hpv16 positive human cervical cancer cell line caski  (ATCC)

Journal: Scientific Reports

Article Title: PCK1-mediated glycogenolysis facilitates ROS clearance and chemotherapy resistance in cervical cancer stem cells

doi: 10.1038/s41598-024-64255-6

Figure Lengend Snippet: ( A – C ) HCC94, and CaSki human cervical cancer cells were sorted into two fractions: CD133 + and CD133 − . After treatment with cisplatin (50 μM) for 48 h, measurements were taken for cell viability ( A ), NADPH/NADP + ratio ( B ), and reactive oxygen species (ROS) levels ( C ). ( D ) HCC94 and CaSki human cancer stem cells or bulk cells were treated with PBS and cisplatin (50uM) for 48 h. The levels of R5P/Ru5P, S7P and 6-PGA (6-phosphogluconic acid) were measured. ( E – G ) HCC94, and CaSki human cancer stem cells or bulk cells were treated with cisplatin (50 μM) or G6PDi (50 μM) for 48 h. NADPH ( E ), ROS ( F ), and cell viability ( G ) levels were measured. Data are presented as mean ± SEMs of three independent experiments. ***P < 0.001. Two-tailed student’s t -test ( A – D ) or one-way analysis of variance (ANOVA) followed by Bonferroni’s test ( E – G ).

Article Snippet: The HCC94 and CaSki human cervical cancer cell lines were obtained from the American type culture collection (Manassas, VA, USA).

Techniques: Two Tailed Test

Journal: Scientific Reports

Article Title: PCK1-mediated glycogenolysis facilitates ROS clearance and chemotherapy resistance in cervical cancer stem cells

doi: 10.1038/s41598-024-64255-6

Figure Lengend Snippet: ( A – B ) HCC94 and CaSki human cervical cancer cells then sorted into two fractions: CD133 + (CSC), CD133 − (bulk cell). Glycogen levels was measured ( A ). The expression of p-PYGL and PYGL in CSC or bulk cell was measured ( B ). ( C ) The level of glycogen was measured in patients with chemotherapy-resistant or -sensitive cancers. ( D ) The level of p-PYGL was measured in patients with chemotherapy-resistant or -sensitive cancers by IF. In ( C – D ), n = 6 patients. The data were presented as means ± SEMs of three independent experiments. ***P < 0.001. Two-tailed student’s t -test ( A , C , D ).

Article Snippet: The HCC94 and CaSki human cervical cancer cell lines were obtained from the American type culture collection (Manassas, VA, USA).

Techniques: Expressing, Two Tailed Test

Journal: Scientific Reports

Article Title: PCK1-mediated glycogenolysis facilitates ROS clearance and chemotherapy resistance in cervical cancer stem cells

doi: 10.1038/s41598-024-64255-6

Figure Lengend Snippet: ( A – E ) HCC94 and CaSki human cancer stem cells, which was transfected with PYGL siRNA, were treated with PBS or cisplatin for 48 h. The level of glycogen ( A ), m+5 R5P, m+7 S7P, m+6 6-PGA ( B ), NADPH/NADP + ratio ( B ), ROS ( C ) and cell viability ( D ) was measured. ( F – J ) HCC94 and CaSki human cancer stem cells, which was transfected with GYS1 siRNA, were treated with PBS or cisplatin for 48 h. The level of glycogen ( F ), m+5 R5P, m+7 S7P, m+6 6-PGA ( G ), NADPH/NADP + ratio ( H ), ROS ( I ) and cell viability ( J ) was measured. The data were presented as means ± SEMs of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. One-way analysis of variance (ANOVA) followed by Bonferroni’s test ( A – H ).

Article Snippet: The HCC94 and CaSki human cervical cancer cell lines were obtained from the American type culture collection (Manassas, VA, USA).

Techniques: Transfection

Journal: Scientific Reports

Article Title: PCK1-mediated glycogenolysis facilitates ROS clearance and chemotherapy resistance in cervical cancer stem cells

doi: 10.1038/s41598-024-64255-6

Figure Lengend Snippet: ( A – D ) HCC94 and CaSki human cancer stem cells, which was pretreated with okadaic acid (OA, 1 0 nM), were treated with cisplatin for 48 h. The level of NADPH/NADP + ( A ), m+5 R5P, m+7 S7P, m+6 6-PGA ( B ), ROS ( C ) and cell viability ( D ) was measured. ( E ) The level of p-PYGL and PYGL was measured in HCC94 and CaSki human cancer stem cells, which was pretreated with OA. ( F ) The level of PCK1 was measured in HCC94 and CaSki human cancer stem cells or bulk cell. ( G – K ) HCC94 and CaSki human cancer stem cells, which was transfected with PCK1 siRNA, were treated with cisplatin for 48 h. The level of NADPH/NADP + ratio ( G ), m + 5 R5P, m + 7 S7P, m + 6 6-PGA ( H ), ROS ( I ) and Cell viability ( J ) were measured. The data were presented as means ± SEMs of three independent experiments. **P < 0.01, ***P < 0.001. One-way analysis of variance (ANOVA) followed by Bonferroni’s test ( A – D , G – J ).

Article Snippet: The HCC94 and CaSki human cervical cancer cell lines were obtained from the American type culture collection (Manassas, VA, USA).

Techniques: Transfection

Journal: Scientific Reports

Article Title: PCK1-mediated glycogenolysis facilitates ROS clearance and chemotherapy resistance in cervical cancer stem cells

doi: 10.1038/s41598-024-64255-6

Figure Lengend Snippet: ( A ) HCC94 and CaSki human cancer stem cells, which was transfected with PCK1 siRNA, were treated with cisplatin for 48 h. The level of p-PYGL and PYGL was measured ( A ). ( B ) Immunoblot of immunoprecipitations of PYGL or PCK1 in lysates from cancer stem cells or bulk cells treated with cisplatin for 24 h. ( C ) Immunoblot of immunoprecipitations of PP1c or PYGL in lysates from cancer stem cells was transfected with PCK1 siRNA.

Article Snippet: The HCC94 and CaSki human cervical cancer cell lines were obtained from the American type culture collection (Manassas, VA, USA).

Techniques: Transfection, Western Blot

Journal: Cells

Article Title: Understanding the Combined Effects of High Glucose Induced Hyper-Osmotic Stress and Oxygen Tension in the Progression of Tumourigenesis: From Mechanism to Anti-Cancer Therapeutics.

doi: 10.3390/cells12060825

Figure Lengend Snippet: Figure 1. Tumour cells show efficient adaptation to the hyper-osmotic stress generating property of high glucose and significantly proliferate in normoxia. CaSki/C-33A cervical cancer cells were cultured for 96 h in NG, HG and HG-HO, with or without hypoxia stimulation. (A–C) Tumour cell proliferation, (D–F) Colony-forming analyses were performed, and all comparisons were made with NG condition unless otherwise indicated. Statistical significance is represented by p values: *** p ≤0.001. See related Supplementary Figure S3A.

Article Snippet: Human cervical cancer cell lines CaSki (HPV16+ve and HPV18+ve) and C-33A (HPVve); metastatic colon cancer cell line HCT116; metastatic pancreatic cancer cell line MIAPaCa2; metastatic breast cancer cell line MDA-MB-231 and human normal keratinocytes cell line HaCaT were obtained from and authenticated by American Tissue Culture Collection, Manassas, VA, USA.

Techniques: Cell Culture

Journal: Cells

Article Title: Understanding the Combined Effects of High Glucose Induced Hyper-Osmotic Stress and Oxygen Tension in the Progression of Tumourigenesis: From Mechanism to Anti-Cancer Therapeutics.

doi: 10.3390/cells12060825

Figure Lengend Snippet: Figure 2. Acute hypoxia attenuates high glucose hyper-osmotic stress pro-proliferative mechanoadaptive effects and generates dormant tumour cell phenotypes, which can recover upon intercepting normoxia. (A) CaSki/C-33A cervical cancer cells were subjected to soft agar-based anchorage-independent NG, HG and HG-HO conditions, with or without hypoxia stimulation. The number of spheroids formed showed substrate-independent self-renewable capacity of a single cell.

Article Snippet: Human cervical cancer cell lines CaSki (HPV16+ve and HPV18+ve) and C-33A (HPVve); metastatic colon cancer cell line HCT116; metastatic pancreatic cancer cell line MIAPaCa2; metastatic breast cancer cell line MDA-MB-231 and human normal keratinocytes cell line HaCaT were obtained from and authenticated by American Tissue Culture Collection, Manassas, VA, USA.

Techniques:

Journal: Cells

Article Title: Understanding the Combined Effects of High Glucose Induced Hyper-Osmotic Stress and Oxygen Tension in the Progression of Tumourigenesis: From Mechanism to Anti-Cancer Therapeutics.

doi: 10.3390/cells12060825

Figure Lengend Snippet: Figure 3. Acute hypoxia attenuates high-glucose hyper-osmotic stress pro-proliferative mechanoadaptive effects by promoting mitotic M-phase arrest. CaSki/C-33A cervical cancer cells were cultured for 96 h in NG, HG and HG-HO with or without hypoxia stimulation. (A,B) FACS- assisted cell cycle analysis was performed. (C,D) Average mitotic cell counts over the total cell number per ROI were obtained to estimate whether more cells were stalled in mitosis over total cells in each condition. (E,F) The images of mitotic cells per ROI were further scored into different mitotic (pro, meta and ana/telo) phases manually to estimate whether the distribution of mitotic cells in these phases could correlate with total cell numbers or not. Statistical significance is represented by p values: *** p ≤0.001.

Article Snippet: Human cervical cancer cell lines CaSki (HPV16+ve and HPV18+ve) and C-33A (HPVve); metastatic colon cancer cell line HCT116; metastatic pancreatic cancer cell line MIAPaCa2; metastatic breast cancer cell line MDA-MB-231 and human normal keratinocytes cell line HaCaT were obtained from and authenticated by American Tissue Culture Collection, Manassas, VA, USA.

Techniques: Cell Culture, Cell Cycle Assay

Journal: Cells

Article Title: Understanding the Combined Effects of High Glucose Induced Hyper-Osmotic Stress and Oxygen Tension in the Progression of Tumourigenesis: From Mechanism to Anti-Cancer Therapeutics.

doi: 10.3390/cells12060825

Figure Lengend Snippet: Figure 4. Acute hypoxia restricts the localization of SUMO2, AURKB and pH3(Ser10) on the mitotic chromosomes of the tumour cells. CaSki/C-33A cervical cancer cells were cultured for 96 h in NG, HG and HG-HO, with or without hypoxia stimulation. (A) Cells were immunostained with SUMO2, AURKB and pH3(Ser10). Coloured arrows in the middle panel indicate an example of differences in expression between normoxia and hypoxia. (B) An example of free-hand ROI selection

Article Snippet: Human cervical cancer cell lines CaSki (HPV16+ve and HPV18+ve) and C-33A (HPVve); metastatic colon cancer cell line HCT116; metastatic pancreatic cancer cell line MIAPaCa2; metastatic breast cancer cell line MDA-MB-231 and human normal keratinocytes cell line HaCaT were obtained from and authenticated by American Tissue Culture Collection, Manassas, VA, USA.

Techniques: Cell Culture, Expressing, Selection

Journal: Cells

Article Title: Understanding the Combined Effects of High Glucose Induced Hyper-Osmotic Stress and Oxygen Tension in the Progression of Tumourigenesis: From Mechanism to Anti-Cancer Therapeutics.

doi: 10.3390/cells12060825

Figure Lengend Snippet: Figure 5. Proximity ligation assay shows that acute hypoxia triggers cytoplasmic sequestration of SUMO2-modified pH3(Ser10) in mitotic cells. CaSki cervical cancer cells were cultured in the general experimental set-up for 96 h. (A) Protein–protein interaction detection assay, the proximity ligation assay (PLA), showed enhanced localization of SUMO2-pH3(Ser10) PLA signal (green dots) in both HG and HG-HO conditions vs NG in normoxia. (B) On the contrary, the PLA signal was prominent in the cytoplasm and around the chromosome periphery in hypoxia-treated cells. Images shown in panel (B) (i) and (ii) represent observations across treatment conditions, irrespective of HG and HG-HO. See related Supplementary Figure S7 for correlated results in acute hypoxia-exposed non-mitotic tumour cells.

Article Snippet: Human cervical cancer cell lines CaSki (HPV16+ve and HPV18+ve) and C-33A (HPVve); metastatic colon cancer cell line HCT116; metastatic pancreatic cancer cell line MIAPaCa2; metastatic breast cancer cell line MDA-MB-231 and human normal keratinocytes cell line HaCaT were obtained from and authenticated by American Tissue Culture Collection, Manassas, VA, USA.

Techniques: Proximity Ligation Assay, Cell Culture, Detection Assay

Journal: Cells

Article Title: Understanding the Combined Effects of High Glucose Induced Hyper-Osmotic Stress and Oxygen Tension in the Progression of Tumourigenesis: From Mechanism to Anti-Cancer Therapeutics.

doi: 10.3390/cells12060825

Figure Lengend Snippet: Figure 6. Direct FRET between pH3(Ser10) and SUMO2 confirms cytoplasmic sequestration of SUMOylated pH3(Ser10) in acute hypoxia exposed mitotic cells. CaSki cervical cancer cells were cultured in the general experimental set-up for 96 h. (A,B) Cells were immunostained with pH3(Ser10) and SUMO2 primary antibodies and were further tagged with Cy3 and Alexa flour 647 conju- gated secondary antibodies, respectively, as Cy3-Alexafluor 647 are established FRET compatible donor–acceptor pairs. Analysis showed enhanced chromosomal localization of SUMO2-pH3(Ser10) FRET signal in both HG and HG-HO conditions vs NG in normoxia. On the contrary, the FRET signal was prominent in the cytoplasm of hypoxia-treated cells. Statistical significance is represented by p values: * p ≤0.05, ** p ≤0.01 and *** p ≤0.001. See related Supplementary Figure S8 for correlated results in acute hypoxia-exposed non-mitotic tumour cells.

Article Snippet: Human cervical cancer cell lines CaSki (HPV16+ve and HPV18+ve) and C-33A (HPVve); metastatic colon cancer cell line HCT116; metastatic pancreatic cancer cell line MIAPaCa2; metastatic breast cancer cell line MDA-MB-231 and human normal keratinocytes cell line HaCaT were obtained from and authenticated by American Tissue Culture Collection, Manassas, VA, USA.

Techniques: Cell Culture