luad cell line pc-9  (Procell Inc)

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: Immunohistochemical staining for ARID1A expression in EGFR-mutant LUAD tissues (50×and 200×). (A) ARID1A low expression. (B) ARID1A high expression.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Immunohistochemical staining, Staining, Expressing, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: SWI/SNF subunit mutations in lung adenocarcinoma (LUAD). (A) The frequencies of EGFR and SWI/SNF subunit mutations in LUAD. (B) The frequency of SWI/SNF subunit mutations in EGFR-mutant LUAD. (C) The types of SWI/SNF subunit mutations in EGFR-mutant LUAD.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: The role of SWI/SNF subunit mutations in the prognosis of EGFR-mutant LUAD. (A-F) ARID1A, ARID1B, ARID2, ARID5B, SMARCA4, and SMARCB1.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: ARID1A mutation confers a poor prognosis for patients with EGFR-mutant LUAD. (A) The frequencies of genes exhibiting coexisting mutations with EGFR. Survival analysis for patients with the EGFR mutation, ARID1A/EGFR comutation and (B) TP53/EGFR, (C) KRAS/EGFR, (D) CDKN2A/EGFR, (E) PIK3CA/EGFR, (F) RB1/EGFR, and (G) PTEN/EGFR comutations.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: The role of ARID1A expression in the prognosis of EGFR-mutant LUAD patients receiving EGFR-TKIs as a first-line treatment after postoperative progression. (A) Comparison of PFS between patients with low ARID1A expression and patients with high ARID1A expression. (B) Forest plots for the univariate and multivariate analyses of PFS.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Pharmacology

Article Title: ARID1A deficiency attenuates the response to EGFR-TKI treatment in lung adenocarcinoma

doi: 10.3389/fphar.2025.1582005

Figure Lengend Snippet: The role of ARID1A in the prognosis of EGFR-mutant LUAD patients receiving postoperative adjuvant EGFR-TKI treatments. (A) Comparison of DFS between patients with low ARID1A expression and patients with high ARID1A expression. (B) Forest plots for univariate and multivariate analyses of DFS. (C) Nomogram for the prediction of 1-, 2- and 3-year survival. (D) Calibration curves of the nomogram.

Article Snippet: LUAD cell line PC-9 (Procell Life Science, Wuhan, China) was maintained in RPMI-1640 medium supplemented with 10% FBS under standard humidified conditions (37°C, 5% CO 2 ).

Techniques: Mutagenesis, Adjuvant, Comparison, Expressing

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: The effects of LINC01087 knockdown on lung adenocarcinoma (LUAD) cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT). (A) LINC01087 expression in LUAD tissues and adjacent normal tissues was predicted using the starBase database. (B) Real‐time quantitative polymerase chain reaction was used to determine LINC01087 expression levels in LUAD cell lines (H1975, A549, PC‐9, Calu‐3, and H358 cells) and human normal bronchial epithelial cells (BEAS‐2B). H1975 and A549 cells were transfected with sh‐NC or sh‐LINC01087. (C) Cell viability was determined using the cell counting kit‐8 assay. (D) Apoptosis was determined by Annexin V‐PI staining via using flow cytometry. (E, F) Cell migration and invasion were assessed by the transwell assay. (G) Protein levels of EMT‐related proteins (E‐cadherin and vimentin) and Ras homolog gene family, member A (RhoA)/Rho‐associated protein kinase1 (ROCK1) signaling pathway markers (GTP‐RhoA, total‐RhoA, and ROCK1) were determined by Western blot. Data are presented as means ± standard deviation (SD). N = 3/group. All data were obtained from three independent replicates. GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase. * p <0.05; ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

Techniques: Knockdown, Migration, Expressing, Real-time Polymerase Chain Reaction, Transfection, Cell Counting, Staining, Flow Cytometry, Transwell Assay, Western Blot, Standard Deviation

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: The effects of RNA‐binding motif protein 15 (RBM15) on the m 6 A modification level and stability of LINC01087. (A) Total m 6 A modification levels in lung adenocarcinoma (LUAD) cells and human bronchial epithelial cells were determined using the methylated RNA binding protein immunoprecipitation (Me‐RIP) assay. (B) The binding relationship between RBM15 and LINC01087 was determined using the RNA immunoprecipitation (RIP) assay. RBM15 antibody was used for RIP assay to isolate RBM15‐bonded RNAs, followed by real‐time quantitative polymerase chain reaction (RT‐qPCR) using LINC01087 specific primers. (C) Levels of m 6 A modification in LINC01087 after RBM15 knockdown in LUAD cells were determined with the MeRIP assay. (D) m 6 A modification sites in LINC01087 were predicted using the SRAMP database. (E) Schematic diagram showing m 6 A modification site and synonymous mutation in LINC01087. (F) The expression level of LINC01087 in LUAD cells cotransfected with sh‐RBM15 and LINC01087‐WT/LINC01087‐MUT was determined by RT‐qPCR. (A–C) Mut, adenine residues substituted by cytosine. (G) Levels of m 6 A modification in LINC01087 in LUAD cells cotransfected with sh‐RBM15 and LINC01087‐WT or LINC01087‐MUT were determined using the Me‐RIP assay. (H) LINC01087 mRNA stability in LUAD cells after RBM15 knockdown was determined with the RNA stability assay. Data are presented as means ± SD. N = 3/group. All data were obtained from three independent replicates. * p <0.05; ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

Techniques: RNA Binding Assay, Modification, Methylation, Immunoprecipitation, Binding Assay, RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown, Mutagenesis, Expressing, Stability Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: The target binding relationship between LINC01087 and miR‐514a‐3p in lung adenocarcinoma (LUAD) cells. (A) The binding relationship between LINC01087 and Ago2 was determined by RNA binding protein immunoprecipitation assay. (B) The potential binding site between LINC01087 and miR‐514a‐3p was predicted using the starBase database. (C) The interaction between LINC01087 and miR‐514a‐3p was determined using the dual‐luciferase reporter assay. (D) miR‐514a‐3p expression levels following sh‐NC or sh‐LINC01087 transfection in LUAD cells were determined using real‐time quantitative polymerase chain reaction. Data are presented as means ± SD. N = 3/group. All data were obtained from three independent replicates. ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

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

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: The effects of miR‐514a‐3p overexpression on lung adenocarcinoma (LUAD) cell proliferation, migration, invasion, and epithelial–mesenchymal transition. (A) Real‐time quantitative polymerase chain reaction was used to determine miR‐514a‐3p expression levels in LUAD cell lines (H1975, A549, PC‐9, Calu‐3, and H358) and human normal bronchial epithelial cells (BEAS‐2B). LUAD cells were transfected with miR‐514a‐3p mimics to overexpress miR‐514a‐3p. (B) LUAD cell viability was determined using the cell counting kit‐8 assay. (C) Apoptosis was determined by Annexin V‐PI staining with flow cytometer. (D, E) The transwell assay was used to analyze cell migration and invasion. (F) E‐cadherin, vimentin, GTP‐RhoA, total‐RhoA, and Rho‐associated protein kinase1 (ROCK1) levels in cells were determined using Western blot. Data are presented as means ± SD. N = 3/group. All data were obtained from three independent replicates. GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; RhoA, Ras homolog gene family, member A. ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

Techniques: Over Expression, Migration, Real-time Polymerase Chain Reaction, Expressing, Transfection, Cell Counting, Staining, Flow Cytometry, Transwell Assay, Western Blot

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: LINC01087 targeted miR‐514a‐3p to upregulate centrosome protein 55 (CEP55) expression in lung adenocarcinoma (LUAD) cells. (A) Common target genes binding to miR‐514a‐3p were predicted using the starBase, TargetScan, and miRTar databases. (B) Potential binding sites between miR‐514a‐3p and CEP55 were predicted using the starBase database. (C) The interaction between miR‐514a‐3p and CEP55 mRNA 3′UTR was determined using the dual‐luciferase reporter assay. (D) The interaction between miR‐514a‐3p and CEP55 mRNA 3′UTR was determined using RNA–RNA pull‐down assay with a biotinylated miR‐514a‐3p. RNA complexes bound to the miR‐514a‐3p were eluted, and the relative expression levels of CEP55 in the RNA complexes were determined using real‐time quantitative polymerase chain reaction (RT‐qPCR). (E, F) CEP55 expression levels in LUAD cells overexpressing miR‐514a‐3p were determined using RT‐qPCR and Western blot. (G) miR‐514a‐3p expression levels in LUAD cells following sh‐LINC01087 and miR‐514a‐3p inhibitor cotransfection were determined with RT‐qPCR. (H, I) CEP55 expression levels in LUAD cells after sh‐LINC01087 and miR‐514a‐3p inhibitor cotransfection were determined using RT‐qPCR and Western blot. Data are presented as means ± SD. N = 3/group. All data were obtained from three independent replicates. GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase. * p <0.05; ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Pull Down Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Cotransfection

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: The inhibitory effects of miR‐514a‐3p overexpression on lung adenocarcinoma (LUAD) cell proliferation, migration, invasion, and epithelial–mesenchymal transition as well as Ras homolog gene family, member A (RhoA)/Rho‐associated protein kinase1 (ROCK1) signaling were abolished by centrosome protein 55 (CEP55) overexpression. LUAD cells were cotransfected with miR‐514a‐3p and CEP55 overexpression plasmids. (A) Cell viability was examined using cell counting kit‐8 assay. (B) Annexin V‐PI staining with flow cytometer was used to determine apoptosis. (C, D) Cell migration and invasion were determined using the transwell assay. (E) Western blot was used to determine E‐cadherin, vimentin, GTP‐RhoA, total‐RhoA, and ROCK1 levels in cells. Data are presented as means ± SD. N = 3/group. All data were obtained from three independent replicates. GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase. * p <0.05; ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

Techniques: Over Expression, Migration, Cell Counting, Staining, Flow Cytometry, Transwell Assay, Western Blot

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: miR‐514a‐3p inhibition or centrosome protein 55 (CEP55) overexpression reversed the inhibitory effects of LINC01087 knockdown on lung adenocarcinoma (LUAD) cell proliferation, migration, invasion, and epithelial–mesenchymal transition. LUAD cells were cotransfected with miR‐514a‐3p knockdown/CEP55 overexpression and LINC01087 knockdown plasmids. (A, B) CEP55 expression levels in LUAD cells were determined with real‐time quantitative polymerase chain reaction and Western blot. (C) Cell viability was determined using cell counting kit‐8 assay. (D) Apoptosis was determined by flow cytometry. (E, F) The transwell assay was employed to determine cell migration and invasion. (G) E‐cadherin and vimentin levels in LUAD cells were determined using Western blot. Data are presented as means ± SD. N = 3/group. All data were obtained from three independent replicates. GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase. * p <0.05; ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

Techniques: Inhibition, Over Expression, Knockdown, Migration, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting, Flow Cytometry, Transwell Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: N6‐methyladenosine ‐mediated LINC01087 promotes lung adenocarcinoma progression by regulating miR ‐514a‐3p to upregulate centrosome protein 55

doi: 10.1002/kjm2.12879

Figure Lengend Snippet: The effects of LINC01087 knockdown on lung adenocarcinoma (LUAD) tumor growth in vivo. Nude mice were injected with LUAD cells with stable LINC01087 knockdown. (A–C) LINC01087, miR‐514a‐3p, and centrosome protein 55 (CEP55) expression levels in tumor tissues were determined by real‐time quantitative polymerase chain reaction. (D–F) Tumor tissues were collected, and tumor volume and weight were measured. (G) Ki67 and CEP55 expression levels in tumor tissues were examined using immunohistochemistry. (H) Western blot was employed to determine GTP‐RhoA, total‐RhoA, and Rho‐associated protein kinase1 (ROCK1) levels in tumor tissues. Data are presented as means ± SD. GAPDH, Glyceraldehyde 3‐phosphate dehydrogenase; RhoA, Ras homolog gene family, member A. N = 5/group. ** p <0.01; *** p <0.001.

Article Snippet: The LUAD cell line PC‐9 was purchased from iCell (Shanghai, China).

Techniques: Knockdown, In Vivo, Injection, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: Upregulated LINC00511 accelerated tumorigenesis in LUAD. (a) LINC00511 expression is up-regulated in tumor samples than in the normal samples in TCGA data by GEPIA webtool. (b-c) OS (b) and RFS (c) in LUAD. OS: overall survival; RFS: disease-free survival. (d) LINC00511 expression in LUAD cell lines. (e) Transfection of si-NC or si-LINC00511 to PC-9 and A549 cell. LINC00511 downregulations in two LUAD cell lines via si-LINC00511 treatment were realized. (f-g) Cell viability of PC-9 (f) and A549 (g) cells with or without si-LINC00511 treatment. LUAD cell viability was suppressed after LINC00511 expression being inhibited. (h) Colony formation of PC-9 and A549 cells treated with si-NC or si-LINC00511. The colony number in LINC00511-transfected LUAD cells was less than negative control group. (i) Flow cytometry results indicate an upregulation of LUAD cells apoptosis occurred when LINC00511 expression was blocked. * P < .05, ** P < .01, compared with the normal group or si-NC group.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

Techniques: Expressing, Transfection, Negative Control, Flow Cytometry

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: Upregulated LINC00511 in LUAD cells after irradiation treatment. (a) 24 h later, LINC00511 expression in PC-9 and A549 cells treated with different doses of IR was detected by qRT-PCR. (b) LINC00511 expression in PC-9 and A549 cells treated with 4 Gy IR was detected every 4 hours for 24 hours by qRT-PCR. ** P < .01, compared with 0 Gy group.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

Techniques: Irradiation, Expressing, Quantitative RT-PCR

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: LINC00511 knockdown potentiated radiosensitivity of LUAD cells. (a) Survival fraction of PC-9 and A549 cells exposed to gradient dose of radiation. LUAD cell survival stepped down along with the radiation dose rising. (b) Survival fraction of PC-9 and A549 cells at different LINC00511 levels under gradient IR. The decline of LUAD cell survival via radiation treatment intensified further when LINC00511 expression was blocked. (c) Cell viability of PC-9 and A549 cells under different treatments. Radiation treatment and si-LINC00511 both decreased cell viability in two cell lines. And the combination of radiation treatment and si-LINC00511 transfection further inhibited LUAD cell viability compared to the groups with single treatment. (d) Colony formation of PC-9 and A549 cells in different groups. Colony numbers in si-LINC00511 group as well as si-NC+4 Gy radiation group were both smaller than the normal control. Si-LINC00511 + 4 Gy radiation presented additional suppression in colony formation. (e) Apoptosis rate of PC-9 and A549 cells with different treatments. Based on the flow cytometry, the apoptosis was activated in si-LINC00511-transfected or radiation-treated LUAD cells. In the group treated with si-LINC00511 and 4 Gy radiation, the apoptosis activation was further promoted. (f) γ-H2AX staining of PC-9 and A549 cells under different treatments. Cells treated by si-LINC00511 exhibited enhanced DNA damage in malignant cells treated by radiation. * P < .05, ** P < .01.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

Techniques: Knockdown, Expressing, Transfection, Control, Flow Cytometry, Activation Assay, Staining

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: Cytoplasmic LINC00511 was an endogenous sponge of miR-497-5p in LUAD. (a) LINC00511 information in lncATLAS. LINC00511 was predicted to locate in cytoplasm. (b) Subcellular location of LINC00511. Both in PC-9 and A549 cells, LINC00511 was identified to be located in cytoplasm. (c) Targeting relationship between LINC00511 and miR-497-5p. miR-497-5p was predicted binding to LINC00511. (d) Transfection of miR-NC or miR-497-5p to PC-9 and A549 cells. miR-497-5p expression was successfully upregulated in two LUAD cell lines after transfection. (e) Luciferase assays in PC-9 and A549. (f) LINC00511 enrichment in PC-9 and A549. Luciferase assays and miRNA array testing both proved miR-497-5p binds to LINC00511. (g) miR-497-5p expression in LUAD cell lines. (h) miR-497-5p expression in PC-9 and A549 cells treated with 4 Gy IR was detected every 4 hours for 24 hours by qRT-PCR. (i) MiR-497-5p expression in PC-9 and A549 cells transfected with si-NC or si-LINC00511. LINC00511 upregulation in LUAD cells inhibited miR-497-5p expression. ** P < .01, compared with the NC group or 0 Gy group.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

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

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: miR-497-5p overexpression promoted the radiosensitivity of LUAD cells. (a) Cell viability of PC-9 and A549 cells under different treatments. Radiation treatment and miR-497-5p both decreased cell viability in two cell lines. The combination of radiation treatment and miR-497-5p transfection further inhibited PC-9 and A549 cells viability compared to the groups with single treatment. (b-c) Colony formation of PC-9 and A549 cells in different groups. Colony numbers in miR-497-5p or 4 Gy radiation treated LUAD cells were both lower than the normal control. And the miR-497-5p+4 Gy radiation presented additional suppression in colony formation. (d-e) Apoptosis rate of PC-9 and A549 cells with different treatments. Flow cytometry results reveal that the apoptosis was activated in miR-497-5p group or 4 Gy radiation group. In miR-497-5p+4 Gy radiation group, apoptosis activation was further enhanced. * P < .05, ** P < .01.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

Techniques: Over Expression, Transfection, Control, Flow Cytometry, Activation Assay

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: LINC00511 regulated SMAD3 expression via miR-497-5p. (a) Targeting relationship between miR-497-5p and SMAD3. (b) Luciferase assays in PC-9 and A549 cells. SMAD3 binding to miR-497-5p was confirmed in LUAD cells. (c) SMAD3 expression in LUAD cell lines. (d) SMAD3 expression in PC-9 and A549 cells treated with 4 Gy IR was detected every 4 hours for 24 hours by qRT-PCR. (e) 24 hours later, SMAD3 expression in PC-9 and A549 cells treated with 4 Gy IR was detected by western blot. (f-g) 48 hours later, the mRNA (f) and protein (g) level of SMAD3 in PC-9 and A549 cells transfected with si-LINC00511 was detected by qRT-PCR and western blot. (h-i) 48 hours later, the mRNA (h) and protein (i) level of SMAD3 in PC-9 and A549 cells transfected with miR-497-5p mimics was detected by qRT-PCR and western blot. * P < .05, ** P < .01, compared with the NC group.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

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

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: SMAD3 suppression enhanced the radiosensitivity of LUAD cells. (a) SMAD3 mRNA expression in PC-9 and A549 cells treated with si-NC or si-SMAD3. (b) Western blot of SMAD3 in cells with or without si-SMAD3 treatment. (c) PC-9 and A549 cells’ survival fraction at different SMAD3 levels under gradient radiation treatment. The decline of LUAD cell survival via radiation dose rising was expanded after SMAD3 downregulation. (d-e) Apoptosis rate of PC-9 and A549 cells under different treatments. In accordance with flow cytometry results, apoptosis was activated in si-SMAD3-transfected or 4 Gy radiation-treated LUAD cells. In the group treated with the combination of si-SMAD3 and 4 Gy radiation, cell apoptosis was further promoted. ** P < .01, compared with the NC group.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

Techniques: Expressing, Western Blot, Flow Cytometry, Transfection

Journal: Cancer Biology & Therapy

Article Title: Knockdown of LINC00511 enhances radiosensitivity of lung adenocarcinoma via regulating miR-497-5p/SMAD3

doi: 10.1080/15384047.2023.2165896

Figure Lengend Snippet: LINC00511 inhibition activated miR-497-5p and mediately downregulated SMAD3, which further promoted radiosensitivity in LUAD cells. (a) SMAD3 mRNA expression in PC-9 and A549 cells of different groups. (b) SMAD3 protein expression in PC-9 and A549 cells with different treatments. Compared to the NC, si-LINC00511 transfection induced a sharp decline in SMAD3 expression, which was reversed with additional anti-miR-497-5p or SMAD3 treatment. (c) Cell viability of 4 Gy radiation-treated PC-9 and A549 cells with different treatments. Downregulated LINC00511 decreased LUAD cell viability, and along with the prolonged radiation time the decreasing was more significant, which was reversed with miR-497-5p inhibition or SMAD3 activation. (d) Colony formation of 4 Gy radiation-treated PC-9 and A549 cells with different treatments. LINC00511 downregulation suppressed colony formation of IR-treated LUAD cells while additional miR-497-5p inhibition or SMAD3 activation reactivated it. (e) Apoptosis rate of 4 Gy radiation-treated PC-9 and A549 cells in different groups. 4 Gy radiation-triggered apoptosis was further promoted via si-LINC00511 transfection, which was repressed with additional anti-miR-497-5p or SMAD3 treatment. ** P < .01, compared with the NC group.

Article Snippet: Human normal lung epithelial cell-line BEAS-2B and 4 LUAD cell lines (NCI-H1975; NCI-H441; PC-9; A549) were obtained from Shanghai iCell Bioscience Inc. BEAS-2B were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA) and 1% Pen/Strep solution.

Techniques: Inhibition, Expressing, Transfection, Activation Assay

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: CLCN3 was upregulated in human LUAD and facilitated tumor proliferation and migration. (a, b) Through IHC and IF analysis, the expression of CLCN3 was examined in a tissue microarray of 30 paraffin-embedded LUAD tissues and adjacent normal tissues (ANT) ( n = 30). (c, d) In human LUAD cell lines as well as in human bronchial epithelial cell lines, the basic protein expression of CLCN3 was measured ( n = 3). (e) The basic RNA level of CLCN3 was detected in human LUAD cell lines and a human bronchial epithelial cell line ( n = 3). (f) The protein expression of CLCN3 was inhibited after CLCN3 knockdown in H1299 and A549 cells. (g) RNA-seq was constructed after CLCN3 knockdown in H1299 cells. Locomotion and growth were significantly enriched as illustrated by GO analysis. (h) Knockdown of CLCN3 suppressed the clonogenicity of H1299 and A549 cells ( n = 3). (i) CLCN3 knockdown suppressed the invasion of H1299 and A549 cells ( n = 3). * P < 0.05.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Migration, Expressing, Microarray, RNA Sequencing Assay, Construct

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: HNRNPK was identified and validated as a CLCN3 promoter-binding transcription factor. (a) To identify putative transcription factors that bind to the CLCN3 promoter, nuclear protein extracts were subjected to incubation with a biotin-labeled DNA probe that included the promoter region. Following the SDS-PAGE separation and silver staining, a protein band with differential expression was identified after which it was excised and subjected to mass spectrometry (MS) analysis. (b) By employing either a synthesized probe or a nonspecific probe (NSP) in the nuclear protein/DNA complex, western blot analysis detected binding between HNRNPK and the CLCN3 promoter. (c) ChIP assays were performed and an HNRNPK antibody was used to immunoprecipitate the fragmented DNA in H1299 cells. The HNRNPK protein band was substantially enriched in the IP group in contrast to the IgG control. (d) The ChIP-seq data showed that the recruitment of HNRNPK was mostly enriched in the upstream and downstream TSS of the CLCN3 promoter region (NM_001829, chr4: 169620033-169620797, region -538/+226 bp). (e) The CLCN3 RNA level was inhibited following HNRNPK knockdown ( n = 3). (f) HNRNPK knockdown disrupted the promoter activities of the pGL4.10-CLCN3-538 reporter plasmid (region -538/+226 bp) in H1299 cells, and the binding motifs 'GCGAGG/CTAATG' from ChIP-seq data were enriched in this region ( n = 3). (g) GO analysis revealed that cell proliferation and migration were the biological functions of HNRNPK. (h) KEGG pathway analysis illustrated that cell growth and death, cell motility, transcription, translation, and other signaling pathways were mainly enriched after HNRNPK knockdown. (i) The luciferase activity was considerably reduced in HNRNPK knockdown cells transfected with a pGL4.10-CLCN3-538 plasmid (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Binding Assay, Incubation, Labeling, SDS Page, Silver Staining, Expressing, Mass Spectrometry, Synthesized, Western Blot, ChIP-sequencing, Plasmid Preparation, Migration, Luciferase, Activity Assay, Transfection

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: HNRNPK/CLCN3 axis facilitated LUAD progression through interaction between tumor cells and CAFs. (a) Primary human CAFs and paired NFs were extracted from fresh LUAD samples. The expression of CAF markers was increased in CAFs compared to NFs, which was in accord with the typical characteristic of CAFs. (b) The levels of CLCN3 were detected in the culture supernatants of LUAD cells, and the data indicated that decreased extracellular CLCN3 secretion could be induced by HNRNPK knockdown. (c-e) The supernatants (HNRNPK knockdown and control) of LUAD cells were incubated with CAFs for 24 h. We confirmed that the PI3K-AKT signaling pathway was significantly enriched in CAFs. (f) The levels of p-AKT, α-SMA, FAP, and COL1A1 were decreased when CAFs were stimulated with the supernatants of HNRNPK-knockdown cells. After CLCN3 knockdown in CAFs, the levels of p-AKT, α-SMA, FAP, and COL1A1 were also effectively inhibited. (g) Due to the activation inhibition, the CAFs co-cultured with HNRNPK-knockdown LUAD cells were then considered as inhibited CAFs. The analysis by ELISA revealed that the TGF-β1 production of inhibited CAFs was significantly decreased ( n = 7). (h) The supernatants of inhibited CAFs attenuated the expression of HNRNPK protein in the H1299 nucleus, and the attenuation effect was reversed after TGF-β1 treatment. (i) The supernatants of inhibited CAFs attenuated the fluorescence intensity of HNRNPK in the H1299 nucleus, which was also reversed after the addition of exogenous TGF-β1. (j, k) The supernatants of inhibited CAFs markedly attenuated the clonogenicity and invasion of LUAD cells, and this phenomenon was further reversed after the addition of TGF-β1 ( n = 3). *P < 0.05.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Expressing, Incubation, Activation Assay, Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: HNRNPK regulated the expression and function of CLCN3 in vivo . (a) In mouse xenograft models, stable HNRNPK-knockdown cells (H1299) and rescue model cells were administered to the nude mice via subcutaneous injection into their left flank. The representative figure of tumors formed in each group is shown ( n = 7). (b, c) The tumor weight and tumor growth curve were suppressed when HNRNPK was knocked down, and this inhibition was reversed by CLCN3 overexpression ( n = 7). (d) In tumor xenografts, the expression of CLCN3 was decreased following HNRNPK knockdown, however, this reduction was reversed by CLCN3 overexpression ( n = 7). (e) Nude mice received an injection with stable HNRNPK-knockdown cells (H1299) and rescue model cells into their tail veins. HNRNPK knockdown effectively decreased the average radiance of lung metastatic lesions, and the reduction was rescued by CLCN3 overexpression ( n = 7). (f) In paraffin sections of nude mouse lung metastases, we found that HNRNPK knockdown caused a reduction of lung metastasis area, which was rescued by CLCN3 overexpression ( n = 7). * P < 0.05.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Expressing, In Vivo, Injection, Inhibition, Over Expression

Journal: International Journal of Biological Sciences

Article Title: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction

doi: 10.7150/ijbs.76083

Figure Lengend Snippet: A schematic representation of the relationship between CLCN3 and HNRNPK in LUAD progression. Firstly, CLCN3 was upregulated in human LUAD and facilitated tumor proliferation and migration. Secondly, HNRNPK was validated as a CLCN3 promoter-binding transcription factor, and the binding motif 'GCGAGG' and binding site '-538/-248 bp' were identified. Finally, the expression and function of CLCN3 were regulated by HNRNPK in vitro and in vivo , and the HNRNPK-CLCN3 axis facilitated LUAD progression in a feedback way through CAF-tumor interaction in the TME.

Article Snippet: After acquiring the human LUAD cell lines (H1299, A549, and PC-9), authentication was done using STR profiling from Shanghai GenePharma Co. (Shanghai, China).

Techniques: Migration, Binding Assay, Expressing, In Vitro, In Vivo

Journal: Oncogenesis

Article Title: Aberrant super-enhancer landscape reveals core transcriptional regulatory circuitry in lung adenocarcinoma

doi: 10.1038/s41389-020-00277-9

Figure Lengend Snippet: A Enhancer regions of the normal adult lung, A549, PC-9 cells were plotted in increasing order based on their H3K27Ac ChIP-Seq signal. Enhancers above the inflection point of the curve were defined as SEs. Examples of SE-genes are listed along with their respective rank. B SE-associated gene in A549 and PC-9 cells were systematically compared with SE-associated genes in normal lung tissue. Totally, 1278 and 848 SE-associated genes were acquired in A549 and PC-9 cells, respectively. A total of 1792 SE-associated genes were acquired in A549 and PC-9 cells, in which 375 SE-associated genes were commonly acquired in both cell lines. C The gene ontology enrichment analysis suggested that LUAD-SE genes were significantly associated with biological processes essential to cancer sustainability. D The KEGG pathway enrichment analysis revealed that LUAD-SE genes were significantly enriched in multiple cancer-relating signaling pathways. E Volcano plot illustrating the biological and statistical significance of LUAD-SE genes are highlighted in red, and non-SE-genes are highlighted in gray. F The expression profile of the common LUAD-SE gene was clearly segregated between LUAD samples and NT samples in the unsupervised clustering analysis.

Article Snippet: Human LUAD cell lines PC-9 and A549 were purchased from KeyGen Biotech (KeyGen Biotech, Nanjing, China), and detected for mycoplasma before use.

Techniques: ChIP-sequencing, Expressing

Journal: Oncogenesis

Article Title: Aberrant super-enhancer landscape reveals core transcriptional regulatory circuitry in lung adenocarcinoma

doi: 10.1038/s41389-020-00277-9

Figure Lengend Snippet: A Experimental validation for LUAD cell circuitry. Core regulatory circuit containing ELF3, EHF, and TGIF1 for LUAD cell lines, A549 and PC-9. B Intersections of TFs between two individual LUAD cell lines, A549 and PC-9, identified by CRC models. C The expression levels of selected TFs in tumor or normal tissues of patients with LUAD based on the TCGA illumina HiSeq RNA-Seq data. The x -axis represents the different tissues of LUAD patients and the y -axis represents expression read counts of TCGA. D Kaplan–Meier survival analysis with TCGA datasets indicates that higher ELF3 ( p = 0.011), EHF ( p = 0.00024), and TGIF1 ( p = 0.00028) expression is associated with a worse overall survival in patients with LUAD. E ChIP-seq occupancy profiles of H3K27Ac at the normal adult lung, A549 cell line and PC-9 cell line. H3K27Ac ChIP-seq signal was highly enriched in the ELF3, EHF and TGIF1 genomic loci of A549 and PC-9 cells but not the normal liver. TCGA The Cancer Genome Atlas.

Article Snippet: Human LUAD cell lines PC-9 and A549 were purchased from KeyGen Biotech (KeyGen Biotech, Nanjing, China), and detected for mycoplasma before use.

Techniques: Expressing, RNA Sequencing Assay, ChIP-sequencing

Journal: International Journal of Medical Sciences

Article Title: The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma

doi: 10.7150/ijms.36071

Figure Lengend Snippet: The receiver operating characteristic (ROC) curve analysis and Kaplan-Meier curves of the disease-free survival (DFS) in patients with Stage I LUAD after surgery according to the PRDX4 expression and the EGFR mutation status. (A) We selected 40% as the cut-off point for PRDX4, since the sum of sensitivity and specificity was the highest at this point. (B) Patients with EGFR mutations and high-PRDX4 showed significantly longer postsurgical DFS. (C) Patients with EGFR wild-type and low-PRDX4 showed significantly shorter postsurgical DFS.

Article Snippet: PC-9, a human LUAD cell line with EGFR mutation, was purchased from Riken BioResource Center (Tsukuba, Japan).

Techniques: Expressing, Mutagenesis