cervical cancer cell lines siha  (ATCC)

Journal: bioRxiv

Article Title: Covalent Inhibition of the Human Papillomavirus Type 16 E6 Protein Restores p53 and Suppresses HPV-Driven Tumorigenesis

doi: 10.1101/2025.08.24.671874

Figure Lengend Snippet: A stable clone of HPV-16 E6/ E7 expressing human cervical cancer SiHa cells with a p53-Luciferase (p53-Luc) reporter and a clone of HPV-negative RPE-1 cells expressing the same p53-Luc reporter were incubated with DMSO (0.02% (v/v)) or increasing concentrations of compounds added to the media. SiHa-P53-Luc cells (red) and RPE-luc cells (blue) were treated with increasing concentrations of (A) KTI-218 or (B) KT-240 for 24 hrs. To study the contribution of the covalent warhead, we prepared, KTI-239, an analog of KTI-218 that lacks the acrylamide warhead and tested in SiHa-P53-Luc (C) and RPE-luc cells (D ), respectively. Structures of each compound is shown in the insets. Luciferase and cell viability were measured after 24 hours. Luc signal was normalized to cell viability and p53-Luc induction is expressed as fold-change over DMSO control. Data is expressed as S.E.M and each experiment was completed at least three independent times (n ≥ 3).

Article Snippet: These DNA constructs were transfected into HPV16 expressing human cervical cancer derived cell line SiHa (ATCC-HTB35TM), which was selected because levels of E6 and E7 are directed by the native HPV16 promoter.

Techniques: Stable Transfection, Expressing, Luciferase, Incubation, Control

Journal: bioRxiv

Article Title: Covalent Inhibition of the Human Papillomavirus Type 16 E6 Protein Restores p53 and Suppresses HPV-Driven Tumorigenesis

doi: 10.1101/2025.08.24.671874

Figure Lengend Snippet: SiHa, SiHa C51S, and RPE-1 cells were cultured in KTI-240 (5 µM), DMSO or etoposide (ETO, 25 µM) for 16 hours, cells were lysed, proteins extracted and subjected to Western blot analysis. A) KTI-240 increased p21 levels without affecting p16 protein. (B) E6AP and MDM2 protein levels were not changed after KTI-240 treatment. p53 protein was included as a positive control. KTI-240 decreased Foxm1 and Rb protein expression in SiHa and C51S cells but not RPE-1, while Puma increased only in SiHa cells. The heatmaps in (E) and (F) summarize the detected changes at the mRNA and protein level. (G, H) E6 protein levels were significantly decreased in SiHa cells, while E7 was unchanged in both HPV+ cell lines. Protein expression was normalized to GAPDH and expressed as fold-change. Data was expressed as S.E.M and analyzed using one-way ANOVA with Dunnett’s or Bonferroni post hoc analysis were applicable. Experiments were repeated at least three independent times (n ≥ 3; * p < 0.05).

Article Snippet: These DNA constructs were transfected into HPV16 expressing human cervical cancer derived cell line SiHa (ATCC-HTB35TM), which was selected because levels of E6 and E7 are directed by the native HPV16 promoter.

Techniques: Cell Culture, Western Blot, Positive Control, Expressing

Journal: bioRxiv

Article Title: Covalent Inhibition of the Human Papillomavirus Type 16 E6 Protein Restores p53 and Suppresses HPV-Driven Tumorigenesis

doi: 10.1101/2025.08.24.671874

Figure Lengend Snippet: SiHa, SiHa C51S, and RPE-1 cells were cultured in KTI-240 for 16 hours and RNA was extracted and sequenced from three independent experiments. (A, B) . Parallel cell cultures were analyzed for p53 protein levels by western blots showing a robust induction of p53. Data presented as S.E.M (n=3; * p<0.05). (C-H) . Differentially expressed genes were identified using DESeq2 analysis with a false discovery rate of <0.01 and a fold change of >2. Volcano plots showing significant transcriptional increases (right, red) and decreases (left, blue) comparing vehicle vs. KTI-240 treated cells. HPV-16 E6 and E7 mRNAs were detected and did not differ between WT and C51S SiHa cells lines. ( F, H ). TP53 mRNA was unchanged in all three cell lines upon KTI-240 treatment. Increased expression of p53 regulated genes occurred only in wild-type SiHa but not C51S or RPE-1 cells ( F, G, H ). p53 protein expression was normalized to GAPDH and expressed as fold-change. Data is expressed as S.E.M and was analyzed with one-way ANOVA with Dunnett’s or Bonferroni post hoc analysis. Each experiment was completed three independent times (n = 3; * p < 0.05).

Article Snippet: These DNA constructs were transfected into HPV16 expressing human cervical cancer derived cell line SiHa (ATCC-HTB35TM), which was selected because levels of E6 and E7 are directed by the native HPV16 promoter.

Techniques: Cell Culture, Western Blot, Expressing

Journal: bioRxiv

Article Title: Covalent Inhibition of the Human Papillomavirus Type 16 E6 Protein Restores p53 and Suppresses HPV-Driven Tumorigenesis

doi: 10.1101/2025.08.24.671874

Figure Lengend Snippet: (A) HPV+ cervical cancer cell lines, SiHa and CaSki, the oral cancer derived lines UM-SCC-47 and UM-SCC-104, and RPE-1 were incubated with increasing concentrations of KTI-218 or DMSO for 24 hours and cell viability was analyzed by Calcein-AM assay. (B) HPV negative RPE-1, human foreskin keratinocytes (HFK) and C51S mutant cells were analyzed for their cell viability after KTI-218 treatment. (C, D) cell viability of SiHa, CaSki, SCC-47, SCC-104, SiHa C51S, and RPE-1, HFK and normal oral keratinocytes was assessed after KTI-240 or DMSO treatment with for 48 hours. Cell viability is expressed as a percent change over DMSO control-treated cells. Data expressed as S.E.M and each experiment was completed at least three independent times (n ≥ 3).

Article Snippet: These DNA constructs were transfected into HPV16 expressing human cervical cancer derived cell line SiHa (ATCC-HTB35TM), which was selected because levels of E6 and E7 are directed by the native HPV16 promoter.

Techniques: Derivative Assay, Incubation, Calcein AM Assay, Mutagenesis, Control

Journal: bioRxiv

Article Title: Covalent Inhibition of the Human Papillomavirus Type 16 E6 Protein Restores p53 and Suppresses HPV-Driven Tumorigenesis

doi: 10.1101/2025.08.24.671874

Figure Lengend Snippet: ( A, B ) Nu/Nu mice were injected subcutaneously with HPV-16+ SCC-UM-47 cancer cells. Intraperitoneal injections of 50 mg/kg KTI-218 (n=8, green) or vehicle (VH, n=7 black) began when tumors were ∼50-100 mm 3 . Tumor size was measured by calipers and expressed as S.E.M. and analyzed using two-way ANOVA (p<0.01). Representative mice and extracted tumors are shown in ( B ). Nu/Nu mice were injected subcutaneously with HPV-16+ SiHa cancer cells stably expressing a Luciferase construct ( C ). Intraperitoneal (IP) injections of 50 mg/kg KTI-218 (n=5, green) or vehicle (VH, n=4 black) began when all tumors had a bioluminescent signal. Bioluminescence was measured using a IVIS SpectrumCT optical imaging system ( D ). Representative tumors and tumor weights are shown in ( E ). Nu/Nu mice were injected subcutaneously (SC) with ( F ) HPV-negative cervical cancer cell line C33a or ( G ) HPV + SiHa C51S cells. Intraperitoneal injections of 50 mg/kg KTI-218 began when tumors were ∼50-100 mm 3 . ( H ) Nu/Nu mice were injected SC with HPV-16+ SCC-UM-47 cancer cells. IP injections of 50 mg/kg KTI-240 (n=10, green) or vehicle (VH, n=9 black) began when tumors were ∼50-100 mm 3 . Data expressed as S.E.M and analyzed using two-way ANOVA; * p <0.05.

Article Snippet: These DNA constructs were transfected into HPV16 expressing human cervical cancer derived cell line SiHa (ATCC-HTB35TM), which was selected because levels of E6 and E7 are directed by the native HPV16 promoter.

Techniques: Injection, Stable Transfection, Expressing, Luciferase, Construct, Optical Imaging

Journal: Proteomes

Article Title: Cell Surface Proteomics Reveals Hypoxia-Regulated Pathways in Cervical and Bladder Cancer

doi: 10.3390/proteomes13030036

Figure Lengend Snippet: Volcano plots of differentially abundant proteins under hypoxia in SiHa and UMUC3 cells. ( A ) SiHa cervical cancer cells and ( B ) UMUC3 bladder cancer cells were cultured under hypoxic conditions (0.1% O 2 ) for 48 h. Plasma membrane proteins were isolated using biotin-based surface enrichment, while total cellular proteins were obtained via whole-cell lysis. Following LC-MS/MS analysis, differential protein abundance was determined by comparing hypoxia vs. normoxia, with significance thresholds set at FDR < 0.05 and |log 2 fold change| ≥ 1. Each volcano plot displays log 2 fold change ( x -axis) versus –log 10 FDR ( y -axis) for all quantified proteins. The total number of proteins shown (6029 for SiHa and 6337 for UMUC3) represents the combined set of proteins identified in either the biotin-enriched fraction, the whole-cell lysate fraction, or both for each cell line. Proteins detected in both fractions were counted once, and proteins found in only one fraction were also included, reflecting the full set of proteins evaluated for differential abundance.

Article Snippet: UMUC3 (bladder) and SiHa (cervical) cancer cell lines, obtained from the American Type Culture Collection (ATCC), were cultured in Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle Medium (DMEM), respectively.

Techniques: Cell Culture, Clinical Proteomics, Membrane, Isolation, Lysis, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics

Journal: Proteomes

Article Title: Cell Surface Proteomics Reveals Hypoxia-Regulated Pathways in Cervical and Bladder Cancer

doi: 10.3390/proteomes13030036

Figure Lengend Snippet: Heatmaps of Differentially abundant proteins in Biotin-Enriched and Whole-Cell Fractions of SiHa and UMUC3 Cells Under Hypoxia. SiHa and UMUC3 cells were cultured under normoxia (21% O 2 ) and hypoxia (0.1% O 2 ) for 48 h. Both biotin-enriched membrane proteins and whole-cell lysates were analysed. ( A ) SiHa: proteins with |log 2 FC| ≥ 1 and FDR < 0.05. Gene names shown for the top 40 detected in the biotin-enriched fraction only. ( B ) UMUC3: proteins with |log 2 FC| ≥ 1 and adjusted p < 0.05. Gene names shown for the top 40 detected in the biotin-enriched fraction only. To avoid overlapping, arrows were added from each gene’s original row; gene names may not align directly with their rows.

Article Snippet: UMUC3 (bladder) and SiHa (cervical) cancer cell lines, obtained from the American Type Culture Collection (ATCC), were cultured in Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle Medium (DMEM), respectively.

Techniques: Cell Culture, Membrane

Journal: Proteomes

Article Title: Cell Surface Proteomics Reveals Hypoxia-Regulated Pathways in Cervical and Bladder Cancer

doi: 10.3390/proteomes13030036

Figure Lengend Snippet: GO and Pathways Enrichment Analyses of Differentially abundant proteins in SiHa Cervical Cancer Cells Under Hypoxia. Differentially abundant proteins (log 2 FC| ≥ 1, FDR < 0.05) were identified in SiHa cells cultured under normoxia (21% O 2 ) and hypoxia (0.1% O 2 ) for 48 h, using either a biotin-based plasma membrane enrichment method or conventional whole-cell lysate extraction. Enrichment analyses were conducted using STRING (v12). ( A,B ) GO Cellular Component (CC) enrichment. ( A ) Whole-cell lysate; ( B ) Biotin-based fraction. ( C,D ) GO Biological Process (BP) enrichment. ( C ) Whole-cell lysate; ( D ) Biotin-enriched fraction. ( E,F ) Reactome pathway enrichment. ( E ) Whole-cell lysate; ( F ) Biotin-based fraction. ( G,H ) KEGG pathway enrichment. ( G ) Whole-cell lysate; ( H ) Biotin-based fraction. Signal represents the pathway enrichment score, which reflects the strength of enrichment based on the number of mapped proteins and their statistical significance. Abbreviations: Mito, mitochondrial; diff, differentiation.

Article Snippet: UMUC3 (bladder) and SiHa (cervical) cancer cell lines, obtained from the American Type Culture Collection (ATCC), were cultured in Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle Medium (DMEM), respectively.

Techniques: Cell Culture, Clinical Proteomics, Membrane, Extraction

Journal: Proteomes

Article Title: Cell Surface Proteomics Reveals Hypoxia-Regulated Pathways in Cervical and Bladder Cancer

doi: 10.3390/proteomes13030036

Figure Lengend Snippet: Protein–Protein Interaction (PPI) Networks of Differentially abundant proteins in SiHa and UMUC3 Cancer Cells. ( A ) SiHa biotin-enriched plasma membrane fraction. The network shows differentially abundant proteins (|log 2 FC| ≥ 1, FDR < 0.05) identified from SiHa cells cultured under normoxic (21% O 2 ) and hypoxic (0.1% O 2 ) conditions for 48 h, following a biotin-based membrane enrichment strategy prior to LC-MS/MS. The resulting network contains 172 nodes and 215 edges, with an average node degree of 2.5 and an average local clustering coefficient of 0.249 (PPI enrichment p -value < 1.0 × 10 −16 ). ( B ) UMUC3 biotin-enriched plasma membrane fraction. The network shows differentially abundant proteins (|log 2 FC| ≥ 1, FDR < 0.05) from UMUC3 cells cultured under identical conditions and processed using the same enrichment approach. This network comprises 340 nodes and 225 edges, with an average node degree of 1.32 and a clustering coefficient of 0.19 (PPI enrichment p -value < 1.0 × 10 −16 ). In both panels, nodes represent proteins, and edges represent predicted functional associations. A minimum required interaction scores of 0.7 (high confidence) was applied. Node colours represent distinct functional clusters identified using MCL clustering (inflation = 2). Networks were generated using STRING (v12).

Article Snippet: UMUC3 (bladder) and SiHa (cervical) cancer cell lines, obtained from the American Type Culture Collection (ATCC), were cultured in Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s Modified Eagle Medium (DMEM), respectively.

Techniques: Clinical Proteomics, Membrane, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Generated