c33a hpv human cervical cancer cell line  (ATCC)

Journal: Cancers

Article Title: Physical Interaction between HPV16E7 and the Actin-Binding Protein Gelsolin Regulates Epithelial-Mesenchymal Transition via HIPPO-YAP Axis

doi: 10.3390/cancers13020353

Figure Lengend Snippet: HPV16E7 expression promotes actin cytoskeleton remodeling, cell invasion ability and EMT in human cervical cancer cells. ( A ) IVM analysis after double-cell staining with TRITC-phalloidin (red) and Hoechst (blue) in C33A cells transfected (green) with pAmCyan empty vector (Mock), pAmCyan-HPV16E7 wild-type (E7wt) or deletion (E7mut) mutant constructs pAmCyan-HPV16E7Δ62–66 (E7Δ62–66) or pAmCyan-HPV17E7Δ71–75 (E7Δ71–75). E7wt-expressing cells show different morphological features compared to both Mock- and E7 mutant transfected cells. Arrow indicates protrusive and invasive structures (left). Flow cytometry evaluation of the intracellular amount of F-actin restricted to pAmCyan-positive cells. Histograms obtained in a representative experiment are shown. Bar graph shows the mean ± SD of the median fluorescence intensity obtained in four different experiments. ** p < 0.01 and *** p < 0.001 vs. Mock transfected cells (right). ( B ) Representative images of transwell invasion assay of pAmCyan C33A transfected cells (left). Fluorescence emission of C33A migrating through Matrigel are quantified by IVM analysis and expressed as percentage (right). ( C ) Western blot analysis of the expression of the EMT markers Zeb1 and Snail. β−actin determination was used as loading controls. Bar graph (bottom) shows relative densitometry quantitation of each protein normalized to β-actin obtained in three independent experiments and reported as mean ±SD. ** p < 0.01 vs. Mock transfected cells. Uncropped western blot figure available in . ( D ) Bar graph showing the evaluation of mRNA levels of Zeb1 and Snail performed by qRT-PCR assay. Data are reported as mean ±SD of RNAs relative fold change vs Mock-transfected cells obtained in three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Mock transfected cells.

Article Snippet: C33A (HPV-) human cervical cancer cell line (American Type Culture Collection ATCC; Rockville, MD, USA) was cultured in DMEM (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% FBS (Euroclone, Milan, Italy).

Techniques: Expressing, Staining, Transfection, Plasmid Preparation, Mutagenesis, Construct, Flow Cytometry, Fluorescence, Transwell Invasion Assay, Western Blot, Quantitation Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Physical Interaction between HPV16E7 and the Actin-Binding Protein Gelsolin Regulates Epithelial-Mesenchymal Transition via HIPPO-YAP Axis

doi: 10.3390/cancers13020353

Figure Lengend Snippet: Effect of Cytochalasin D on p-YAP/AMOT1 interaction and expression of EMT markers. ( A ) Quantitative evaluation of p-YAP/AMOT1 molecular association by FRET technique, as revealed by flow cytometry analysis restricted to pAmCyan-positive cells treated or not with CytoD (1 mM for 4 h). Numbers indicate the percentage of FL3 positive events obtained in one experiment representative of three. Bar graphs on the right show FRET efficiency calculated according to the Riemann’s algorithm. Data are reported as mean ± SD from three independent experiments. ** p < 0.01 vs. Mock transfected cells. ( B ) Western blot analysis of the expression of the EMT markers Zeb1 and Snail in C33A expressing E7wt or E7 mutated treated with CytoD. β-actin determination was used as loading controls. Bar graph (right) shows relative densitometry quantitation of each protein normalized to β-actin obtained in three independent experiments and reported as mean ± SD. ** p < 0.01, *** p < 0.001 vs the same sample treated with CytoD. Uncropped western blot images for B are available in .

Article Snippet: C33A (HPV-) human cervical cancer cell line (American Type Culture Collection ATCC; Rockville, MD, USA) was cultured in DMEM (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% FBS (Euroclone, Milan, Italy).

Techniques: Expressing, Flow Cytometry, Transfection, Western Blot, Quantitation Assay

Journal: Discover. Oncology

Article Title: miR-17 ~ 92 suppresses proliferation and invasion of cervical cancer cells by inhibiting cell cycle regulator Cdt2

doi: 10.1007/s12672-023-00775-3

Figure Lengend Snippet: miR-17 ~ 92 over expression decreases Cdt2 expression in cervical cancer cells. A RT-qPCR analysis of mRNA level of Cdt2, 48 h after transfection of miR-17 ~ 92 in HEK293T, C33A and SiHa cell lines B Western blot for analysis of Cdt2 protein expression level, 48 h after transfection of miR-17 ~ 92 in HEK293T, SiHa, HeLa and C33A cell lines. C Quantification of Cdt2 protein level, 48 h after miR-17 ~ 92 transfection in HEK293T, SiHa, HeLa and C33A cell lines compared to vector control. The experiments were done in triplicates. Error bar depicts standard error (S.E.). *p value < 0.05 and **p value < 0.001

Article Snippet: C33A (HPV negative cervical cancer cell line) and HEK293T (Human Embryonic Kidney cell line) were procured from National Centre for Cell Science (NCCS, Pune, India).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Plasmid Preparation

Journal: Discover. Oncology

Article Title: miR-17 ~ 92 suppresses proliferation and invasion of cervical cancer cells by inhibiting cell cycle regulator Cdt2

doi: 10.1007/s12672-023-00775-3

Figure Lengend Snippet: miR-17 ~ 92 was ectopically expressed and the proliferation was observed from day of transfection till day 4. A Growth curve of HEK293T cells with its respective control. B Control and treated HEK293T cells after 48 h of transfection under phase contrast microscopy. C Growth curve of SiHa cells along with its respective control D Control and treated SiHa cells under phase contrast microscopy after 48 h of transfection. E Growth curve of HeLa and its respective control. F Control and treated HeLa cells after 48 h of transfection under phase contrast microscopy. G Growth curve of C33A cells and its respective control. H Control and treated C33A cells after 48 h of transfection under phase contrast microscopy. The experiment was done in triplicate. Error bar represents S.D. *p value- < 0.05, **p value < 0.001

Article Snippet: C33A (HPV negative cervical cancer cell line) and HEK293T (Human Embryonic Kidney cell line) were procured from National Centre for Cell Science (NCCS, Pune, India).

Techniques: Transfection, Microscopy

Journal: bioRxiv

Article Title: miR-17∼92 Suppresses Proliferation and Invasion of Cervical Cancer Cells by Inhibiting Cell Cycle Regulator Cdt2

doi: 10.1101/2022.12.30.522306

Figure Lengend Snippet: miR17-92 over expression decreases Cdt2 expression in cervical cancer cells. A . Different miRNAs from miR 17-92 cluster targets Cdt2/DTL according to different miRNA databases. B . RT-qPCR analysis of mRNA level of Cdt2 48h after transfection of miR-17∼92 in HEK293T, C33A and SiHa C . Western blot for analysis of Cdt2 protein expression level 48 hours after transfection of miR-17∼92 in HEK293T, SiHa, HeLa and C33A cell line. D . Quantification of Cdt2 protein level 48h post miR 17-92 transfection in HEK, SiHa, HeLa and C33A compared to vector control. Quantification experiments were done in triplicates. Error bar depicts standard error (S.E.). *p value <0.05

Article Snippet: C33A (HPV negative cervical cancer cell line) and HEK293T (Human kidney cell line) were procured from National Centre for Cell Science (NCCS, Pune, India).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: miR-17∼92 Suppresses Proliferation and Invasion of Cervical Cancer Cells by Inhibiting Cell Cycle Regulator Cdt2

doi: 10.1101/2022.12.30.522306

Figure Lengend Snippet: miR17-92 suppresses proliferation of cervical cancer cells: miR17-92 ectopically expressed and the proliferation was observed from day of transfection till day 4 A. Growth curve of HEK293T cells with its respective control B. Control and treated HEK293T cells after 48h of transfection under phase contrast microscopy. C. Growth curve of SiHa along with its control D. SiHa cells (control and treated) under phase contrast microscopy after 48h of transfection. E. Growth curve of HeLa and it’s control F. Phase contrast microscopy of treated and control HeLa cells 48h post transfection. G. Growth curve of C33A and its control after transfection F. C33A control and treated cells under phase contrast microscopy after 8h of transfection. The experiment was done in triplicate. Error bar represents S.D. *p value-<0.05, **p value<0.001

Article Snippet: C33A (HPV negative cervical cancer cell line) and HEK293T (Human kidney cell line) were procured from National Centre for Cell Science (NCCS, Pune, India).

Techniques: Transfection, Control, Microscopy

Journal: Oncology reports

Article Title: High resolution analysis of the intracellular proteome of cervical cancer cell lines unveils novel regulators of cervical carcinogenesis.

doi: 10.3892/or.2019.7269

Figure Lengend Snippet: Figure 1. Graphical representation of the experimental workflow and analysis followed. The total cell extract from three cervical cancer cell lines [HeLa (HPV18+), SiHa (HPV16+), C33A (HPV‑)] and normal cervical keratinocytes (HCK1T) was collected and analyzed by SDS‑PAGE, following the GeLC‑MS/MS protocol. The bands were excised from the gel and cut into small pieces where in‑gel trypsinization was performed. The extracted peptides were then analyzed by LC‑MS/MS and the resulting spectra were translated into protein lists. After differential expression analysis, an extensive literature mining was performed in order to shortlist the proteins based on existing studies on cancer and cervical cancer, in particular. The datasets and shortlisted proteins from this study can be used for clinical validation (e.g. immunohistochemistry, multiple reaction monitoring mass spectrometry) and functional investigation with the aim to develop novel biomarkers and drug targets. LC‑MS/MS, liquid chromatography‑tandem mass spectrometry.

Article Snippet: HeLa (HPV 18+), SiHa (HPV 16+) and C33A (HPV-) cervical cancer cell lines, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco/Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin‐streptomycin at 37 ̊C in 5% CO2 as previously described (11).

Techniques: Quantitative Proteomics, Biomarker Discovery, Immunohistochemistry, Targeted Proteomics, Mass Spectrometry, Functional Assay

Journal: Oncology reports

Article Title: High resolution analysis of the intracellular proteome of cervical cancer cell lines unveils novel regulators of cervical carcinogenesis.

doi: 10.3892/or.2019.7269

Figure Lengend Snippet: Figure 4. Reactome pathways enriched by the differentially expressed proteins between the C33A and HCK1T cell lines. Results were simplified based on biological relevance and only the leading term from each group is presented (Bonferroni corrected p‑value ≤0.05, two‑sided hypergeometric test).

Article Snippet: HeLa (HPV 18+), SiHa (HPV 16+) and C33A (HPV-) cervical cancer cell lines, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco/Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin‐streptomycin at 37 ̊C in 5% CO2 as previously described (11).

Techniques: