human lung squamous cell carcinoma cell line nci h520  (ATCC)

Journal: Scientific Reports

Article Title: Transcriptional activation of PPP1R14C by KLF7 unleashes CDK1 activity to promote lung squamous cell carcinoma

doi: 10.1038/s41598-026-39174-3

Figure Lengend Snippet: PPP1R14C is significantly upregulated in LUSC and portends poor patient prognosis. ( A ) Pan-cancer transcriptome analysis of PPP1R14C expression in human cancers using GEPIA2 ( http://gepia2.cancer-pku.cn/#survival ) revealed that PPP1R14C expression was significantly elevated in lung squamous cell carcinoma (LUSC) compared with corresponding normal tissues and other malignancies. ( B ) PPP1R14C mRNA is highly expressed in LUSC. Analysis of TCGA-LUSC data showed that PPP1R14C expression was significantly higher in primary tumor tissue (T) than in matched normal lung tissue (N). ( C ) Correlation between PPP1R14C expression and LUSC clinical stage. TCGA-LUSC data showed that PPP1R14C expression was significantly higher in all tumor stages compared to normal tissue (Normal). Stage comparison revealed significant differences in PPP1R14C expression across different clinical stages of LUSC. ( D ) Transcriptomic data from CPTAC showed that PPP1R14C mRNA levels were significantly higher in lung cancer tissues compared with matched adjacent normal tissues (**** p < 0.0001). ( E ) PPP1R14C protein is upregulated in LUSC. CPTAC proteomic data indicate that PPP1R14C protein levels are significantly higher in LUSC tumor tissues compared with adjacent normal tissues (*** p < 0.001). ( F ) Prognostic value of PPP1R14C in LUSC. Kaplan–Meier survival analysis (TCGA-LUSC) showed that high PPP1R14C expression is significantly associated with shorter overall survival (OS) in patients ( p = 6.56e-03, HR = 1.85).

Article Snippet: Human LUSC cell lines NCI-H520 and SK-MES-1, as well as human embryonic kidney 293 T cells, were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Comparison

Journal: Scientific Reports

Article Title: Transcriptional activation of PPP1R14C by KLF7 unleashes CDK1 activity to promote lung squamous cell carcinoma

doi: 10.1038/s41598-026-39174-3

Figure Lengend Snippet: KLF7 is a direct transcriptional activator of PPP1R14C in LUSC. ( A ) Identification of candidate PPP1R14C transcriptional regulators. A Venn diagram shows the intersection of PPP1R14C promoter-binding transcription factors predicted by JASPAR, GTRD, and ChIP-Atlas with PPP1R14C co-expressed genes based on GTEx and TCGA-LUSC. Three candidate factors—KLF7, SMAD3, and CEBPA—were identified. ( B ) Correlation analysis between KLF7 and PPP1R14C. Expression correlation analysis based on TCGA-LUSC data showed that among the three candidate transcription factors, KLF7 had the strongest correlation with PPP1R14C. ( C , D ) LUSC-specific KLF7–PPP1R14C correlation. Pan-cancer analysis showed that the positive correlation between KLF7 and PPP1R14C was most significant in LUSC, and the TCGA-LUSC scatter plot further confirmed the significant positive correlation between the two in LUSC (R = 0.285, p = 9.78e − 11). ( E ) KLF7 overexpression upregulates PPP1R14C. Analysis of the GSE92507 dataset indicated that in vitro overexpression of KLF7 significantly increases PPP1R14C levels (* p < 0.05). ( F ) Schematic diagram of the PPP1R14C promoter. The diagram shows the structure of the PPP1R14C promoter, with a predicted KLF7 binding motif located in the region − 498 to − 480 bp upstream of the transcription start site (TSS). ( G ) Overexpression of KLF7 increases PPP1R14C expression. qRT-PCR analysis revealed that PPP1R14C mRNA levels significantly increased in NCI-H520 cells following KLF7 overexpression ( **** p < 0.0001). ( H ) KLF7 knockdown reduces PPP1R14C expression. qRT-PCR analysis showed that knockdown of KLF7 using two independent shRNAs (shKLF7-#1 and shKLF7-#2) significantly decreased PPP1R14C mRNA levels in NCI-H520 cells (** p < 0.01, *** p < 0.001). ( I , J ) KLF7 activates the PPP1R14C promoter. Luciferase reporter assays showed that KLF7 significantly activated the wild-type (WT) PPP1R14C promoter in 293 T cells, whereas mutation of the KLF7 binding site (MUT) abolished this activation (* p < 0.05). ( K ) KLF7 directly binds to the PPP1R14C promoter. ChIP-qPCR analysis in NCI-H520 cells showed significant enrichment of KLF7 at the PPP1R14C promoter region containing the predicted binding site compared with the IgG control (** p < 0.01).

Article Snippet: Human LUSC cell lines NCI-H520 and SK-MES-1, as well as human embryonic kidney 293 T cells, were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Binding Assay, Expressing, Over Expression, In Vitro, Quantitative RT-PCR, Knockdown, Luciferase, Mutagenesis, Activation Assay, ChIP-qPCR, Control

Journal: Scientific Reports

Article Title: Transcriptional activation of PPP1R14C by KLF7 unleashes CDK1 activity to promote lung squamous cell carcinoma

doi: 10.1038/s41598-026-39174-3

Figure Lengend Snippet: PPP1R14C enhances proliferation, invasion, and tumorigenicity of LUSC cells. ( A ) Validation of PPP1R14C shRNA knockdown efficiency. Five independent shRNAs were tested in NCI-H520 cells, and their knockdown effects on PPP1R14C were assessed by qRT-PCR. The results showed that shPPP1R14C-#1 and shPPP1R14C-#2 achieved the most effective knockdown and were therefore selected for subsequent experiments (**** p < 0.0001). ( B ) Validation of PPP1R14C protein knockdown. Western blot analysis showed that PPP1R14C protein levels were significantly reduced in NCI-H520 and SK-MES-1 cells transduced with shPPP1R14C-#1 or shPPP1R14C-#2. ( C ) PP1R14C knockdown inhibits LUSC cell proliferation. CCK-8 assays revealed that NCI-H520 and SK-MES-1 cells exhibited significantly inhibited proliferation after PPP1R14C knockdown compared with the control group. ( D ) PPP1R14C knockdown affects DNA synthesis. EdU incorporation assay in NCI-H520 cells showed that DNA synthesis was reduced after PPP1R14C knockdown (** p < 0.01, *** p < 0.001). ( E ) PPP1R14C promotes the clone formation of LUSC cells. The results of the clone formation experiment showed that after PPP1R14C silencing, the clone formation ability of cells was significantly weakened (** p < 0.01, *** p < 0.001). ( F ) PPP1R14C knockdown impairs the invasive ability of LUSC cells. Transwell invasion assays showed that the invasive ability of NCI-H520 cells with PPP1R14C knockdown was significantly weakened compared with the control group (** p < 0.01). ( G ) Knockdown of PPP1R14C significantly increased the overall apoptotic rate of NCI-H520 LUSC cells, as determined by Annexin V-FITC/PI staining followed by flow cytometry (** p < 0.01). ( H ) PPP1R14C knockdown promotes cell apoptosis. Caspase-3/7 activity assay results showed that the apoptosis level of NCI-H520 cells was significantly increased after PPP1R14C knockdown (* p < 0.05). ( I – K ) PPP1R14C promotes LUSC tumor growth in vivo. Xenografts of NCI-H520 cells expressing either PPP1R14C knockdown (shPPP1R14C) or control (shCtrl) were performed. Results showed that tumor growth in the PPP1R14C knockdown group was inhibited ( H ), ultimately leading to a reduction in tumor volume ( I ), and a significant decrease in tumor weight ( J ) (*** p < 0.001, **** p < 0.0001).

Article Snippet: Human LUSC cell lines NCI-H520 and SK-MES-1, as well as human embryonic kidney 293 T cells, were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Biomarker Discovery, shRNA, Knockdown, Quantitative RT-PCR, Western Blot, Transduction, CCK-8 Assay, Control, DNA Synthesis, Staining, Flow Cytometry, Activity Assay, In Vivo, Expressing

Journal: Scientific Reports

Article Title: Transcriptional activation of PPP1R14C by KLF7 unleashes CDK1 activity to promote lung squamous cell carcinoma

doi: 10.1038/s41598-026-39174-3

Figure Lengend Snippet: PPP1R14C promotes LUSC cell proliferation and invasion via CDK1 activation. ( A ) Overexpression of PPP1R14C activates CDK1 through T161-CDK1 phosphorylation. Western blot analysis revealed that compared to the control group, overexpression of PPP1R14C in NCI-H520 cells significantly increased the phosphorylation levels of T161-CDK1 and histone H1 (p-CDK1, P-T161(CDK1), and p-Histone H1). ( B ) PPP1R14C knockdown inhibits CDK1 activation by reducing T161-CDK1 phosphorylation. Western blot analysis showed that compared to the control group, PPP1R14C knockdown (shPPP1R14C) significantly reduced the expression levels of p-CDK1, P-T161(CDK1), and p-Histone H1. ( C ) Interaction between PPP1R14C and PP1α. Co-immunoprecipitation experiments showed that PPP1R14C antibodies pulled down PP1α, while isotype control IgG did not detect PP1α signals, indicating that PPP1R14C can directly bind to PP1α. ( D ) Overexpression of PPP1R14C inhibits the binding of CDK1 to PP1α. Co-immunoprecipitation experiments showed that PP1α could co-precipitate with CDK1 in cells expressing the empty vector, while overexpression of PPP1R14C inhibited the binding of CDK1 to PP1α. ( E ) CDK1 inhibition abolished PPP1R14C-mediated proliferation. CCK-8 proliferation assays showed that the growth advantage conferred by PPP1R14C overexpression was completely abolished by the CDK1 inhibitor RO-3306 (* p < 0.05, ** p < 0.01, **** p < 0.0001). ( F ) CDK1 activity is critical for PPP1R14C-driven clonogenicity. Colony formation experiments showed that inhibition of CDK1 with RO-3306 neutralized PPP1R14C-driven clonogenic growth (* p < 0.05, **** p < 0.0001). ( G ) CDK1 inhibition attenuated the invasive ability enhanced by PPP1R14C. Transwell invasion assays showed that blocking CDK1 with RO-3306 could inhibit the pro-invasive effect of PPP1R14C overexpression (* p < 0.05, **** p < 0.0001).

Article Snippet: Human LUSC cell lines NCI-H520 and SK-MES-1, as well as human embryonic kidney 293 T cells, were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Activation Assay, Over Expression, Phospho-proteomics, Western Blot, Control, Knockdown, Expressing, Immunoprecipitation, Binding Assay, Plasmid Preparation, Inhibition, CCK-8 Assay, Activity Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Transcriptional activation of PPP1R14C by KLF7 unleashes CDK1 activity to promote lung squamous cell carcinoma

doi: 10.1038/s41598-026-39174-3

Figure Lengend Snippet: Schematic model. In normal lung epithelial cells (left panel), the transcription factor KLF7 is expressed at a basal level, leading to low expression of its target, PPP1R14C. Consequently, the protein phosphatase PP1 remains active and available to dephosphorylate key substrates, including the activating phosphorylation site (Thr161) of CDK1. This maintains CDK1 activity in a tightly regulated state, ensuring normal, controlled cell cycle progression. In LUSC cells (right panel), aberrant overexpression of KLF7 drives the transcriptional upregulation of PPP1R14C. The elevated PPP1R14C protein then physically binds to and sequesters the PP1 catalytic subunit, potently inhibiting its phosphatase activity. This “release of the brakes” prevents the dephosphorylation and inactivation of CDK1, resulting in its sustained hyperactivation. Hyperactive CDK1 then promotes an uncontrolled G2/M transition, fueling relentless cell proliferation, invasion, and ultimately, tumorigenesis. This axis represents a key vulnerability, as pharmacological inhibition of CDK1 can block the downstream oncogenic consequences, providing a compelling therapeutic strategy for PPP1R14C-overexpressing LUSC.

Article Snippet: Human LUSC cell lines NCI-H520 and SK-MES-1, as well as human embryonic kidney 293 T cells, were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Phospho-proteomics, Activity Assay, Over Expression, De-Phosphorylation Assay, Inhibition, Blocking Assay