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cervical cancer derived cell line siha (ATCC)

Name: cervical cancer derived cell line siha
Brand: ATCC
Rating: 99 (2610 reviews)

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ATCC cervical cancer derived cell line siha

A stable clone of HPV-16 E6/ E7 expressing <t>human</t> <t>cervical</t> cancer <t>SiHa</t> 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).

Cervical Cancer Derived Cell Line Siha, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 2610 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Covalent Inhibition of the Human Papillomavirus Type 16 E6 Protein Restores p53 and Suppresses HPV-Driven Tumorigenesis"

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

Journal: bioRxiv

doi: 10.1101/2025.08.24.671874

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).

Figure Legend 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).

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

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).

Figure Legend 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).

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

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).

Figure Legend 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).

Techniques Used: Cell Culture, Western Blot, Expressing

(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).

Figure Legend 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).

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

( 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.

Figure Legend 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.

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

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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).

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).

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).

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.

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

Relative expression of GRPR mRNA by real time PCR: Relative expression of GRPR mRNA in (A) normal keratinocytes (PHK) and cervical cancer-derived cell lines (C33, SiHa and HeLa) and (B) keratinocytes transduced with the empty vector (pLXSN Ø) or with the vector containing HPV16 oncogenes E6 and/or E7

Journal: Revista Brasileira de Ginecologia e Obstetrícia

Article Title: Gastrin-releasing peptide receptor: a promising new biomarker to identify cervical precursor lesions and cancer

doi: 10.61622/rbgo/2025rbgo4

Figure Lengend Snippet: Relative expression of GRPR mRNA by real time PCR: Relative expression of GRPR mRNA in (A) normal keratinocytes (PHK) and cervical cancer-derived cell lines (C33, SiHa and HeLa) and (B) keratinocytes transduced with the empty vector (pLXSN Ø) or with the vector containing HPV16 oncogenes E6 and/or E7

Article Snippet: Cervical cancer-derived cell lines SiHa (HPV16, ATCC #HTB-35), HeLa (HPV18, ATCC #CCL-2) and C33 (HPV negative, ATCC #HTB-31) were cultured in Eagle's minimal essential medium (MEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% bovine fetal serum (BFS) (Cultilab, Campinas, SP, Brazil) and maintained at 37°C and 5% CO 2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Transduction, Plasmid Preparation

Metformin induces ZFP36 expression in cervical cancer-derived-cell lines. HeLa and CaSki cells were treated with metformin 20mM for 24 hours, then RNA was extracted for analysis by RT-qPCR. RT-qPCR of ZFP36 after metformin treatment in ( A ) HeLa and ( B ) CaSki. Representative Western Blot of ZFP36 and densitometric analysis in ( C ) HeLa and ( D ) CaSki, ( E ) HaCaT and ( F ) Human Uterine Fibroblast (HUF). Results are shown as means ± SD of at least three independent experiments. * p <0.05 **, p <0.01

Journal: BMC Cancer

Article Title: Metformin induces ZFP36 by mTORC1 inhibition in cervical cancer-derived cell lines

doi: 10.1186/s12885-024-12555-5

Figure Lengend Snippet: Metformin induces ZFP36 expression in cervical cancer-derived-cell lines. HeLa and CaSki cells were treated with metformin 20mM for 24 hours, then RNA was extracted for analysis by RT-qPCR. RT-qPCR of ZFP36 after metformin treatment in ( A ) HeLa and ( B ) CaSki. Representative Western Blot of ZFP36 and densitometric analysis in ( C ) HeLa and ( D ) CaSki, ( E ) HaCaT and ( F ) Human Uterine Fibroblast (HUF). Results are shown as means ± SD of at least three independent experiments. * p <0.05 **, p <0.01

Article Snippet: Primary Human Uterine Fibroblast (HUF) (PCS-460–010) and human cervical cancer-derived cell lines HeLa (RRID: CVCL_0030) and CaSki (RRID: CVCL_1100) were obtained from ATCC (Manassas, VA, USA).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot

Metformin downregulates mTORC1/S6K1/rpS6 pathway and decreases cell viability in cervical cancer cell lines. HeLa and CaSki cells were treated with different metformin concentrations for 24 hours and, the levels of p-P70/P85 S6K1, p-rpS6, and p-AMPK were measured by Western Blot in HeLa cells ( A - C ) and CaSki cells ( D - F ). E and F HeLa cells and CaSki cells were treated with 5, 10, and 20 mM of metformin for 24 h. Then, cell viability was examined using an MTT assay. The plots represent the densitometric and statistical analysis of Western Blot and data expressed as mean + SD of at least three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.001

Journal: BMC Cancer

Article Title: Metformin induces ZFP36 by mTORC1 inhibition in cervical cancer-derived cell lines

doi: 10.1186/s12885-024-12555-5

Figure Lengend Snippet: Metformin downregulates mTORC1/S6K1/rpS6 pathway and decreases cell viability in cervical cancer cell lines. HeLa and CaSki cells were treated with different metformin concentrations for 24 hours and, the levels of p-P70/P85 S6K1, p-rpS6, and p-AMPK were measured by Western Blot in HeLa cells ( A - C ) and CaSki cells ( D - F ). E and F HeLa cells and CaSki cells were treated with 5, 10, and 20 mM of metformin for 24 h. Then, cell viability was examined using an MTT assay. The plots represent the densitometric and statistical analysis of Western Blot and data expressed as mean + SD of at least three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.001

Techniques: Western Blot, MTT Assay

ZFP36 is induced by mTORC1 inhibition in CC cells. HeLa and CaSki cells were treated with 50 nM rapamycin or with vehicle (ethanol) for 24 h, and the levels of ZFP36 and p-rpS6 were measured by Western Blot. A and B Corresponding Western Blot and densitometric analysis of ZFP36/GAPDH in HeLa cells. C and D Corresponding Western Blot and densitometric analysis of ZFP36/GAPDH in CaSki cells. E HeLa cells were transfected with control or AMPK-targeting siRNAs for 24 h, and the levels of AMPK and ZFP36 were measured by Western Blot. F Corresponding densitometric analysis of ZFP36/GAPDH. The plots represent the densitometric and statistical analysis of Western Blot and data expressed as mean + SD of at least three independent experiments. * p < 0.05, ** p < 0.01

Journal: BMC Cancer

Article Title: Metformin induces ZFP36 by mTORC1 inhibition in cervical cancer-derived cell lines

doi: 10.1186/s12885-024-12555-5

Figure Lengend Snippet: ZFP36 is induced by mTORC1 inhibition in CC cells. HeLa and CaSki cells were treated with 50 nM rapamycin or with vehicle (ethanol) for 24 h, and the levels of ZFP36 and p-rpS6 were measured by Western Blot. A and B Corresponding Western Blot and densitometric analysis of ZFP36/GAPDH in HeLa cells. C and D Corresponding Western Blot and densitometric analysis of ZFP36/GAPDH in CaSki cells. E HeLa cells were transfected with control or AMPK-targeting siRNAs for 24 h, and the levels of AMPK and ZFP36 were measured by Western Blot. F Corresponding densitometric analysis of ZFP36/GAPDH. The plots represent the densitometric and statistical analysis of Western Blot and data expressed as mean + SD of at least three independent experiments. * p < 0.05, ** p < 0.01

Techniques: Inhibition, Western Blot, Transfection, Control

Metformin inhibits mTORC1 and cell viability independently of AMPK. HeLa cells were transfected with control or AMPK-targeting siRNAs and treated or not with 20 mM metformin for 24 h. A Cell viability was examined using an MTT assay. B The levels of AMPK, p-rpS6, rpS6 were measured by Western Blot. C Corresponding densitometric analysis of p-rpS6/rpS6. Data expressed as mean + SD of at least three independent experiments. * p < 0.05, ** p < 0.01

Journal: BMC Cancer

Article Title: Metformin induces ZFP36 by mTORC1 inhibition in cervical cancer-derived cell lines

doi: 10.1186/s12885-024-12555-5

Figure Lengend Snippet: Metformin inhibits mTORC1 and cell viability independently of AMPK. HeLa cells were transfected with control or AMPK-targeting siRNAs and treated or not with 20 mM metformin for 24 h. A Cell viability was examined using an MTT assay. B The levels of AMPK, p-rpS6, rpS6 were measured by Western Blot. C Corresponding densitometric analysis of p-rpS6/rpS6. Data expressed as mean + SD of at least three independent experiments. * p < 0.05, ** p < 0.01

Techniques: Transfection, Control, MTT Assay, Western Blot

NEAT1 promotes the proliferation, migration, invasion, and EMT of cervical cancer cells. (A) Expression of NEAT1 in a human cervical squamous epithelial cell line (End1) and human cervical cancer cell lines (HeLa, Caski, C33a, SiHa). Among the four cervical cancer cell lines tested, the expression of NEAT1 was highest in the HeLa cells, and lowest in the SiHa cells. (B, C) Expression of NEAT1 in HeLa and SiHa cells transfected with siRNAs and NEAT1 overexpression plasmid, respectively. NEAT1 was significantly downregulated by siRNAs and overexpressed by plasmids. (D) CCK‐8 and (E) colony formation assays were performed to detect the proliferation ability of HeLa and SiHa cells. The proliferation of HeLa cells was suppressed after NEAT1 knockdown, while that of SiHa cells was promoted after NEAT1 overexpression. (F) The cell cycle distribution was detected by flow cytometry. (G) The expression of cell cycle‐related proteins was detected by western blotting. The cell cycle was arrested in NEAT1‐knockdown HeLa cells but promoted in NEAT1‐overexpressing SiHa cells. (H) Migration and invasion ability were detected by Transwell assays. (I) The expression of EMT‐related proteins was detected by western blotting. EMT, migration and invasion of HeLa cells were inhibited following NEAT1 knockdown, while these effects were promoted in SiHa cells following NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Cancer Medicine

Article Title: Long non‐coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT /β‐catenin/ PDK1 axis

doi: 10.1002/cam4.7221

Figure Lengend Snippet: NEAT1 promotes the proliferation, migration, invasion, and EMT of cervical cancer cells. (A) Expression of NEAT1 in a human cervical squamous epithelial cell line (End1) and human cervical cancer cell lines (HeLa, Caski, C33a, SiHa). Among the four cervical cancer cell lines tested, the expression of NEAT1 was highest in the HeLa cells, and lowest in the SiHa cells. (B, C) Expression of NEAT1 in HeLa and SiHa cells transfected with siRNAs and NEAT1 overexpression plasmid, respectively. NEAT1 was significantly downregulated by siRNAs and overexpressed by plasmids. (D) CCK‐8 and (E) colony formation assays were performed to detect the proliferation ability of HeLa and SiHa cells. The proliferation of HeLa cells was suppressed after NEAT1 knockdown, while that of SiHa cells was promoted after NEAT1 overexpression. (F) The cell cycle distribution was detected by flow cytometry. (G) The expression of cell cycle‐related proteins was detected by western blotting. The cell cycle was arrested in NEAT1‐knockdown HeLa cells but promoted in NEAT1‐overexpressing SiHa cells. (H) Migration and invasion ability were detected by Transwell assays. (I) The expression of EMT‐related proteins was detected by western blotting. EMT, migration and invasion of HeLa cells were inhibited following NEAT1 knockdown, while these effects were promoted in SiHa cells following NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human‐derived cervical cancer cell lines HeLa (RRID: CVCL_0030) and SiHa (RRID: CVCL_0032) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Migration, Expressing, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Knockdown, Flow Cytometry, Western Blot

NEAT1 reprograms metabolism to promote metastasis of cervical cancer cells. (A) Glucose consumption, (B) lactate production, and (C) ATP levels in HeLa cells and SiHa cells were detected after transfection with siRNAs or NEAT1 overexpression plasmids for 48 h. Glucose consumption, lactate production and ATP levels were decreased by NEAT1 knockdown in HeLa cells but increased by NEAT1 overexpression in SiHa cells. (D) The mRNA levels of glycolysis‐related genes (HK2, PFKL, PKM2, LDHA, and GLUT1) were detected by RT–qPCR. (E) mRNA expression of tricarboxylic acid cycle‐related genes (CS, IDHA, and OGDH) was detected. The mRNA expression of HK2, PFKL, PKM2, LDHA, and GLUT1 was increased, and that of CS and IDHA was decreased in NEAT1‐knockdown HeLa cells, while NEAT1 overexpression in SiHa cells had the opposite effect. (F) ROS levels were detected with DCFH‐DA probes through flow cytometry, and representative images are shown. ROS levels were decreased in NEAT1‐knockdown HeLa cells but increased in NEAT1‐overexpressing SiHa cells. (G) The MMP was detected by JC‐1 probes through flow cytometry, and representative images were obtained under a fluorescence microscope. The MMP was increased in NEAT1‐knockdown HeLa cells but decreased in NEAT1‐overexpressing SiHa cells. (H) Glucose consumption and (I) lactate production in the control group, NEAT1‐overexpressing group and 2‐DG‐treated NEAT1‐overexpressing group were detected. 2‐DG counteracted the promotion of glucose consumption and lactate production induced by NEAT1 overexpression. (J) Transwell assays were performed to detect cell migration and invasion, and quantitative analysis of the results is shown. (K) The expression of EMT‐related proteins was detected by western blotting. 2‐DG counteracted the enhanced EMT, migration and invasion induced by NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Cancer Medicine

Article Title: Long non‐coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT /β‐catenin/ PDK1 axis

doi: 10.1002/cam4.7221

Figure Lengend Snippet: NEAT1 reprograms metabolism to promote metastasis of cervical cancer cells. (A) Glucose consumption, (B) lactate production, and (C) ATP levels in HeLa cells and SiHa cells were detected after transfection with siRNAs or NEAT1 overexpression plasmids for 48 h. Glucose consumption, lactate production and ATP levels were decreased by NEAT1 knockdown in HeLa cells but increased by NEAT1 overexpression in SiHa cells. (D) The mRNA levels of glycolysis‐related genes (HK2, PFKL, PKM2, LDHA, and GLUT1) were detected by RT–qPCR. (E) mRNA expression of tricarboxylic acid cycle‐related genes (CS, IDHA, and OGDH) was detected. The mRNA expression of HK2, PFKL, PKM2, LDHA, and GLUT1 was increased, and that of CS and IDHA was decreased in NEAT1‐knockdown HeLa cells, while NEAT1 overexpression in SiHa cells had the opposite effect. (F) ROS levels were detected with DCFH‐DA probes through flow cytometry, and representative images are shown. ROS levels were decreased in NEAT1‐knockdown HeLa cells but increased in NEAT1‐overexpressing SiHa cells. (G) The MMP was detected by JC‐1 probes through flow cytometry, and representative images were obtained under a fluorescence microscope. The MMP was increased in NEAT1‐knockdown HeLa cells but decreased in NEAT1‐overexpressing SiHa cells. (H) Glucose consumption and (I) lactate production in the control group, NEAT1‐overexpressing group and 2‐DG‐treated NEAT1‐overexpressing group were detected. 2‐DG counteracted the promotion of glucose consumption and lactate production induced by NEAT1 overexpression. (J) Transwell assays were performed to detect cell migration and invasion, and quantitative analysis of the results is shown. (K) The expression of EMT‐related proteins was detected by western blotting. 2‐DG counteracted the enhanced EMT, migration and invasion induced by NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human‐derived cervical cancer cell lines HeLa (RRID: CVCL_0030) and SiHa (RRID: CVCL_0032) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Transfection, Over Expression, Knockdown, Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Microscopy, Control, Migration, Western Blot

NEAT1 promotes glycolysis by upregulating PDK1. (A) The mRNA and (B) protein levels of PDK1 were detected. (C) The localization and expression of PDK1 in HeLa and SiHa cells were detected by immunofluorescence. Representative images are shown. PDK1 is expressed in both the nucleus and cytoplasm and is positively regulated by NEAT1 in HeLa and SiHa cells. (D) mRNA expression and (E) protein expression of PDK1 in HeLa and SiHa cells transfected with siRNA were detected. (F) Glucose consumption and (G) lactate production in HeLa and SiHa cells were detected. (H) The mRNA expression of glycolysis‐related genes was detected. Glucose consumption, lactate production and the mRNA expression of glycolysis‐related genes were suppressed after PDK1 knockdown in HeLa and SiHa cells. (I) Glucose consumption, (J) lactate production and mRNA expression of glycolysis‐related genes in the control group, NEAT1‐overexpressing group and NEAT1+ si‐PDK1 group were detected. PDK1 knockdown reversed the promoting effect of NEAT1 on glucose consumption, lactate production and the mRNA expression of glycolysis‐related genes. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Cancer Medicine

Article Title: Long non‐coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT /β‐catenin/ PDK1 axis

doi: 10.1002/cam4.7221

Figure Lengend Snippet: NEAT1 promotes glycolysis by upregulating PDK1. (A) The mRNA and (B) protein levels of PDK1 were detected. (C) The localization and expression of PDK1 in HeLa and SiHa cells were detected by immunofluorescence. Representative images are shown. PDK1 is expressed in both the nucleus and cytoplasm and is positively regulated by NEAT1 in HeLa and SiHa cells. (D) mRNA expression and (E) protein expression of PDK1 in HeLa and SiHa cells transfected with siRNA were detected. (F) Glucose consumption and (G) lactate production in HeLa and SiHa cells were detected. (H) The mRNA expression of glycolysis‐related genes was detected. Glucose consumption, lactate production and the mRNA expression of glycolysis‐related genes were suppressed after PDK1 knockdown in HeLa and SiHa cells. (I) Glucose consumption, (J) lactate production and mRNA expression of glycolysis‐related genes in the control group, NEAT1‐overexpressing group and NEAT1+ si‐PDK1 group were detected. PDK1 knockdown reversed the promoting effect of NEAT1 on glucose consumption, lactate production and the mRNA expression of glycolysis‐related genes. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human‐derived cervical cancer cell lines HeLa (RRID: CVCL_0030) and SiHa (RRID: CVCL_0032) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Immunofluorescence, Transfection, Knockdown, Control

NEAT1 upregulates PDK1 by activating WNT/β‐catenin signaling pathway. (A) The expression of PDK1, β‐catenin and downstream molecules of the WNT/β‐catenin signaling pathway was detected. A CTNNB1 overexpression plasmid was used in HeLa cells to establish a WNT signaling activation model, and iCRT3 was used in SiHa cells to establish a WNT signaling inactivation model. Inactivation of WNT signaling led to a decrease in PDK1 and downstream molecules of the WNT signaling pathway, while activation of WNT signaling led to the opposite effect. (B) The correlation between the mRNA expression of PDK1 and CTNNB1 in our local cervical cancer specimens was analyzed. The mRNA expression of PDK1 was positively correlated with the mRNA expression of CTNNB1. (C) The localization of β‐catenin was detected through immunofluorescence. (D) The expression of PDK1, β‐catenin and downstream molecules in the nucleus and cytoplasm was detected. β‐catenin was located in both the nucleus and cytoplasm in HeLa and SiHa cells. After NEAT1 knockdown, nuclear β‐catenin was decreased, and cytoplasmic β‐catenin was increased. NEAT1 overexpression led to an opposite result. (E) The expression of β‐catenin, phosphorylated β‐catenin and component proteins of the WNT pathway was detected. The expression of β‐catenin and components of the WNT pathway decreased in NEAT1‐knockdown HeLa cells but increased in NEAT1‐overexpressing SiHa cells. The phosphorylation of β‐catenin was enhanced in NEAT1‐knockdown HeLa cells and reduced in NEAT1‐overexpressing SiHa cells. (F) CTNNB1 mRNA expression in HeLa and SiHa cells after treatment with Actinomycin D (Act D, 5 μM) was detected. (G) The protein expression of β‐catenin in HeLa and SiHa cells treated with CHX (20 μg/mL) was detected. NEAT1 inhibited the degradation of β‐catenin at the protein but not at the mRNA level. (H) MG132 was used in NEAT1‐knockdown HeLa cells and NEAT1‐overexpressing SiHa cells. MG132 reversed the degradation of β‐catenin caused by NEAT1 knockdown. (I) Ubiquitination of β‐catenin was detected. NEAT1 suppressed the ubiquitination of β‐catenin. (J) An RNA pull‐down assay was performed to detect the interaction between NEAT1 and β‐catenin. NEAT1 and β‐catenin cannot interact with each other. (K) The expression of PDK1 and downstream proteins of the WNT pathway was detected. CTNNB1 overexpression reversed the decrease in PDK1 caused by NEAT1 knockdown, while iCRT3 counteracted the increase in PDK1 induced by NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Cancer Medicine

Article Title: Long non‐coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT /β‐catenin/ PDK1 axis

doi: 10.1002/cam4.7221

Figure Lengend Snippet: NEAT1 upregulates PDK1 by activating WNT/β‐catenin signaling pathway. (A) The expression of PDK1, β‐catenin and downstream molecules of the WNT/β‐catenin signaling pathway was detected. A CTNNB1 overexpression plasmid was used in HeLa cells to establish a WNT signaling activation model, and iCRT3 was used in SiHa cells to establish a WNT signaling inactivation model. Inactivation of WNT signaling led to a decrease in PDK1 and downstream molecules of the WNT signaling pathway, while activation of WNT signaling led to the opposite effect. (B) The correlation between the mRNA expression of PDK1 and CTNNB1 in our local cervical cancer specimens was analyzed. The mRNA expression of PDK1 was positively correlated with the mRNA expression of CTNNB1. (C) The localization of β‐catenin was detected through immunofluorescence. (D) The expression of PDK1, β‐catenin and downstream molecules in the nucleus and cytoplasm was detected. β‐catenin was located in both the nucleus and cytoplasm in HeLa and SiHa cells. After NEAT1 knockdown, nuclear β‐catenin was decreased, and cytoplasmic β‐catenin was increased. NEAT1 overexpression led to an opposite result. (E) The expression of β‐catenin, phosphorylated β‐catenin and component proteins of the WNT pathway was detected. The expression of β‐catenin and components of the WNT pathway decreased in NEAT1‐knockdown HeLa cells but increased in NEAT1‐overexpressing SiHa cells. The phosphorylation of β‐catenin was enhanced in NEAT1‐knockdown HeLa cells and reduced in NEAT1‐overexpressing SiHa cells. (F) CTNNB1 mRNA expression in HeLa and SiHa cells after treatment with Actinomycin D (Act D, 5 μM) was detected. (G) The protein expression of β‐catenin in HeLa and SiHa cells treated with CHX (20 μg/mL) was detected. NEAT1 inhibited the degradation of β‐catenin at the protein but not at the mRNA level. (H) MG132 was used in NEAT1‐knockdown HeLa cells and NEAT1‐overexpressing SiHa cells. MG132 reversed the degradation of β‐catenin caused by NEAT1 knockdown. (I) Ubiquitination of β‐catenin was detected. NEAT1 suppressed the ubiquitination of β‐catenin. (J) An RNA pull‐down assay was performed to detect the interaction between NEAT1 and β‐catenin. NEAT1 and β‐catenin cannot interact with each other. (K) The expression of PDK1 and downstream proteins of the WNT pathway was detected. CTNNB1 overexpression reversed the decrease in PDK1 caused by NEAT1 knockdown, while iCRT3 counteracted the increase in PDK1 induced by NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human‐derived cervical cancer cell lines HeLa (RRID: CVCL_0030) and SiHa (RRID: CVCL_0032) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Over Expression, Plasmid Preparation, Activation Assay, Immunofluorescence, Knockdown, Phospho-proteomics, Ubiquitin Proteomics, Pull Down Assay

NEAT1 enhances proliferation, metastasis and glycolysis in cervical cancer cells through WNT/β‐catenin pathway. (A) Colony formation assays and (B) CCK‐8 assays were performed to detect the proliferation of HeLa and SiHa cells. CTNNB1 overexpression reversed the suppression of proliferation caused by NEAT1 knockdown, while iCRT3 counteracted the promotion of proliferation induced by NEAT1 overexpression. (C) Cell cycle analysis through flow cytometry and (D) Western blotting analyses of cell cycle‐related proteins were performed to detect the cell cycle progression of HeLa and SiHa cells. CTNNB1 overexpression reversed the cell cycle arrest mediated by NEAT1 knockdown, while iCRT3 counteracted the acceleration of the cell cycle resulting from NEAT1 overexpression. (E) Cell migration and invasion were detected by Transwell assays. (F) The expression of EMT‐related proteins was detected. CTNNB1 overexpression reversed the inhibition of migration and invasion induced by NEAT1 knockdown, while iCRT3 counteracted the enhancement of migration and invasion mediated by NEAT1 overexpression. (G) Glucose consumption, (H) lactate production and (I) mRNA expression of glycolysis‐related genes were detected. CTNNB1 overexpression reversed the suppression of glycolysis mediated by NEAT1 knockdown, while iCRT3 counteracted the promotion of glycolysis induced by NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Cancer Medicine

Article Title: Long non‐coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT /β‐catenin/ PDK1 axis

doi: 10.1002/cam4.7221

Figure Lengend Snippet: NEAT1 enhances proliferation, metastasis and glycolysis in cervical cancer cells through WNT/β‐catenin pathway. (A) Colony formation assays and (B) CCK‐8 assays were performed to detect the proliferation of HeLa and SiHa cells. CTNNB1 overexpression reversed the suppression of proliferation caused by NEAT1 knockdown, while iCRT3 counteracted the promotion of proliferation induced by NEAT1 overexpression. (C) Cell cycle analysis through flow cytometry and (D) Western blotting analyses of cell cycle‐related proteins were performed to detect the cell cycle progression of HeLa and SiHa cells. CTNNB1 overexpression reversed the cell cycle arrest mediated by NEAT1 knockdown, while iCRT3 counteracted the acceleration of the cell cycle resulting from NEAT1 overexpression. (E) Cell migration and invasion were detected by Transwell assays. (F) The expression of EMT‐related proteins was detected. CTNNB1 overexpression reversed the inhibition of migration and invasion induced by NEAT1 knockdown, while iCRT3 counteracted the enhancement of migration and invasion mediated by NEAT1 overexpression. (G) Glucose consumption, (H) lactate production and (I) mRNA expression of glycolysis‐related genes were detected. CTNNB1 overexpression reversed the suppression of glycolysis mediated by NEAT1 knockdown, while iCRT3 counteracted the promotion of glycolysis induced by NEAT1 overexpression. The data are shown as the mean ± SD. Three individual experiments were performed. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The human‐derived cervical cancer cell lines HeLa (RRID: CVCL_0030) and SiHa (RRID: CVCL_0032) were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: CCK-8 Assay, Over Expression, Knockdown, Cell Cycle Assay, Flow Cytometry, Western Blot, Migration, Expressing, Inhibition

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