human colon cancer cell lines SW480 and SKCO1 (KRAS mutant) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Construct, Luciferase, Reporter Assay, Incubation, Labeling

Journal: Cell Death & Disease

Article Title: ZNF32 contributes to the induction of multidrug resistance by regulating TGF- β receptor 2 signaling in lung adenocarcinoma

doi: 10.1038/cddis.2016.328

Figure Lengend Snippet: ZNF32 overexpression in AC cells confers MDR in AC cells. ( a ) ZNF32-overexpressing (Lv-ZNF32), control (Lv-Vector), ZNF32-knockout (Sh-ZNF32) and control (Sh-NC) A549 and PC9 cells were treated with gradually increasing concentrations of CIS and GEF for 3 days, and the IC50 values of CIS and GEF were compared among the groups. ( b ) The cells were mixed with Matrigel and cultured for 7 days. The colonies were then treated with CIS or GEF (at two times the IC50 values) for 3 days, and the colony inhibition ratios were compared. ( c ) The ratio of dead cells was detected by flow cytometric analysis. Each column and bar represents the mean±S.D. of three independent experiments. The photograph shows a representative result from three independent experiments

Article Snippet: The human lung adenocarcinoma cell lines A549, PC9 (EGFR mutant) and the human colon cancer cell lines SW480 and SKCO1 (KRAS mutant) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Over Expression, Control, Plasmid Preparation, Knock-Out, Cell Culture, Inhibition

Journal: Cell Death & Disease

Article Title: ZNF32 contributes to the induction of multidrug resistance by regulating TGF- β receptor 2 signaling in lung adenocarcinoma

doi: 10.1038/cddis.2016.328

Figure Lengend Snippet: TGF- β signaling is essential for drug resistance induced by ZNF32 overexpression. ( a ) Western blot analysis of MEK/ERK signaling in PC9 cells in both the absence and presence of GEF (10 μ M). ( b ) qRT-PCR detection of TGF- β R2 and TGF- β target gene (CDH2, TAGLN and CYR61) expression in A549 and PC9 cells. ( c ) Western blot analysis of TGF- β R2 expression and SMAD2 (pSMAD2) phosphorylation in PC9 cells. ( d ) In PC9 cells, the combination of ZNF32 overexpression and recombinant TGF- β (10 ng/ml) activates TGF- β and MEK/ERK signaling, and LY2157299 (1 μ M) inhibits TGF- β and the majority of MEK/ERK signaling. ( e ) and ( f ) A 3D colony-forming assay and a flow cytometric analysis confirm that TGF- β can induce resistance in AC cells, whereas LY2157299 can counteract the effect of ZNF32 and cancel this resistance. NS, non-significant difference. Each column and bar represents the mean±S.D. of three independent experiments. The photograph shows a representative result from three independent experiments

Article Snippet: The human lung adenocarcinoma cell lines A549, PC9 (EGFR mutant) and the human colon cancer cell lines SW480 and SKCO1 (KRAS mutant) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Over Expression, Western Blot, Quantitative RT-PCR, Expressing, Phospho-proteomics, Recombinant

Journal: Cell Death & Disease

Article Title: ZNF32 contributes to the induction of multidrug resistance by regulating TGF- β receptor 2 signaling in lung adenocarcinoma

doi: 10.1038/cddis.2016.328

Figure Lengend Snippet: ZNF32 deficiency might exhibit synergistic effects with a TGF- β R inhibitor to augment the anti-tumor effect of drugs and improve patient survival in vivo . Twenty-nine days after the injection of A549 and PC9 cells into the mice, the tumor mass was obtained. ( a ) Volume and weight of the tumor. ( b ) Growth curve of the tumor. ( c ) These samples were sliced and stained with HE to measure the necrosis area. ( d ) When the mice died, the survival time of each group was recorded, and the Kaplan–Meier survival curves for each group were analyzed ( n =10 per group; * P <0.05). NS, non-significant difference. Each column and bar represents the median±S.D. of three independent experiments. The photograph shows a representative result from three independent experiments

Article Snippet: The human lung adenocarcinoma cell lines A549, PC9 (EGFR mutant) and the human colon cancer cell lines SW480 and SKCO1 (KRAS mutant) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: In Vivo, Injection, Staining

Journal: Cancer Management and Research

Article Title: miR-124-3p Regulates FGF2–EGFR Pathway to Overcome Pemetrexed Resistance in Lung Adenocarcinoma Cells by Targeting MGAT5

doi: 10.2147/CMAR.S274192

Figure Lengend Snippet: Characteristics of anti-pemetrexed (PC9-MTA and H1993-MTA) and parent (PC9 and H1993) lung adenocarcinoma cells. ( A ) MTS was used to analyze cell viability. ( B ) IC 50 values for lung adenocarcinoma cells. ( C ) Morphological observation of anti-pemetrexed and parent lung adenocarcinoma cells. ( D ) RT-PCR was used to analyze the expression of miR-124-3p in lung adenocarcinoma cells. *p<0.05 compared with PC9 cells; # p<0.05 compared with H1993 cells.

Article Snippet: Human lung adenocarcinoma cell lines PC9 (mutant EGFR, EGFR-DelE746_A750, BIV10058, BioInnovatise) and H1993 (wild type) were purchased from BNCC (BNCC331211) and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (HyClone, USA) in a constant-temperature incubator (Thermo, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Cancer Management and Research

Article Title: miR-124-3p Regulates FGF2–EGFR Pathway to Overcome Pemetrexed Resistance in Lung Adenocarcinoma Cells by Targeting MGAT5

doi: 10.2147/CMAR.S274192

Figure Lengend Snippet: Effects of differential miR-124-3p expression on the expression of FGF2–EGFR signaling pathway-related proteins in PC9-MTA cells ( A ) and H1993-MTA cells ( B ). *p<0.05 compared with control group; # p<0.05 compared with miR-124-3p group.

Article Snippet: Human lung adenocarcinoma cell lines PC9 (mutant EGFR, EGFR-DelE746_A750, BIV10058, BioInnovatise) and H1993 (wild type) were purchased from BNCC (BNCC331211) and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (HyClone, USA) in a constant-temperature incubator (Thermo, USA).

Techniques: Expressing, Control

Journal: Cancer Management and Research

Article Title: miR-124-3p Regulates FGF2–EGFR Pathway to Overcome Pemetrexed Resistance in Lung Adenocarcinoma Cells by Targeting MGAT5

doi: 10.2147/CMAR.S274192

Figure Lengend Snippet: Dual-luciferase reporter assay system. ( A ) The expression of MGAT5 protein was detected by Western blotting in PC9-MTA cells. ( B ) The expression of MGAT5 protein was detected by Western blotting in H1993-MTA cells. ( C ) Predicted binding sites of miR-124-3p and MGAT5 3′UTR by TargetScan ( http://www.targetscan.org ). ( D ) Dual-luciferase reporter results for recombinant vector of miR-124-3p and target gene MGAT5. *p<0.05 compared with control group; # p<0.05, **p<0.01 compared with miR-124-3p group.

Article Snippet: Human lung adenocarcinoma cell lines PC9 (mutant EGFR, EGFR-DelE746_A750, BIV10058, BioInnovatise) and H1993 (wild type) were purchased from BNCC (BNCC331211) and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (HyClone, USA) in a constant-temperature incubator (Thermo, USA).

Techniques: Luciferase, Reporter Assay, Expressing, Western Blot, Binding Assay, Recombinant, Plasmid Preparation, Control

Journal: Cancer Management and Research

Article Title: miR-124-3p Regulates FGF2–EGFR Pathway to Overcome Pemetrexed Resistance in Lung Adenocarcinoma Cells by Targeting MGAT5

doi: 10.2147/CMAR.S274192

Figure Lengend Snippet: Effects of miR-124-3p targeting MGAT5 on the expression of FGF2–EGFR signaling pathway-related proteins in PC9-MTA cells ( A ) and H1993-MTA cells ( B ). *p<0.05 compared with control group; # p<0.05 compared with miR-124-3p group; & p<0.05 compared with sh-NC group; ^ p<0.05 compared with miR+M-NC group.

Article Snippet: Human lung adenocarcinoma cell lines PC9 (mutant EGFR, EGFR-DelE746_A750, BIV10058, BioInnovatise) and H1993 (wild type) were purchased from BNCC (BNCC331211) and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (HyClone, USA) in a constant-temperature incubator (Thermo, USA).

Techniques: Expressing, Control

Journal: Cancer Management and Research

Article Title: miR-124-3p Regulates FGF2–EGFR Pathway to Overcome Pemetrexed Resistance in Lung Adenocarcinoma Cells by Targeting MGAT5

doi: 10.2147/CMAR.S274192

Figure Lengend Snippet: Effects of miR-124-3p on apoptosis of transplanted tumors in nude mice and expression of FGF2–EGFR signaling pathway-related proteins by targeting MGAT5. ( A ) Tumor cell apoptosis detected using TUNEL kit (×400); ( B ) FGF2–EGFR signaling pathway-related protein expression in PC9-MTA tumor tissue detected by Western blot; ( C ) FGF2–EGFR signaling pathway-related protein expression in H1993-MTA tumor tissue detected by Western blot. *p<0.05 vs Control group; # p<0.05 vs miR-124-3p group.

Article Snippet: Human lung adenocarcinoma cell lines PC9 (mutant EGFR, EGFR-DelE746_A750, BIV10058, BioInnovatise) and H1993 (wild type) were purchased from BNCC (BNCC331211) and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (HyClone, USA) in a constant-temperature incubator (Thermo, USA).

Techniques: Expressing, TUNEL Assay, Western Blot, Control