Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Increased Cx26 is positively correlated with gefitinib resistance in NSCLC cells. ( a ) Differential expression of Cx26, Cx31.1, Cx32, and Cx43 in different gefitinib-sensitive NSCLC cell lines was determined by RT-PCR. ( b and c ) High level of Cx26 in gefitinib-insensitive A549 and H1299 cells than that in gefitinib-sensitive HCC827 and PC9 cells was detected by RT-PCR and western blotting. GAPDH or β -actin was used as internal loading control

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Cx26 induces acquired gefitinib resistance in NSCLC cells via GJIC-independent manner. ( a ) Functional GJIC was detected by parachute assay and no detectable GJIC was found in HCC827 GR, PC9 GR, and their parental cells. Top: fluorescence images. Bottom: overlaid the corresponding phase-contrast images. Original magnification, × 200. ( b ) No enhancement of GJIC in these cells incubated with 10, 20, and 40 μ M of RA (a well-defined GJIC enhancer) for 4, 8, 12, 24, and 48 h, respectively. Top: fluorescence images. Bottom: overlaid the corresponding phase-contrast images. Original magnification, × 200. ( c and d ) Immunofluorescence staining of the cellular localization of Cx26 with or without RA treatment. All scare bars represent 50 μ m

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Functional Assay, Fluorescence, Incubation, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

doi: 10.1038/cddis.2015.197

Figure Lengend Snippet: Cx26 and PI3K/Akt pathway functionally interplay to promote EMT and gefitinib resistance in NSCLC cells. ( a and b ) Effect of LY294002 or Akt overexpression on Cx26 expression in HCC827, PC9, and their GR cells was determined by western blotting. ( c ) Effects of Akt overexpression alone or combined with Cx26 overexpression or Cx26 depletion on cell morphology changes in HCC827 and PC9 cells. Original magnification, × 400. ( d – f ) Effects of Akt overexpression alone or combined with Cx26 overexpression or Cx26 depletion on the expression of EMT markers (E-cadherin, vimentin, and slug), cell migration, and invasion, as well as cell sensitivity to gefitinib in HCC827 and PC9 cells, respectively. Error bars are mean±S.D. from four independent experiments, ** P <0.01 versus vector group. # P <0.05 and ## P <0.01 versus Akt-overexpressing group

Article Snippet: Human NSCLC cell lines (HCC827, PC9, A549, and H1299) were originally obtained from the ATCC (Manassas, VA, USA).

Techniques: Over Expression, Expressing, Western Blot, Migration, Plasmid Preparation

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Gefitinib enhances the cytotoxicity of PBMCs in EGFR-mutated NSCLC cells, but not in NSCLC cells with wild-type EGFR. Cell lysis measured by Calcein staining in four NSCLC lines: (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC-9. NSCLC cells were cultured with PBMCs at different E/T ratios. * P<0.05 vs. control. E/T, effector/target; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Lysis, Staining, Cell Culture, Control

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Expression of B7H5 following treatment with Gefitinib in NSCLC cells. (A-D) Reverse transcription-quantitative PCR and western blot analysis of B7H5 expression after treatment with gefitinib in NSCLC cells (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC-9. (E) Immunofluorescence determined B7H5 expression (green colour) following treatment with or without gefitinib in NSCLC cells (magnification, ×200). ** P<0.01 vs. control. NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Control

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Changes in B7H5 expression in mutant and wild-type NSCLC cell lines after EGFR siRNA interference. (A-D) Reverse transcription-quantitative PCR and western blot analysis of B7H5 and EGFR expression following transfection with an EGFR siRNA in NSCLC cells. (A) NCI-H358, (B) NCI-H1299, (C) HCC827 and (D) PC9. (E) Immunofluorescence analysis B7H5 (green colour) and EGFR (red colour) expression following transfection with EGFR siRNA in NSCLC cells (magnification, ×200). ** P<0.01, *** P<0.001 vs. negative control. siRNA, small interfering RNA; NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Mutagenesis, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Immunofluorescence, Negative Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Gefitinib treatment increases B7H5 expression in wild-type NSCLC cell lines expressing EGFR mutants. The levels of EGFR and B7H5 as determined by western blot analysis in (A) NCI-H1299 and (B) NCI-H358 cells. (C) Immunofluorescence analysis of B7H5 expression (green colour) after transfection with the EGFR mutation plasmid or the EGFR mutation plasmid combined with gefitinib in wild-type EGFR NSCLC cells (magnification, ×200). ** P<0.01, *** P<0.001. NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Immunofluorescence, Transfection, Mutagenesis, Plasmid Preparation

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: B7H5 affects the killing effect of gefitinib combined with PBMCs in EGFR-mutated NSCLC cells. (A and B) Cell Counting Kit-8 assay and cytotoxicity test models of mixed cell culture to determine the cell viability and killing effect of PBMCs after treatment with or without B7H5 siRNA in (A) NCI-H358 and (B) NCI-H1299 cells. (C) Expression of B7H5 in NCI-H358 and NCI-H1299 cells transfected with or without B7H5 siRNA by immunofluorescence analysis (magnification, ×200; B7H5 expression: Green colour). (D and E) The mixed cell culture toxicity test model was used to test the efficacy of gefitinib against EGFR-mutated NSCLC cells prior to and after B7H5 siRNA interference in (D) HCC827 and (E) PC-9 cells. (F) Expression of B7H5 in HCC827 and PC-9 cells transfected with or without B7H5 siRNA by immunofluorescence analysis (magnification, ×200). (G) Killing effect of gefitinib combined with PBMCs with and without transfection with B7H5 siRNA on EGFR-mutated NSCLC cells in HCC827 and PC-9 cells. * P<0.05 vs. Negative control (A-E); * P<0.05 vs. control (G). siRNA, small interfering RNA; E/T, effector/target; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Cell Counting, Cell Culture, Expressing, Transfection, Immunofluorescence, Negative Control, Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Gefitinib affects PBMC-mediated cytolysis in cells transfected with EGFR mutants and wild-type EGFR. (A) The killing effect of gefitinib combined with PBMCs was observed with and without transfection of the EGFR mutation plasmid. (B and C) Western blot analysis detected EGFR levels and the killing effect of PBMCs after transfection with or without the EGFR mutation plasmid in wild-type EGFR NSCLC (B) NCI-H358 and (C) NCI-H1299 cells. (D) Immunofluorescence analysis of EGFR expression (red colour) after transfection with or without the EGFR mutation plasmid in wild-type EGFR NSCLC cells (magnification, ×200). * P<0.05 vs. control. siRNA, small interfering RNA; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer; E/T, effector/target.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Immunofluorescence, Expressing, Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Gefitinib enhances the anti-tumor immune response against EGFR-mutated NSCLC by upregulating B7H5 expression and activating T cells via CD28H

doi: 10.3892/ijo.2022.5436

Figure Lengend Snippet: Interference with CD28 expression markedly decreases the killing activity against EGFR-mutant NSCLC cells. (A) CD28 expression was determined by western blot analysis and flow cytometry. (B) Mixed cell culture toxicity assay to determine the cytotoxic activity after transfection with or without CD28H siRNA; (C) combined efficacy of PBMCs and gefitinib, (D) combined efficacy of PBMCs and gefitinib with and without CD28H siRNA interference in EGFR-mutant NSCLC cells HCC827. (E) Mixed cell culture toxicity assay to determine the cytotoxic activity after transfection with or without CD28H siRNA; (F) combined efficacy of PBMCs and gefitinib, (G) combined efficacy of PBMCs and gefitinib with and without CD28H siRNA interference in EGFR-mutant NSCLC cells PC-9. * P<0.05 vs. Negative control. siRNA, small interfering RNA; PBMC, peripheral blood mononuclear cell; NSCLC, non-small cell lung cancer; E/T, effector/target; NC, negative control.

Article Snippet: NSCLC cell lines (NCI-H1299, NCI-H358, HCC827 and PC-9) were purchased from the American Type Culture Collection.

Techniques: Expressing, Activity Assay, Mutagenesis, Western Blot, Flow Cytometry, Cell Culture, Transfection, Negative Control, Small Interfering RNA

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) was highly expressed in non‐small cell lung cancer (NSCLC) tissues and cells. (a) USP22 expression in NSCLC tissues was evaluated by immunohistochemistry (IHC) assay. (b, c) USP22 mRNA and protein levels in NSCLC tissues and adjacent normal tissues were measured by qRT‐PCR and western blot assays. (d, e) The mRNA and protein levels of USP22 in 16HBE, PC9, and HCC827 cells were determined by qRT‐PCR assay and western blot assay, respectively. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Deficiency of ubiquitin‐specific peptidase 22 (USP22) repressed non‐small cell lung cancer (NSCLC) cell migration and aggravated apoptosis and ferroptosis. (a) The protein level of USP22 in sh‐NC or sh‐USP22‐transfected PC9 and HCC827 cells was measured by western blot. (b) The migration of PC9 and HCC827 cells with sh‐NC or sh‐USP22 transfection was evaluated by transwell assay. (c) The apoptosis of PC9 and HCC827 cells transfected with sh‐NC or sh‐USP22 was analyzed by flow cytometry analysis. (d–g) The levels of reactive oxygen species (ROS), MDA, Fe 2+ , and GSH in sh‐NC or sh‐USP22 transfected PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Migration, Transfection, Western Blot, Transwell Assay, Flow Cytometry

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ferroptosis promoted the effect of gefitinib on non‐small cell lung cancer (NSCLC) cells. (a) The migration ability of PC9 and HCC827 cells treated with DMSO or 2.5‐μM gefitinib was assessed by transwell assay. (b) The apoptosis of DMSO or 2.5‐μM gefitinib‐treated PC9 and HCC827 cells was analyzed by flow cytometry analysis. (c, d) After PC9 and HCC827 cells were treated with DMSO, gefitinib, Fer‐1, or gefitinib+Fer‐1, the oxidized C11‐BODIPY fluorescence intensity was examined by C11‐BODIPY staining. (e–g) After PC9 and HCC827 cells were treated with dimethyl sulfoxide (DMSO), gefitinib, Fer‐1, or gefitinib+Fer‐1, the levels of MDA, Fe 2+ , and glutathione (GSH) were examined by relevant commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) overexpression promoted gefitinib resistance and repressed ferroptosis in gefitinib‐treated non‐small cell lung cancer (NSCLC) cells. (a) USP22 protein level in gefitinib or dimethyl sulfoxide (DMSO)‐treated PC9 and HCC827 cells was measured by western blot. (b) The effect of USP22 on gefitinib resistance was assessed by CCK‐8 assay. (c–i) PC9 and HCC827 cells were treated with DMSO+pcDNA, gefitinib+pcDNA, or gefitinib+USP22. (c, d) The migration and apoptosis of PC9 and HCC827 cells were evaluated by transwell assay and flow cytometry analysis, respectively. (e, f) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (g–i) The levels of MDA, Fe 2+ , and glutathione (GSH )in PC9 and HCC827 cells were examined by relevant commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Over Expression, Western Blot, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) stabilized murine double minute 2 (MDM2) and modulated MDM2 expression. (a) Ubibrowser software predicted the relation between USP22 and MDM2. (b) Pull‐down assay was conducted to analyze the level of MDM2 ubiquitination. (c) Co‐IP assay was performed to explore the interaction between USP22 and MDM2. (d) The expression of MDM2 in non‐small cell lung cancer (NSCLC) tissues and normal tissues was estimated through IHC assay. (e, f) The mRNA and protein levels of MDM2 in NSCLC tissues and normal tissues were measured by qRT‐PCR and western blot, respectively. (g, h) The mRNA and protein levels of MDM2 in 16HBE, PC9, and HCC827 cells were examined by qRT‐PCR and western blot, respectively. (i) After PC9 and HCC827 cells were treated with DMSO, gefitinib, gefitinib+USP22, or gefitinib+USP22 + sh‐MDM2, MDM2 protein level was measured by western blot. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Expressing, Software, Pull Down Assay, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Murine double minute 2 (MDM2) knockdown reversed the effects of Ubiquitin‐specific peptidase 22 (USP22) overexpression on gefitinib sensitivity and ferroptosis in gefitinib‐treated NSCLC cells. (a) IC50 of gefitinib in PC9 and HCC827 cells transfected with pcDNA, USP22, or USP22 + sh‐MDM2 was estimated through CCK‐8 assay. (b–h) PC9 and HCC827 cells were treated with dimethyl sulfoxide (DMSO), gefitinib, gefitinib+USP22, gefitinib+USP22 + sh‐NC, or gefitinib+USP22 + sh‐MDM2. (b, c) The migration and apoptosis of PC9 and HCC827 cells were explored by transwell assay and flow cytometry analysis. (d, e) The oxidized C11‐BODIPY fluorescence intensity in PC9 and HCC827 cells was examined by C11‐BODIPY staining. (f–h) The levels of MDA, Fe 2+ , and glutathione (GSH) in PC9 and HCC827 cells were determined with commercial kits. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: Knockdown, Over Expression, Transfection, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry, Fluorescence, Staining

Journal: Thoracic Cancer

Article Title: USP22 promotes gefitinib resistance and inhibits ferroptosis in non‐small cell lung cancer by deubiquitination of MDM2

doi: 10.1111/1759-7714.15439

Figure Lengend Snippet: Ubiquitin‐specific peptidase 22 (USP22) enhanced gefitinib resistance of non‐small cell lung cancer (NSCLC) cells in vivo. (a) The protein levels of USP22 and murine double minute 2 (MDM2) in PBS, gefitinib, and gefitinib+USP22 treated PC9 cells were measured by western blot. (b) Xenograft tumor volume was monitored every 5 days. (c) Xenograft tumor weight was examined after 25 days. (d) The expression of USP22 and MDM2 in xenograft tumors was examined through immunohistochemistry (IHC) assay. * p < 0.05.

Article Snippet: NSCLC cells (PC9 and HCC827) were purchased from Procell (Wuhan, China).

Techniques: In Vivo, Western Blot, Expressing, Immunohistochemistry

Journal: International Journal of General Medicine

Article Title: Identification of Prognostic Factors Related to Super Enhancer-Regulated ceRNA Network in Metastatic Lung Adenocarcinoma

doi: 10.2147/IJGM.S332317

Figure Lengend Snippet: Identification of super enhancers in LUAD cells. ( A ) Enhancers were ranked according to the H3K27ac signals in HSC4 and A549 cells based on GSE143653. ( B ) Overlapping analysis of SE-associated genes in HSC4 and A549 cells. ( C ) Functional distribution of the overlapping genes.

Article Snippet: One normal human bronchial epithelial cell line (BEAS-2B) and six LUAD cell lines (H460, HCC827, A549, H1299, PC9 and Calu3) were obtained from American Type Culture Collection (ATCC, VA, USA).

Techniques: Functional Assay

Journal: International Journal of General Medicine

Article Title: Identification of Prognostic Factors Related to Super Enhancer-Regulated ceRNA Network in Metastatic Lung Adenocarcinoma

doi: 10.2147/IJGM.S332317

Figure Lengend Snippet: Effect of AC074117.1 on proliferation of LUAD cells. ( A ) qRT-PCR was used to detect the relative expression of AC074117.1 in BEAS-2B, H460, HCC827, A549, H1299, PC9 and Calu3 cells. ( B ) qRT-PCR was used to measure the knockdown efficiency of AC074117.1 in A549 and H1299 cells. ( C and D ) MTT assay was performed to evaluate changes in cell proliferation following AC074117.1 silencing in A549 and H1299 cells. **P<0.01.

Article Snippet: One normal human bronchial epithelial cell line (BEAS-2B) and six LUAD cell lines (H460, HCC827, A549, H1299, PC9 and Calu3) were obtained from American Type Culture Collection (ATCC, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, Knockdown, MTT Assay

Journal: Computational and Mathematical Methods in Medicine

Article Title: Upregulation of NDUFAF2 in Lung Adenocarcinoma Is a Novel Independent Prognostic Biomarker

doi: 10.1155/2023/2912968

Figure Lengend Snippet: The expression of NDUFAF2 at the protein level in LUAD tissues was confirmed using clinical tissue and NSCLC cell lines. (a) Validation of the expression level of NDUFAF2 in LUAD and paired adjacent lung tissues was conducted using western blotting analysis. (b) Western blotting analysis was used to detect the protein expression level of NDUFAF2 in different NSCLC cell lines and normal lung epithelial cell line 2B. (c) IHC staining of NDUFAF2 was performed in adenocarcinoma tissue and paired adjacent normal lung tissue from typical clinical specimens. Representative images are shown. Scale bar, left, 200 μ m; right, 50 μ m.

Article Snippet: Six types of human LUAD cell lines (PC9, H358, A549, HCC827, H1299, and H1975) and pulmonary epithelial cell line (BEAS-2B) were provided by the Procell Life Science &Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Biomarker Discovery, Western Blot, Immunohistochemistry

Journal: Thoracic Cancer

Article Title: A novel molecular mechanism mediated by circ CCDC134 regulates non‐small cell lung cancer progression

doi: 10.1111/1759-7714.14942

Figure Lengend Snippet: The expression of circCCDC134 in non‐small cell lung cancer (NSCLC) tissues and cells. (a) The circCCDC134 expression was measured by quantitative real‐time PCR (qRT‐PCR) in NSCLC tumor tissues and adjacent normal tissues. (b) The circCCDC134 expression in the tumor tissues of NSCLC patients with different stages was detected by qRT‐PCR. (c) QRT‐PCR was used to determine the circCCDC134 expression in NSCLC cells and BEAS‐2B cells. Subcellular localization analysis (d, e), RNase R assay (f, g), and ActD assay (h–i) were used to assess the circular features of circCCDC134. * p < 0.05.

Article Snippet: Human NSCLC cells (H1299, HCC827, PC9, and A549) and a normal bronchial epithelial cell line (BEAS‐2B) were purchased from Biovector NTCC (Beijing, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Thoracic Cancer

Article Title: A novel molecular mechanism mediated by circ CCDC134 regulates non‐small cell lung cancer progression

doi: 10.1111/1759-7714.14942

Figure Lengend Snippet: The regulation of circCCDC134 knockdown on non‐small cell lung cancer (NSCLC) cell functions. PC9 and A549 cells were transfected with sh‐NC or sh‐circCCDC134#1/#2. (a) The circCCDC134 expression was assessed by qRT‐PCR. Colony formation assay (b), 5‐ethynyl‐2′‐deoxyuridine (EdU) assay (c), transwell assay (d, e), wound healing assay (f) and flow cytometry (g) were used to measure cell proliferation, migration, invasion and apoptosis. (h, i) Protein expression was detected by western blot (WB) analysis. (j–l) Glucose consumption, lactate production and ATP level were measured to assess cell glycolysis ability. * p < 0.05.

Article Snippet: Human NSCLC cells (H1299, HCC827, PC9, and A549) and a normal bronchial epithelial cell line (BEAS‐2B) were purchased from Biovector NTCC (Beijing, China).

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Colony Assay, EdU Assay, Transwell Assay, Wound Healing Assay, Flow Cytometry, Migration, Western Blot

Journal: Thoracic Cancer

Article Title: A novel molecular mechanism mediated by circ CCDC134 regulates non‐small cell lung cancer progression

doi: 10.1111/1759-7714.14942

Figure Lengend Snippet: CircCCDC134 sponged miR‐625‐5p. (a) The binding sites between circCCDC134 and miR‐625‐5p are shown. (b) The transfection efficiency of miR‐625‐5p mimic and inhibitor was confirmed by quantitative real‐time PCR (qRT‐PCR). Dual‐luciferase reporter assay (c, d) and RNA immunoprecipitation (RIP) assay (e, f) were used to assess RNA interaction. (g) The miR‐625‐5p expression was measured by qRT‐PCR in non‐small cell lung cancer (NSCLC) tumor tissues and adjacent normal tissues. (h) QRT‐PCR was used to determine the miR‐625‐5p expression in NSCLC cells and BEAS‐2B cells. (i) The miR‐625‐5p expression was detected by qRT‐PCR in NSCLC cells transfected with sh‐circCCDC134#1 and in‐miR‐625‐5p. * p < 0.05.

Article Snippet: Human NSCLC cells (H1299, HCC827, PC9, and A549) and a normal bronchial epithelial cell line (BEAS‐2B) were purchased from Biovector NTCC (Beijing, China).

Techniques: Binding Assay, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Luciferase, Reporter Assay, RNA Immunoprecipitation, Expressing

Journal: Thoracic Cancer

Article Title: A novel molecular mechanism mediated by circ CCDC134 regulates non‐small cell lung cancer progression

doi: 10.1111/1759-7714.14942

Figure Lengend Snippet: The regulation of sh‐circCCDC134#1 and in‐miR‐625‐5p on non‐small cell lung cancer (NSCLC) cell behaviors. PC9 and A549 cells were transfected with sh‐circCCDC134#1 and in‐miR‐625‐5p. Colony formation assay (a), 5‐ethynyl‐2′‐deoxyuridine (EdU) assay (b), transwell assay (c, d), wound healing assay (e) and flow cytometry (f) were performed to detect cell proliferation, migration, invasion, and apoptosis. (g, h) Western blot (WB) analysis was used to test protein expression. (i–k) Cell glycolysis ability was measured by detecting glucose consumption, lactate production and ATP level. * p < 0.05.

Article Snippet: Human NSCLC cells (H1299, HCC827, PC9, and A549) and a normal bronchial epithelial cell line (BEAS‐2B) were purchased from Biovector NTCC (Beijing, China).

Techniques: Transfection, Colony Assay, EdU Assay, Transwell Assay, Wound Healing Assay, Flow Cytometry, Migration, Western Blot, Expressing

Journal: Thoracic Cancer

Article Title: A novel molecular mechanism mediated by circ CCDC134 regulates non‐small cell lung cancer progression

doi: 10.1111/1759-7714.14942

Figure Lengend Snippet: MiR‐625‐5p could target NFAT5. (a) The binding sites between NFAT5 3'UTR and miR‐625‐5p are shown. (b, c) Dual‐luciferase reporter assay was used to assess RNA interaction. (d, e) The NFAT5 mRNA and protein expression in non‐small cell lung cancer (NSCLC) tumor tissues and adjacent normal tissues was measured by qRT‐PCR and western blot (WB) analysis. (f) WB analysis was used to determine the NFAT5 protein expression in NSCLC cells and BEAS‐2B cells. (g, h) The NFAT5 protein expression was detected by WB analysis under the specified transfection conditions. * p < 0.05.

Article Snippet: Human NSCLC cells (H1299, HCC827, PC9, and A549) and a normal bronchial epithelial cell line (BEAS‐2B) were purchased from Biovector NTCC (Beijing, China).

Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: Thoracic Cancer

Article Title: A novel molecular mechanism mediated by circ CCDC134 regulates non‐small cell lung cancer progression

doi: 10.1111/1759-7714.14942

Figure Lengend Snippet: The regulation of miR‐625‐5p and NFAT5 on non‐small cell lung cancer (NSCLC) cell behaviors. PC9 and A549 cells were transfected with miR‐625‐5p mimic and pcDNA NFAT5 overexpression vector. Cell proliferation, migration, invasion and apoptosis were determined using colony formation assay (a), 5‐ethynyl‐2′‐deoxyuridine (EdU) assay (b), transwell assay (c, d), wound healing assay (e) and flow cytometry (f). (g, h) Western blot (WB) analysis was performed to examine protein expression. (i–k) Glucose consumption, lactate production, and ATP level were analyzed to evaluate cell glycolysis ability. * p < 0.05.

Article Snippet: Human NSCLC cells (H1299, HCC827, PC9, and A549) and a normal bronchial epithelial cell line (BEAS‐2B) were purchased from Biovector NTCC (Beijing, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Migration, Colony Assay, EdU Assay, Transwell Assay, Wound Healing Assay, Flow Cytometry, Western Blot, Expressing

Journal: Thoracic Cancer

Article Title: A novel molecular mechanism mediated by circ CCDC134 regulates non‐small cell lung cancer progression

doi: 10.1111/1759-7714.14942

Figure Lengend Snippet: The regulation of circCCDC134 knockdown on non‐small cell lung cancer (NSCLC) tumor growth. A549 cells transfected with sh‐circCCDC134#1/sh‐NC were injected into nude mice. Tumor volume (a) and weight (b) were detected. (c, d) The circCCDC134 and miR‐625‐5p expression was examined by quantitative real‐time PCR (qRT‐PCR). (e) The NFAT5 protein expression was tested by western blot (WB) analysis. (f) Immunohistochemical (IHC) staining was used to assess NFAT5 positive cells. * p < 0.05.

Article Snippet: Human NSCLC cells (H1299, HCC827, PC9, and A549) and a normal bronchial epithelial cell line (BEAS‐2B) were purchased from Biovector NTCC (Beijing, China).

Techniques: Knockdown, Transfection, Injection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Nutlin-3a induces KRAS mutant/ p53 wild type lung cancer specific methuosis-like cell death that is dependent on GFPT2

doi: 10.1186/s13046-023-02922-8

Figure Lengend Snippet: Nutlin-3a reduces KRAS mutant NSCLC cell viability via the KRAS-PI3K/Akt pathways. A Cell viability was determined using MTT assay after nutlin-3a treatment for 48 h. B Cell proliferation examined using the colony formation assay after nutlin-3a treatment (2 μM) for 14 days (top); quantification of colony formation (bottom). C KRAS-GTP form detected by the KRAS activation assay after nutlin-3a treatment for 24 h. D KRAS protein stability was detected by western blotting after treatment of MG132 (10 μM) for 6 h before harvest. E KRAS downstream molecules were detected by western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control

Article Snippet: One normal human bronchial epithelial cell line (BEAS-2B) and 12 NSCLC cell lines (HCC827, H1299, H1975, PC9, PC9GR, H820, A427, A549, H460, H358, H23, and H1573) were purchased from the American Type Culture Collection (Manassas, VA, USA) and the Korean Cell Line Bank (Seoul, South Korea).

Techniques: Mutagenesis, MTT Assay, Colony Assay, Activation Assay, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Nutlin-3a induces KRAS mutant/ p53 wild type lung cancer specific methuosis-like cell death that is dependent on GFPT2

doi: 10.1186/s13046-023-02922-8

Figure Lengend Snippet: GFPT2 is a target of nutlin-3a-induced cell death. GFPT2 was inhibited by exogenously introduced siGFPT2 for indicated times in KRAS MT/ p53 WT NSCLC cells ( A - F ). A , B Expression of GFPT2, O-GlcNAc ( A ), and the KRAS-PI3K/Akt-mTOR pathway ( B ) were detected by western blotting. C KRAS-GTP form was detected through the KRAS activation assay. D Cell viability was measured by MTT assay. E Apoptotic cell death number (top), and total cell death number (bottom) were analyzed via flow cytometry using annexin V/PI staining. F Expression of caspase-9, caspase-3, and PARP detected by western blotting. Etoposide (ETO) treatment (100 μM) for 24 h was used as a positive control for inducing caspase cleavage. GFPT2 was re-expressed by transfection of GFPT2-HA for 24 h into GFPT2 knockout A549 cells ( G - L ). G KRAS-GTP form and O-GlcNAc were verified by KRAS activation assay and western blotting. H , I Cell viability was detected by cell counting ( H ), and MTT assays ( I ). J , K Cytoplasmic vacuoles were examined under the light microscope ( J ), and confocal microscope after incorporation of dextran (green) ( K ). L Autophagic flux was detected after transfection of the mRFP-GFP-LC3 tandem vector ( n ≥ 10). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control

Article Snippet: One normal human bronchial epithelial cell line (BEAS-2B) and 12 NSCLC cell lines (HCC827, H1299, H1975, PC9, PC9GR, H820, A427, A549, H460, H358, H23, and H1573) were purchased from the American Type Culture Collection (Manassas, VA, USA) and the Korean Cell Line Bank (Seoul, South Korea).

Techniques: Expressing, Western Blot, Activation Assay, MTT Assay, Flow Cytometry, Staining, Positive Control, Transfection, Knock-Out, Cell Counting, Light Microscopy, Microscopy, Plasmid Preparation, Control