cervical cancer cell line  (ATCC)

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of EGF binding in ME180 wild type cells and CRISPR/Cas9 mutant clones. ( A ) Confocal imaging of EGF binding, was done on a laser scan confocal Nikon Ti microscope at 100x magnification. Fluorescent staining was done for nucleus (Hoechst 33342), baseline endocytosis was assessed as binding of fluorescently labeled transferrin while binding of fluorescently labeled EGF showed differences in EGFR activity. The scale bars represent 50 μm. ( B ) Quantification of EGF-Alexa flour TM 555 fluorescence intensity in ME180 and mutant CRISPR/Cas9 clones was done by a plate reader. Relative fluorescence intensity of EGF-Alexa fluor TM 555 was normalized to uptake of Transferrin-Alexa fluor TM 488 which served as a control. Relative fluorescence intensity is presented as % of value obtained for wild type ME180 cells set as 100%. Results are presented as the mean ± SD of four independent experiments, each performed in five technical replicates. ( C ) quantification of Transferrin-Alexa Fluor TM 488 fluorescence intensity in ME180 and mutant CRISPR/Cas9 clones read by a plate reader. Relative fluorescence intensity for Alexa Fluor TM 488 was normalized to ME180 cells. Relative fluorescence intensity is presented as % of value obtained for wild type ME180 cells set as 100%. Results are presented as the mean ± SD of four independent experiments performed as five technical replicates.

Article Snippet: ME180 cervical cancer cell line was purchased from ATCC ® (HTB-33) and maintained in McCoy’s medium (Sigma-Aldrich, MO, United States) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, United States), 1x antibiotic antimycotic (Sigma-Aldrich, MO, United States), 1x essential amino acids, 1mM glutamine.

Techniques: Binding Assay, CRISPR, Mutagenesis, Clone Assay, Imaging, Microscopy, Staining, Labeling, Activity Assay, Fluorescence, Control

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: EGFR expression and subcellular localization in ME180 cells and CRISPR/Cas9 clones. ( A ) EGFR expression and subcellular localization visualized by ICC. Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification. Staining was done for nuclei (Hoechst 33342), F-actin (Phalloidin, Alexa Fluor 594) and EGFR (antibody recognizing non-phosphorylated and phosphorylated intracellular part of EGFR, the residues around Tyr1068: ab76153, Abcam, ). The scale bars represent 50 μm. ( B ) Relative EGFR mRNA expression analyzed by qRT-PCR. Relative EGFR expression is compared to ME180 value, which was set as 1. Results are presented as the means ± SD of at least three independent experiments. ( C ) Western blot of EGFR expression in mutant clones and quantification of its expression in cells from three different cell passages. Western blot was done with anti-EGFR antibody (ab76153, Abcam). Relative EGFR expression is compared to ME180 value, which was set as 100%. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: ME180 cervical cancer cell line was purchased from ATCC ® (HTB-33) and maintained in McCoy’s medium (Sigma-Aldrich, MO, United States) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, United States), 1x antibiotic antimycotic (Sigma-Aldrich, MO, United States), 1x essential amino acids, 1mM glutamine.

Techniques: Expressing, CRISPR, Clone Assay, Imaging, Microscopy, Staining, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Distribution of EGF and EGFR and phosphorylation status of EGFR in EGF treated cells. ( A ) ICC for EGF and EGFR in untreated cells and cells treated with EGF for 30 min Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification; staining was done for nuclei (Hoechst 33342), EGF (ab9595 Abcam, Cambridge, United Kingdom) and EGFR (antibody recognizing extracellular domain of human EGFR: epitope 6-273 aa, antibody clone sc-101; Santa Cruz Biotechnology, TX, United States). The scale bars are 50 μm. ( B ) Phosphorylation of EGFR upon treatment with EGF was determined by Western blot. Whole cell lysates were obtained from cells without treatment or 30 min after treatment with recombinant EGF. Three independent experiments were done and imaged using Li-Cor system; representative Western blots are shown. Normalization was done using α-Tubulin as a loading control (Catalog number 926-42213 Li-Cor, NE, United States). Antibodies for phosphorylated EGFR included Y1068 (Catalog number #3777, Cell Signaling, MA, United States), Y1086 (Catalog number #2220S, Cell Signaling, MA, United States) and Y1173 (Catalog number ab32578 Abcam, Cambridge, United Kingdom). ( C ) Western blot quantification was done using software provided by Li-Cor and plotted using Graph Pad Prizm. This bar graph represents relative quantification of phosphorylation of EGFR (for tyrosine positions Y1068, Y1086 and Y1173) for wild type ME180 cells and mutant clone VII11 at 30 min after EGF treatment. To show relative phosphorylation values, the highest phosphorylation signal (that for Tyrosine 1068 in wild type cells) was set as 1. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: ME180 cervical cancer cell line was purchased from ATCC ® (HTB-33) and maintained in McCoy’s medium (Sigma-Aldrich, MO, United States) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, United States), 1x antibiotic antimycotic (Sigma-Aldrich, MO, United States), 1x essential amino acids, 1mM glutamine.

Techniques: Phospho-proteomics, Imaging, Microscopy, Staining, Western Blot, Recombinant, Control, Software, Quantitative Proteomics, Mutagenesis

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of cell cycle distribution and expression of cell cycle regulator proteins. ( A ) Representative cell cycle profiles of wild type ME180 cells and mutant clones based on DAPI staining and incorporation of EdU. The usual horseshoe pattern cell distribution allowed separation of cell populations into G1, S and G2/M stage of the cell cycle. ( B ) Histogram showing cell cycle distribution in wild type cells and mutant clones based on flow cytometry data done on 5 independent experiments using cells from 5 different cell passages. The data are shown as means ± SD from five independent experiments including cells from three different cell cycle passages. ( C ) Western blots performed on whole cell lysates from wild type cells and mutant clones growing in cell culture or additionally treated with recombinant EGF for 30 min. Experiments were performed independently using at least three different cell passages and representative blots are shown. ( D ) Quantification of Western blot results was performed using Li-Cor quantification software and the results are presented values relative to protein expression in untreated wild type cells set as 100%. Data are presented as means ± SD of at least three independent experiments.

Article Snippet: ME180 cervical cancer cell line was purchased from ATCC ® (HTB-33) and maintained in McCoy’s medium (Sigma-Aldrich, MO, United States) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, United States), 1x antibiotic antimycotic (Sigma-Aldrich, MO, United States), 1x essential amino acids, 1mM glutamine.

Techniques: Expressing, Mutagenesis, Clone Assay, Staining, Flow Cytometry, Western Blot, Cell Culture, Recombinant, Software

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Possible protein coding differences between clones generated in this study based on WGS data comparisons. ( A ) Venn diagram summary of sequence variants either unique to individual clones or overlapping between different mutant clones based on their genomic positions, highlighting only those differences that were found in comparison with the wild type ME180 cells. Left: comparison of the three separate homozygous Y45 mutant clones generated in the first round of CRISPR/Cas9. Right: comparison between clones isolated after second round of CRISPR/Cas9 and their parental clone VII11. ( B ) Diagram summarizing genes with possible high impact mutations in mutant clones VII11, A13 and A40; for a list of genes other than EGFR that acquired high impact mutations please see Supplemental Table 6.

Article Snippet: ME180 cervical cancer cell line was purchased from ATCC ® (HTB-33) and maintained in McCoy’s medium (Sigma-Aldrich, MO, United States) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, United States), 1x antibiotic antimycotic (Sigma-Aldrich, MO, United States), 1x essential amino acids, 1mM glutamine.

Techniques: Clone Assay, Generated, Sequencing, Mutagenesis, Comparison, CRISPR, Isolation

Journal: BMC Molecular and Cell Biology

Article Title: Mitochondrial HMGCS1 mediates cisplatin resistance in cervical cancer through regulation of mitochondrial transcription

doi: 10.1186/s12860-026-00566-y

Figure Lengend Snippet: HMGCS1 regulates cisplatin sensitivity and resistance in cervical cancer cells through mitochondrial localization. ( A - B ) Dose-response curves showing cell viability of wild-type (WT) and HMGCS1 knockout (KO) HeLa ( A ) and C33A ( B ) cells treated with increasing concentrations of cisplatin for 72 h. IC50 values are indicated. Cell viability was determined using SRB assay. The corresponding Western blots below validate HMGCS1 protein knockout, using TUBA1A as a loading control. ( C - D ) Dose-response curves showing cell viability of parental and cisplatin-resistant (CisR) HeLa ( C ) and C33A ( D ) cells treated with increasing concentrations of cisplatin for 72 h. IC50 values are indicated. ( E ) Western blot analysis of HMGCS1 protein expression in parental and cisplatin-resistant HeLa and C33A cells. TUBA1A was used as a loading control. ( F - G ) Dose-response curves showing cell viability of wild-type (WT) and HMGCS1 knockout (KO) cisplatin-resistant HeLa_CisR ( F ) and C33A_CisR ( G ) cells treated with increasing concentrations of cisplatin for 72 h. IC50 values are indicated. ( H - I ) Western blot analysis of HMGCS1 protein expression in nuclear and mitochondrial fractions of parental and cisplatin-resistant HeLa ( H ) and C33A ( I ) cells. Lamin B1 and Tomm20 were used as nuclear and mitochondrial markers, respectively. ( A - D , F - G ) Data are presented as mean ± SD. Statistical significance of the difference between IC50 values was determined by the Extra Sum-of-Squares F-test

Article Snippet: Cervical cancer cell lines HeLa and C33A were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C with 5% CO2.

Techniques: Knock-Out, Sulforhodamine B Assay, Western Blot, Control, Expressing

Journal: BMC Molecular and Cell Biology

Article Title: Mitochondrial HMGCS1 mediates cisplatin resistance in cervical cancer through regulation of mitochondrial transcription

doi: 10.1186/s12860-026-00566-y

Figure Lengend Snippet: Subcellular localization of HMGCS1 determines its role in cisplatin resistance. ( A ) Immunofluorescence microscopy of HeLa and C33A cells expressing 6xHis-tagged NLS-HMGCS1 or MTS-HMGCS1. Cells were stained with DAPI (blue) for nuclei, anti-6xHis antibody (green) for tagged HMGCS1, and MitoTracker (red) for mitochondria. Merged images show the distinct subcellular localization of each construct. ( B- E ) Dose-response curves showing cell viability after cisplatin treatment (72 h) in: parental HeLa ( B ) and C33A ( C ) cells; and HMGCS1-knockout cisplatin-resistant HeLa_CisR ( D ) and C33A_CisR ( E ) cells expressing empty vector (EV), MTS-HMGCS1, or NLS-HMGCS1. Cell viability was determined using SRB assay. Data are presented as mean ± SD. IC50 values are indicated. Statistical significance was determined by pairwise comparison of the LogIC50 value for each construct against the EV control using the Extra sum-of-squares F-test, with a Bonferroni correction for multiple comparisons (*** p < 0.001, ns: not significant)

Article Snippet: Cervical cancer cell lines HeLa and C33A were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C with 5% CO2.

Techniques: Immunofluorescence, Microscopy, Expressing, Staining, Construct, Knock-Out, Plasmid Preparation, Sulforhodamine B Assay, Comparison, Control

Journal: BMC Molecular and Cell Biology

Article Title: Mitochondrial HMGCS1 mediates cisplatin resistance in cervical cancer through regulation of mitochondrial transcription

doi: 10.1186/s12860-026-00566-y

Figure Lengend Snippet: Mitochondrial HMGCS1 enhances oxidative phosphorylation and regulates redox homeostasis in cisplatin-resistant cervical cancer cells. ( A , C ) Mitochondrial respiration profiles showing oxygen consumption rate (OCR) in parental and cisplatin-resistant (CisR) HeLa ( A ) and C33A ( C ) cells. Sequential addition of oligomycin (O), FCCP (F), and rotenone/antimycin A (R/A) is indicated by arrows. ( B , D ) Quantification of basal and maximal respiratory capacity in parental and cisplatin-resistant HeLa ( B ) and C33A ( D ) cells. ( E ) Relative intracellular cholesterol levels in parental and cisplatin-resistant HeLa and C33A cells as measured by a commercial cholesterol assay kit. ( F , H ) Mitochondrial respiration profiles of wild-type (WT), HMGCS1-knockout (KO), and KO cells expressing NLS-HMGCS1 (KO + NLS) or MTS-HMGCS1 (KO + MTS) in HeLa_CisR ( F ) and C33A_CisR ( H ) cells. ( G , I ) Quantification of basal and maximal respiratory capacity in WT, KO, and rescued HeLa_CisR ( G ) and C33A_CisR ( I ) cells. ( J) Relative ROS levels in wild-type, HMGCS1-knockout, and rescued HeLa_CisR and C33A_CisR cells measured by measured by ROS Deep Red fluorescence. Data are presented as mean ± SD. Statistical significance for panels G , I , and J was determined by One-way ANOVA with Dunnett’s multiple comparisons test. Comparisons in panels B , D , and E were made using Student’s t-test: ** p < 0.01, *** p < 0.001, ns: not significant, by Student’s t-test

Article Snippet: Cervical cancer cell lines HeLa and C33A were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C with 5% CO2.

Techniques: Phospho-proteomics, Cholesterol Assay, Knock-Out, Expressing, Fluorescence

Journal: BMC Molecular and Cell Biology

Article Title: Mitochondrial HMGCS1 mediates cisplatin resistance in cervical cancer through regulation of mitochondrial transcription

doi: 10.1186/s12860-026-00566-y

Figure Lengend Snippet: Mitochondrial HMGCS1 enhances mtDNA-encoded gene expression without affecting mtDNA copy number in cisplatin-resistant cells. ( A - B ) Relative mRNA expression levels of mtDNA-encoded respiratory complex genes (MT-ND1, MT-ND5, MT-CO1, MT-CYB, and MT-ATP6) in HeLa_CisR ( A ) and C33A_CisR ( B ) cells with indicated HMGCS1 status: wild-type (WT), knockout (KO), nuclear-localized HMGCS1 (NLS), or mitochondria-targeted HMGCS1 (MTS). ( C ) Relative mtDNA copy number quantification in HeLa_CisR and C33A_CisR cells with different HMGCS1 status. Data are presented as mean ± SD. Statistical significance was determined using Two-way ANOVA ( A , B ) or One-way ANOVA ( C ), followed by Dunnett’s multiple comparisons test: *** p < 0.001, ns: not significant

Article Snippet: Cervical cancer cell lines HeLa and C33A were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C with 5% CO2.

Techniques: Gene Expression, Expressing, Knock-Out

Journal: BMC Molecular and Cell Biology

Article Title: Mitochondrial HMGCS1 mediates cisplatin resistance in cervical cancer through regulation of mitochondrial transcription

doi: 10.1186/s12860-026-00566-y

Figure Lengend Snippet: HMGCS1 inhibitor and mitochondrial transcription inhibitor synergize with cisplatin in drug-resistant cervical cancer cells. ( A - D ) Dose-response curves showing cell viability of cisplatin-resistant cells treated with increasing concentrations of cisplatin alone (DMSO control) or in combination with 2 µM hymeglusin or 1 µM IMT1B. Curves are shown for parental HeLa_CisR ( A ) and C33A_CisR ( B ) cells, and for their HMGCS1 knockout (KO) counterparts, HeLa_CisR HMGCS1 KO ( C ) and C33A_CisR HMGCS1 KO ( D ). IC50 values are indicated for each treatment condition. Statistical significance was determined by pairwise comparison of the LogIC50 value for each construct against the EV control using the Extra sum-of-squares F-test, with a Bonferroni correction for multiple comparisons (*** p < 0.001, ns: not significant). ( E - H ) Synergy analysis of drug combinations in resistant cells using the Bliss independence model. Heat maps show synergy scores for various concentration combinations of cisplatin with hymeglusin in ( E ) HeLa_CisR and ( F ) C33A_CisR cells, or with IMT1B in ( G ) HeLa_CisR and ( H ) C33A_CisR cells. Positive values (blue) indicate synergistic interactions, values near zero (white) indicate additive effects, and negative values (red) indicate antagonistic interactions

Article Snippet: Cervical cancer cell lines HeLa and C33A were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C with 5% CO2.

Techniques: Control, Knock-Out, Comparison, Construct, Concentration Assay