human lung cancer cell derived a549  (ATCC)

Journal: Oncology Research

Article Title: Cisplatin-induced activation of TGF-β signaling contributes to drug resistance

doi: 10.32604/or.2023.030190

Figure Lengend Snippet: Effect of cisplatin on various types of cancer cell lines. (A) Cisplatin enhanced N-cadherin expression in A549 but not in DLD-1, HCT116, HT29, SW48, and LNCaP cells. Cells were treated with 1 μM cisplatin for the indicated periods (0, 3, 6, 9, and 12 days). The protein expression of N-cadherin and β-actin was detected by western blotting. Images are representative of two independent experiments. (B) Cisplatin decreased E-cadherin mRNA, and increased N-cadherin and Slug mRNAs in A549 cells. A549 cells were treated with 1 μM cisplatin for the indicated periods. The amount of E-cadherin, N-cadherin, and Slug mRNAs were evaluated using qPCR. The presented data are normalized to Day 0. All data are representative of three independent experiments and presented as mean SD ± (n = 3). * p < 0.05 and ** p < 0.01 (two-tailed Student’s test). (C) The TβRI kinase inhibitor, SB431452, partially suppressed cisplatin-induced Slug upregulation in A549 cells. A549 cells were treated with 10 μM cisplatin and/or 1 μM SB431452. Twenty-four hours later, the cells were collected and subjected to qPCR analysis to detect Slug mRNA. The presented data are normalized to control cells. All data are representative of three independent experiments and presented as mean SD ± (n = 3). * p < 0.05 and ** p < 0.01 (two-tailed Student’s test). (D) TGF-β1 induces and decreases N-cadherin and E-cadherin in LoVo and A549 cells. Cells were stimulated with the indicated concentrations of TGF-β1 (0, 0.1, 1, 10 ng/mL). Two days later, the cells were collected and subjected to western blot analysis to detect E-cadherin, N-cadherin, and β-actin.

Article Snippet: Human colorectal adenocarcinoma-derived cell line HCT116, HT29, and DLD-1, human non-small cell lung cancer (NSCLC) adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell line LNCaP were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Expressing, Western Blot, Two Tailed Test, Control

Journal: Oncology Research

Article Title: Cisplatin-induced activation of TGF-β signaling contributes to drug resistance

doi: 10.32604/or.2023.030190

Figure Lengend Snippet: Cisplatin enhances the secretion of TGF-β1. (A) Cisplatin increased the amount of TGF-β1 in the culture media from LoVo and A549 cells. LoVo and A549 cells were treated with 1 μM cisplatin for 72 h. The culture media from cells were collected and subjected to ELISA to measure the amount of TGF-β1. The amounts of TGF-β1 from LoVo and A549 cells without cisplatin were used to calculate relative values. All data are presented as mean SD ± (n = 3). *** p < 0.001 (two-tailed Student’s test). (B) The amount of TGF-β1 in the culture media from cisplatin r /LoVo cells were increased. The culture media from LoVo or cisplatin r /LoVo (#10, #19, and #54) cells were collected and subjected to ELISA to measure the amount of TGF-β1. The amount of TGF-β1 from LoVo cells without cisplatin was used to calculate relative values. All data are presented as mean SD ± (n = 3). ** p < 0.01 and *** p < 0.001 (two-tailed Student’s test). (C) The effect of cisplatin on the expression of TGF-β1 mRNA in LoVo cells. LoVo cells were treated with 1 μM cisplatin. Three days later, the total RNA was extracted and subjected to qPCR analysis to measure the amount of TGF-β1 mRNA. The presented data are normalized to control cells. All data are representative of three independent experiments. (D) The comparison of the expression levels of TGF-β1 mRNA in LoVo and cisplatin r /LoVo cells. Total RNA from exponentially growing LoVo or cisplatin r /LoVo cells (#10, #19, and #54) was extracted and subjected to qPCR analysis to measure the amount of TGF-β1 mRNA. The presented data are normalized to LoVo cells. All data are representative of three independent experiments.

Article Snippet: Human colorectal adenocarcinoma-derived cell line HCT116, HT29, and DLD-1, human non-small cell lung cancer (NSCLC) adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell line LNCaP were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Expressing, Control, Comparison

Journal: Cancer Cell International

Article Title: EF24 exerts cytotoxicity against NSCLC via inducing ROS accumulation

doi: 10.1186/s12935-021-02240-z

Figure Lengend Snippet: EF24 inhibits the growth of NSCLC cells in-vitro. A A549, SPC-A1, H460 and H520 cells were treated with the indicated concentrations of EF24 for 24 h and 48 h, and then subjected to MTT assay. The absorbance value was calculated and standardized to the control group. B The cells were treated with 0 μM, 1 μM, 2 μM and 4 μM EF24 respectively for 2 h and subjected to the cell colony formation assay. Surviving fraction is presented as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.01

Article Snippet: Human non-small cell lung cancer (NSCLC) derived cell lines A549, SPC-A1, H460 and H520 were purchased from American Type Culture Collection (Manassas, VA, USA) and China Center for Type Culture Collection (Wuhan, China).

Techniques: In Vitro, MTT Assay, Control, Colony Assay

Journal: Cancer Cell International

Article Title: EF24 exerts cytotoxicity against NSCLC via inducing ROS accumulation

doi: 10.1186/s12935-021-02240-z

Figure Lengend Snippet: EF24 inhibits tumor growth in-vivo. A A brief flowchart of the experiment design in-vivo. A549 xenograft tumor was established and divided into the following four groups and received 17-day treatment: control, EF24 (5 mg/kg/d, 10 mg/kg/d and 20 mg/kg/d). B The weight of tumors. C The curves of tumor growth. D The body weight of mice. E Representative images of xenografts from different groups. F H&E staining tumor tissue sections of different groups. Magnification: 100×. G Anti-Ki-67 staining of tumor tissues in different groups. Magnification: 200×. The results shown are means ± SD; * P < 0.05

Article Snippet: Human non-small cell lung cancer (NSCLC) derived cell lines A549, SPC-A1, H460 and H520 were purchased from American Type Culture Collection (Manassas, VA, USA) and China Center for Type Culture Collection (Wuhan, China).

Techniques: In Vivo, Control, Staining

Journal: Cancer Cell International

Article Title: EF24 exerts cytotoxicity against NSCLC via inducing ROS accumulation

doi: 10.1186/s12935-021-02240-z

Figure Lengend Snippet: Histopathological analyses of major organs. Histopathological analyses of liver, lung, spleen, kidney and heart tissues of A549 xenograft. Scale bars: 50 μm

Article Snippet: Human non-small cell lung cancer (NSCLC) derived cell lines A549, SPC-A1, H460 and H520 were purchased from American Type Culture Collection (Manassas, VA, USA) and China Center for Type Culture Collection (Wuhan, China).

Techniques:

Journal: Cancer Cell International

Article Title: EF24 exerts cytotoxicity against NSCLC via inducing ROS accumulation

doi: 10.1186/s12935-021-02240-z

Figure Lengend Snippet: EF24 induces apoptosis in NSCLC cells. A A549, SPC-A1, H460 and H520 cells were treated with or without indicated concentrations of EF24 respectively for 48 h and subjected to apoptosis assay using Annexin-V & PI staining. B The expression of apoptotic marker Fas in cells were determined. Three independent experiments were performed and the results statistically analyzed as means ± SD, * P < 0.05, ** P < 0.01, *** P < 0.01

Article Snippet: Human non-small cell lung cancer (NSCLC) derived cell lines A549, SPC-A1, H460 and H520 were purchased from American Type Culture Collection (Manassas, VA, USA) and China Center for Type Culture Collection (Wuhan, China).

Techniques: Apoptosis Assay, Staining, Expressing, Marker

Journal: Cancer Cell International

Article Title: EF24 exerts cytotoxicity against NSCLC via inducing ROS accumulation

doi: 10.1186/s12935-021-02240-z

Figure Lengend Snippet: EF24 induces ROS accumulation, mitochondrial morphological changes and autophagy. A – B EF24 induces ROS accumulation. C EF24 induced mitochondrial fragmentation of the mitochondrial network in A549 cells. The arrow indicates fragmented mitochondria. D Western blot analysis demonstrated SQSTM1, LC3B and ACTB expression in EF24-treated A549 cells. E Autophagic vacuoles in A549 cells treated with various concentrations of EF24 were observed by TEM. The arrow indicates autophagic vacuoles. Number of autophagic vacuoles were calculated. F Changes in the localization of LC3B in cells assessed by immunofluorescence under confocal laser microscopy. The results are presented as mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.01

Article Snippet: Human non-small cell lung cancer (NSCLC) derived cell lines A549, SPC-A1, H460 and H520 were purchased from American Type Culture Collection (Manassas, VA, USA) and China Center for Type Culture Collection (Wuhan, China).

Techniques: Western Blot, Expressing, Immunofluorescence, Microscopy

Journal: Cancer Cell International

Article Title: EF24 exerts cytotoxicity against NSCLC via inducing ROS accumulation

doi: 10.1186/s12935-021-02240-z

Figure Lengend Snippet: EF24 induces cytotoxicity is restored by ROS scavengers. A549 and H520 cells were treated with 4 μM EF24 for 48 h with or without CAT and NAC employed 2 h before EF24 treatment, after that cell viability using MTT assay ( A ) and apoptotic cells using Annexin-V & PI staining ( B ) were detected. Three independent experiments were performed and the results statistically analyzed as means ± SD, * P < 0.05, ** P < 0.01, *** P < 0.01

Article Snippet: Human non-small cell lung cancer (NSCLC) derived cell lines A549, SPC-A1, H460 and H520 were purchased from American Type Culture Collection (Manassas, VA, USA) and China Center for Type Culture Collection (Wuhan, China).

Techniques: MTT Assay, Staining

Journal: Nature Communications

Article Title: Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS -mutant lung adenocarcinoma

doi: 10.1038/s41467-019-12164-y

Figure Lengend Snippet: PPP blockade abrogates the growth of KEAP1 -mutant LUAD. a Schematic of spontaneous tumor treatment study. Briefly, KK or KP mice were randomized into 20 mg/kg 6-AN or vehicle treated 40 days post Ad5-CMV-Cre intranasal infection. Lungs were harvested for analysis on day 64. b Representative H&E images of KK vehicle or 6-AN-treated lungs. Scale, 1 mm. c Quantification of KK superior lobe lung weight (milligrams, mg) following vehicle ( n = 6) or 6-AN ( n = 8) treatment. Unpaired t test * p = 0.0106. Mean ± SEM. d Ratio of KK hyperplasia relative to large airway size in vehicle ( n = 6) and 6-AN ( n = 8) treated lungs. Unpaired t test * p = 0.0116. Mean ± SEM. e Colony assay 72 h following treatment with 62.5 μM 6-AN vs vehicle in KEAP1 MUT and KEAP1 WT NSCLC cell lines. Scale, 5 mm. f Relative change in tumor size of xenograft models ( n = 3/cell line) of KEAP1 MUT and KEAP1 WT 48 h following treatment with 20 mg/kg 6-AN or vehicle. Change in size relative to vehicle of each cell line. Two-way ANOVA/Sidak’s multiple comparisons test: A549 ** p = 0.0012; H460 *** p = 0.001. Mean ± SD. g Tumor measurements of A549 cell line xenograft following treatment with 20 mg/kg 6-AN ( n = 6) or vehicle ( n = 6) every 10 days. Survival log-rank (Mantel-Cox) test ** p = 0.0023. h Kaplan–Meier survival curve of A549 xenograft NSG mice treated with 20 mg/kg 6-AN ( n = 6) or vehicle ( n = 6) every 10 days. Mantel–Cox test ** p = 0.0017. i Tumor measurements of H358 xenograft following treatment with 20 mg/kg 6-AN ( n = 8) or vehicle ( n = 4) every 10 days. j Kaplan–Meier survival curve of H358 xenograft NSG mice treated with 20 mg/kg 6-AN ( n = 8) or vehicle ( n = 4) every 10 days

Article Snippet: The human lung cancer derived cell lines A549, H460, H441 and H358 were obtained from ATCC and cultured in RPMI-1640 + GlutaMAX medium (Gibco) supplemented with 10 % FBS (Sigma-Aldrich), 100 U/mL penicillin 100 μg/mL streptomycin (Gibco).

Techniques: Mutagenesis, Infection, Colony Assay

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: β-catenin is essential for CUG2-induced CSC-like phenotypes. (A) A549-VEC and A549-CUG2 cells were separated into cytosolic and nuclear fractions. β-catenin expression was determined by performing western blot analysis with an anti-β-catenin antibody. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. The cytosolic and nuclear fractions were confirmed by detecting actin and Sp1, respectively. (B) Total RNAs (3 µ g) were isolated from the A549-VECand A549-CUG2 cells, and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (C) A549-VEC and A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector and were harvested at 48 h after the transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results are an average of three experiments; bars indicate the means ± SD ( *** P<0.001, A549-VEC vs A549-CUG2 cells). (D) Transfection of efficiency of β-catenin siRNAs (#1 or #2) was confirmed by western blot analysis. (E) Migration of A549-CUG2 cells was measured with the wound healing assay at 24 h after control or β-catenin siRNAs (#1 and #2) transfection. Wound closure areas were monitored using a phase-contrast microscope at ×100 magnification, and the assay was repeated twice. (F) Invasion of A549-CUG2 cells transfected with control or β-catenin siRNAs (#1 and #2) was determined using the 48-well Boyden chamber. The chamber was assembled using Matrigel-coated polycarbonate filters. Scale bar indicates 100 µ m, and the assay was repeated twice. Each assay was performed in triplicate, and error bars indicate the means ± SD ( *** P<0.001, control vs. β-catenin siRNA). (G) A549-CUG2 cells (1,000 cells per well) transfected with control or β-catenin siRNAs (#1 and #2) were seeded in ultra-low attachment plates for 2, 4, or 6 days. The assay was performed in triplicate, and error bars indicate the means ± SD ( ** P<0.01 and *** P<0.001, control vs. β-catenin siRNA). Scale bars indicate 50 µ m. CUG2, cancer-upregulated gene 2.

Article Snippet: Human lung cancer-derived A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Migration, Wound Healing Assay, Microscopy

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-induced β-catenin phosphorylation is unique and differs from that induced by Wnt3a. (A) A549-VEC and A549-CUG2 cells were treated with wortmannin (10 µ M), H89 (10 µ M), PP2 (2.5 µ M) and DMSO (mock) in the presence of Wnt3a (100 ng/ml) or PBS as a vehicle for 12 h, and cell lysates were prepared. Proteins present in the cell lysates were separated by performing SDS-PAGE on 10% gels, and β-catenin and α-actin were detected using corresponding antibodies. (B and C) Proteins present in the lysates of A549-VEC cells treated with Wnt3a (100 ng/ml) or PBS, and of A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were determined using antibodies against phosphorylated Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (D) Lysates of A549-VEC and A549-CUG2 cells treated with a control or GSK3β siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. GSK3β and phosphorylated GSK3β were detected by performing western blot with corresponding antibodies. CUG2, cancer-upregulated gene 2; GSK3β, glycogen synthase kinase 3β.

Article Snippet: Human lung cancer-derived A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Phospho-proteomics, SDS Page, Control, Western Blot

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CUG2-activated NEK2 is responsible for the phosphorylation of β-catenin at Ser33/Ser37. (A) Lysates of A549-VEC and A549-CUG2 cells were separated by performing SDS-PAGE on 10% gels, and NEK2 was detected by performing western blot analysis with anti-NEK2 antibody. (B) Levels of NEK2 and β-catenin phosphorylated at Ser33/Ser37 (p-β-catenin; p-Ser33/Ser37) in A549-VEC and A549-CUG2 cells were determined by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) β-catenin was pulled down from the lysates of A549-VEC and A549-CUG2 cells by using an anti-β-catenin antibody. NEK2 present in the immunoprecipitates was detected using an anti-NEK2 antibody, and β-catenin present in the immunoprecipitates was detected as a loading control by using an anti-β-catenin antibody. (D) NEK2 was pulled down from A549-VEC and A549-CUG2 cells by using an anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay, including immunoprecipitates as NEK2 kinase, recombinant GST-β-catenin as a substrate, ATP, and a reaction buffer, was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by determining the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing immunoblotting, and β-catenin levels were examined as a loading control. (E) Lysates of A549-CUG2 cells transfected with the control or NEK2 siRNAs (#1 and #2) were separated by performing SDS-PAGE on 10% gels. Phosphorylation states of β-catenin following treatment were detected using antibodies against Ser33/Ser37/Thr41, Ser45 and Ser675 of β-catenin. (F) Following transfection with control or NEK2 siRNA#1, the levels of p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells were detected by performing immunofluorescence microscopy with Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Human lung cancer-derived A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Recombinant, Incubation, Activity Assay, Transfection

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment destabilizes β-catenin in a proteasome- and PKCα- dependent manner. (A) A549-CUG2 cells were treated with CGK062 (0, 1, 10, 50 and 100 µ M) for 24 h, and β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (B) A549-CUG2 cells were transfected with the Top-Flash (1 µ g) or Fop-Flash (1 µ g) luciferase reporter vector in the presence of CGK062 (50 µ M) and were harvested at 48 h following transfection. Transfection efficiency was normalized with that of the β-galactosidase reporter vector pGK-βgal (1 µ g) during the measurement of luciferase activity. Results shown are an average of 3 experiments; bars indicate the means ± SD ( *** P<0.001, 0 vs. 10 µ M, 0 vs. 50 µ M). (C) Total RNAs (3 µ g) were isolated from A549-CUG2 cells treated with or without CGK062 (50 µ M), and cDNAs were synthesized using reverse transcriptase II. β-catenin gene sequences were amplified using specific primers by using an optimized PCR cycle and were visualized on 1.5% agarose gels following ethidium bromide staining. β-actin was used as an internal control. (D) A549-CUG2 cells were treated with MG132 (1 µ M) for 8 h in the absence or presence of CGK062 (50 µ M) and were harvested. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. (E) A549-CUG2 cells were treated with BMI (7.5 µ M), a specific PKCα inhibitor, or were transfected with a control and PKCα siRNAs (#1 or #2) in the absence or presence of CGK062 (50 µ M) for 24 h. Transfection efficiency of PKCα siRNAs (#1 or #2) was confirmed by western blot analysis. β-catenin levels were measured by performing western blot analysis with an anti-β-catenin antibody. CUG2, cancer-upregulated gene 2; PKC, protein kinase C.

Article Snippet: Human lung cancer-derived A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Isolation, Synthesized, Reverse Transcription, Amplification, Staining, Control

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment decreases NEK2 expression and kinase activity in A549-CUG2 cells. (A) Lysates of A549-CUG2 cells treated with CGK062 (0, 10, 30, 40 and 50 µ M) for 24 h were separated by performing SDS-PAGE on 10% gels. The expression of NEK2, β-catenin, E-cadherin, N-cadherin and vimentin was detected by performing western blot analysis with corresponding antibodies. (B) Levels of NEK2 and p-β-catenin (p-Ser33/Ser37) in A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO as Mock were detected by performing immunofluorescence microscopy with Alexa Fluor 594-conjugated goat anti-mouse IgG (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), respectively. For performing nuclear staining, DAPI was added before mounting in glycerol. Scale bar indicates 10 µ m. (C) NEK2 was pulled down from A549-VEC and A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO by using the anti-NEK2 antibody or isotype IgG as a control. The reaction mixture for the NEK2 kinase assay was incubated at 30°C for 1 h. NEK2 kinase activity was analyzed by detecting the phosphorylation of GST-β-catenin at Ser33/Ser37 by performing western blot analysis, and β-catenin levels were examined as a loading control. CUG2, cancer-upregulated gene 2; NEK2, never in mitosis gene A-related kinase 2.

Article Snippet: Human lung cancer-derived A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Activity Assay, SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Control, Kinase Assay, Incubation, Phospho-proteomics

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment inhibits CUG2-induced CSC-like phenotypes. (A) Migration of A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO (control) was measured by performing wound healing assay. Wound closure areas were monitored using a phase-contrast microscope at ×100 magnification, and the assay was repeated twice. (B) Invasion of A549-CUG2 cells treated with CGK062 (50 µ M) or DMSO was determined using the 48-well Boyden chamber. The chamber was assembled using Matrigel-coated polycarbonate filters. Scale bar indicates 100 µ m, and the assay was repeated twice. Each assay was performed in triplicate, and error bars indicate the means ± SD ( ** P<0.01, DMSO vs. CGK062). (C, D and F) Lysates of A549-CUG2 cells treated with CGK062 (0, 10, 30, 40 and 50 µ M) or DMSO were separated by performing SDS-PAGE on 10% gels, and expression of phosphorylated Smad2, Smad2/3, Twist, Snail, Oct4, Bmi1, Klf4, Sox2, Nanog and ALDH1 was determined by performing western blot analysis with corresponding antibodies. (E) A549-CUG2 cells treated with CGK062 (0, 10, 30, 40 and 50 µ M) or DMSO (1,000 cells per well) were seeded in ultra-low attachment plates for 2, 4 or 6 days. The assay was performed in triplicate, and error bars indicate the means ± SD (ns; not significant, DMSO vs. CGK062 at 10 µ M, *** P<0.001, DMSO vs. CGK062 at 30, 40 and 50 µ M). Scale bars indicate 50 µ m. CUG2, cancer-upregulated gene 2.

Article Snippet: Human lung cancer-derived A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Control, Wound Healing Assay, Microscopy, SDS Page, Expressing, Western Blot

Journal: International Journal of Oncology

Article Title: CGK062, a small chemical molecule, inhibits cancer upregulated gene 2-induced oncogenesis through NEK2 and β-catenin

doi: 10.3892/ijo.2019.4724

Figure Lengend Snippet: CGK062 treatment inhibits CUG2-induced tumor development in vivo. (A and B) Balb/C nude mice were subcutaneously transplanted with A549-CUG2 cells (1×10 6 cells/mouse) for 2 weeks and were intraperitoneally injected with CGK062 (100 mg/kg body weight) for 4 weeks. Tumor sizes and body weights were measured for 4 weeks [ ** P<0.01, mock (n=6) vs. CGK062 (n=6); ns; not significant]. (C) At 4 weeks after the treatment, tumor tissues harvested from the mice were embedded in paraffin and were sectioned for performing the TUNEL assay. Green fluorescence indicates apoptotic cells.

Article Snippet: Human lung cancer-derived A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vivo, Injection, TUNEL Assay, Fluorescence