Journal: Frontiers in Cell and Developmental Biology

Article Title: Förster Resonance Energy Transfer-Based Single-Cell Imaging Reveals Piezo1-Induced Ca 2+ Flux Mediates Membrane Ruffling and Cell Survival

doi: 10.3389/fcell.2022.865056

Figure Lengend Snippet: Piezo1-mediated extracellular Ca 2+ influx induced by Yoda1 in various cell lines. (A) Viability of MCF-7 cells exposed to control [0.2% (v/v) DMSO] and Yoda1 (0.1–25 μM) for 24 h, measured using the WST-8 assay. The bar graphs describe mean values of cell viability with error bars indicating the standard error of the mean (S.E.M) ( n = 4, ** p < 0.01, student t -test). (B,D) Piezo1 expression in MCF-7 cells with or without control/Piezo1 shRNA as analysed by (B) RT-PCR and (D) western blotting. GAPDH was used as an internal control. (C,E) The bar graphs describe the cumulative densitometric analysis of (C) RT-PCR and (E) western blot bands normalized to the GAPDH. Data are presented as the means ± S.E.M ( n = 4, ** p < 0.01 and *** p < 0.001, student t -test). (F,H) Time-lapse FRET images of the Cytosolic-D3cpv in (F) MCF-7 cells, (H) HEK293A and HeLa cells introduced with or without exogenous Piezo1. MCF-7 cells were exposed to control [0.2% (v/v) DMSO] and Yoda1 (1 μM) dissolved in CO 2 -independent culture medium (Yoda1 group), Ca 2+ -free medium (Yoda1 + w/o Ca 2+ group), and Ca 2+ medium (Yoda1 + w/Ca 2+ group), respectively. (F) Yoda1 + Piezo1 KD group and (H) HEK293A and HeLa cells were incubated in CO 2 -independent culture medium. The color scale bars represent the range of the FRET/CFP emission ratio determined using the biosensor. Hot and cold colors indicate high and low Ca 2+ concentration, respectively. (G,I,J) The time courses represent the average of the normalized FRET/CFP emission ratio changes of cytosolic-D3cpv in (G) MCF-7, (I) HEK293A, and (J) HeLa cells. The lines are mean values of normalized emission ratios, with diluted colors indicating the S.E.M ( n = 7).

Article Snippet: The MCF-7, SiHa, and BeWo cell lines were purchased from the Korean Cell Line Bank (KCLB; Seoul, Republic of Korea), and HEK293A and HeLa cell lines were provided by Dr. Jihye Seong (Korea Institute of Science and Technology, Seoul, South Korea).

Techniques: Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Förster Resonance Energy Transfer-Based Single-Cell Imaging Reveals Piezo1-Induced Ca 2+ Flux Mediates Membrane Ruffling and Cell Survival

doi: 10.3389/fcell.2022.865056

Figure Lengend Snippet: Piezo1-dependent ER-stored Ca 2+ release in response to Yoda1. (A,D) Time-lapse FRET images of the ER Ca 2+ sensor in (A) MCF-7 and (D) HeLa cells. The cells were exposed to control [0.2% (v/v) DMSO] and Yoda1 (1 μM) dissolved in CO 2 -independent culture medium (Yoda1 group), Ca 2+ -free medium (Yoda1 + w/o Ca 2+ group), and Ca 2+ medium (Yoda1 + w/Ca 2+ group), respectively. The color scale bars represent the range of the FRET/CFP emission ratio determined using the biosensor. Hot and cold colors indicate high and low Ca 2+ concentration, respectively. (D) The red fluorescence protein images confirmed the expression of Piezo1-tdTomato. (B,E) The time courses represent the average of the normalized FRET/CFP emission ratio changes of the ER Ca 2+ sensor. The lines are mean values of the normalized emission ratios, with diluted colors indicating the S.E.M ( n = 7). (C,F) The bar graph describes the mean values of the normalized FRET/CFP emission ratios of the ER Ca 2+ sensor at the described time with error bars indicating the S.E.M ( n = 7, * p < 0.05 and ** p < 0.01, Student’s t -test).

Article Snippet: The MCF-7, SiHa, and BeWo cell lines were purchased from the Korean Cell Line Bank (KCLB; Seoul, Republic of Korea), and HEK293A and HeLa cell lines were provided by Dr. Jihye Seong (Korea Institute of Science and Technology, Seoul, South Korea).

Techniques: Concentration Assay, Fluorescence, Expressing

Journal: Nanomaterials

Article Title: Anticancer Potential of L-Histidine-Capped Silver Nanoparticles against Human Cervical Cancer Cells (SiHA)

doi: 10.3390/nano11113154

Figure Lengend Snippet: ( I ) Antiproliferative effect of L-HAgNPs against SiHa cells; ( II ) phase-contrast microscopic images to assess structural changes in cells treated with L-HAgNPs; ( III ) fluorescent images illustrating intracellular ROS level; ( IV ) bar graph depicting quantitative measurement of green fluorescent intensity proportional to ROS level. * p < 0.05 denotes statistical significance between control vs. treated groups.

Article Snippet: SiHa cells were purchased from National Centre for Cell Science (NCCS), Pune, India.

Techniques: Control

Journal: Journal of Translational Medicine

Article Title: RGS1 and related genes as potential targets for immunotherapy in cervical cancer: computational biology and experimental validation

doi: 10.1186/s12967-022-03526-0

Figure Lengend Snippet: Effect of RGS1 knockdown on cervical cancer cells. A qRT-PCR was used to detect the expression of RGS1 after knockdown of RGS1 in HeLa and SiHa cells. B Western Blot was used to detect the expression of RGS1 after knockdown of RGS1 in HeLa and SiHa cells. C CCK8 detected the effect of RGS1 on cell proliferation. D Transwell examined the effects of RGS1 on cell migration and invasion. E FCM was used to detect the effect of RGS1 on apoptosis. F The localization of RGS1 in cells was determined by immunofluorescence

Article Snippet: HeLa (CL-0101, Procell, China) and SiHa (CL-0210, Procell, China) were cultured in RPMI 1640 (SH30255.01, HyClone, USA) with 10% fetal bovine serum (FBS) (164,210, Procell, China) at 37 °C and 5% CO 2 .

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Western Blot, Migration, Immunofluorescence

Journal: PLoS ONE

Article Title: Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action

doi: 10.1371/journal.pone.0191523

Figure Lengend Snippet: Cytotoxicity % (CT) was assessed in cervical cell lines (A) HeLa, (B) Ca Ski, (C) SiHa cells on different concentrations of P N treatments as determined by MTT reduction assay at different incubation period. The bar graphs represent the percentage of cytotoxicity of P N in the cells. Cytotoxicity is shown as mean ± SD derived from at least three separate experiments in triplicate wells. Ordinary two-way ANOVA (multiple comparisons) was performed to calculate the statistical difference ( p≤0 . 05 ) among all treated groups as compared to vehicle treated group. * represents p ≤0.05, ∞ p ≤0.01, $ p ≤0.001 and # p ≤0.0001.

Article Snippet: HeLa (human adeno cervical carcinoma cells), Ca Ski (human epidermoid cervical carcinoma cells), SiHa (Grade II, squamous cervical carcinoma cell) and HEK293 (Human embryonic kidney cells) cell lines were procured from culture collection at National Centre for Cell Science (NCCS, Pune) India.

Techniques: MTT Reduction Assay, Incubation, Derivative Assay

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A). Clonogenic cell survival assays for CaSki, C33A, SiHa, and SiHa PTEN−/− in buffered media with and without glutamine (Gln+ and Gln−). Cells were grown in glutamine and pyruvate-free media (CaSki-5 days and C33A, SiHa and SiHa PTEN−/− - 10 days) and plated for clonogenic cell survival assay in media supplemented with 2 mM glutamine. Cell lines with PI3K pathway mutation are marked by #. (B). TCGA primary cervical cancer (TCGA-CESC) samples with PTEN-inactivating alterations, including 4.8% with deep copy number loss (−2), 22.8% with shallow copy number loss (−1), and ~7.2% with single-nucleotide variants (mut). “−2” (or deep deletion) indicates a deep loss, possibly a homozygous deletion, and “−1” (or shallow deletion) indicates a shallow loss, possibly a heterozygous deletion. (C). PTEN alterations significantly down-regulate PTEN gene expression (FPKM) in cervix tumor samples from the TCGA-CESC cohort. Samples with mutations and shallow deletions show lower PTEN expression compared to samples with wild type (WT) PTEN. Samples with deep deletions show the lowest PTEN expression levels. (D). Changes in glutamine and glutamate metabolism related gene expression after PTEN loss in SiHa cell lines. SiHa_1, SiHa_2, and SiHa_3 (biological replicates of SiHa) cells have wild-type PTEN, while PTEN−/−_1, PTEN−/−_2, and PTEN−/−_3 (biological replicates of SiHa PTEN−/−) cells are derived through knocking out PTEN in SiHa cells (See Methods and Supplementary Figure 1). (E). Changes in glycolysis and gluconeogenesis related gene expression after PTEN loss in SiHa cell lines.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Clonogenic Cell Survival Assay, Mutagenesis, Gene Expression, Expressing, Derivative Assay

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A-C) For clonogenic cell survival assays in CaSki, SiHa, and SiHa PTEN−/− cells were treated with 200 nM telaglenastat monotherapy (CB-839), 25 nM auranafin and 125 μM L-buthionine sulfoximine for 96h. Plating efficiencies for CaSki cells were Ctrl - 109±2, CB-839 - 76±6 , CB-839+AUR - 44±3, CB-839+BSO - 0±0.56, CB-839+BA - 0±0, BA- 39±3), SiHa cells Ctrl - 130±4, CB-839 - 131±12, CB-839+AUR - 124±5, CB-839+BSO - 10±4, CB-839+BA - 6±1.5, BA - 114±4 and, SiHa PTEN−/− cells Ctrl - 65±3., CB-839 - 48±1.5, CB-839+AUR - 35±4, CB-839+BSO - 2±1, CB-839+BA - 2±0.5, BA- 56±3. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05. (D-F) Clonogenic cell survival assays in CaSki, SiHa, and SiHa PTEN−/− with CB839 (200nM) plus increasing single fraction doses of radiation. (G). Cell viability study of normal cervix epithelial cells HCvEpC with and without 200 nM telaglenastat (CB-839) and increasing doses of radiation. (H) Tumor growth delay of SiHa xenografts treated with 75mg/kg of telaglenastat (CB-839) and 4 Gy single fraction dose of tumor directed radiation delivered via SARRP. Statistical Analysis for xenograft studies was performed using Linear mixed model fit by REML (Restricted Maximum Likelihood) as described in the Materials and Methods. Ctrl vs 4 Gy, Ctrl vs CB-839, Ctrl vs CB-839+ 4 Gy, p<0.05; CB-839+ 4 Gy vs CB-839, p<0.05; 4 Gy vs CB-839+ 4 Gy p<0.05. (I) Schematic diagram of proposed primary mechanism of action of CB-839 in cervical cancer.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A) Consumption rate of Glutamine (Gln) (nmol/cell/h) for CaSki, SiHa and SiHa PTEN−/− cells in media with 13C5 labeled Gln. Results are normalized to glucose uptake. (B) Glucose uptake quantified by 18F-Fluoro-deoxy-glucose (FDG) uptake for CaSki, SiHa and SiHa PTEN−/− cells. (C) Glucose consumption from media for cells cultured in the presence and absence of glutamine starvation. (D-G) Cell proliferation assays for CaSki, C33A, SiHa and SiHa PTEN−/− cells in media with and without (Gln+ and Gln−) glutamine. Cells were seeded in 12 well plate at the density of 4000 cells per well. Relative cell density is plotted in Y axis with time as a function in X-axis. (H-K) Cell viability assays for CaSki, C33A, SiHa and SiHa PTEN−/− cells in media with (Gluc+) and without glucose (Gluc−) and or glutamine (Gln+ and Gln−). Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Labeling, Cell Culture, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: A-D Glutamine carbon is used to generate reduced glutathione (GSH) and tricarboxylic acid (TCA) cycle intermediates in PI3K activated cervical cancer cells. CaSki SiHa, and SiHa PTEN−/− cells were grown with 13C5 labeled glutamine (Gln) for 18 hrs, and isotope incorporation into (A) glutamate (M+5 fraction), (B) α-ketoglutarate (α-KG M+5 fraction) and reduced glutathione (GSH represented as relative intensity of M+5 fraction (C) and peak areas of 13C5Gln labeled GSH (D) were quantified by liquid chromatography mass spectroscopy as described in the materials and methods. (E-F) SiHa cells divert glucose carbon into the tricarboxylic acid cycle (TCA) intermediates under conditions of glutamine deprivation (Gln−). SiHa, SiHa PTEN−/− and CaSki cells were grown with 13C6 glucose for 18hrs, followed by 48hrs of media with glutamine (Gln+) or without glutamine (Gln−), and 13C6 glucose isotope incorporation in m+3 tricarboxylic acid (TCA) cycle intermediates (E) Malate and (F) Citrate were quantified by liquid chromatography mass spectrometry as described in the materials and methods. (G-H) Glutamine deprivation results in decreased generation of nucleotide precursors (G) adenosine and (H) dGMP in cervical cancer cells. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Labeling, Liquid Chromatography, Mass Spectrometry, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A-B). Total Glutathione (GSH) level (moles of GSH/mg of protein) and percent GSSG (% of total cellular GSH present in the form of GSSG) for CaSki, SiHa and SiHa PTEN−/− cells grown with (Gln+) and without (Gln−) glutamine for 48 hrs. (C). Reactive oxygen species (ROS) quantified by 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) oxidation in CaSki, SiHa and SiHa PTEN−/− cells with (Gln+) and without (Gln−) glutamine. (D). NADPH/NADP ratio for CaSki, SiHa and SiHa PTEN−/− cells grown with (Gln+) and without (Gln−) glutamine. (E). Thioredoxin reductase (TR) activity (mU/mg of protein) for CaSki, SiHa and SiHa PTEN−/− cells grown with (Gln+) and without (Gln−) glutamine for 48 hrs. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Activity Assay, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: Glutaminase inhibitors induce thiol-mediated oxidative stress and radio-sensitization in treatment resistant cervical cancers

doi: 10.1158/1535-7163.MCT-20-0271

Figure Lengend Snippet: (A) Dose response curve for telaglenastat monotherapy in CaSki, SiHa and SiHa PTEN−/− cells. (B-D). Telaglenastat alteration of intracellular redox pools were measured by quantifying levels of reduced glutathione (GSH), percent oxidized glutathione (%GSSG) and thioredoxin reductase (TR) activity after treating cells with 50 nM of telaglenastat for 48hr. (E-F). Cytotoxic effects of telaglenastat (CB-839) can be rescued by the additional of the thiol antioxidant, N-acetylcysteine (NAC). Clonogenic cell survival assays were performed in CaSki (E) and SiHa PTEN−/− (F) cells 96 h after incubation with 200 nM telaglenastat (CB-839) with (CB + NAC) and without NAC rescue. Statistical analysis: Error bars represent ± SD of N=3 experiments performed on different days. Two tailed paired student’s test was performed and asterisks represents * p <0.05. (G-H) In vivo efficacy of telaglenastat (CB-839) monotherapy in mice with (G) CaSki and SiHa (H) xenografts treated with 50 and 100 mg/Kg for 37 days. Statistical analysis was performed by Linear mixed model fit by REML (Restricted Maximum Likelihood). For CaSki xenograft Ctrl vs CB-839 50 mg/KG, Ctrl vs CB-839 100 mg/KG, the tumor volumes of both treated groups were smaller than Ctrl group with p<0.05. There was no significant difference between CaSki CB-839 50 mg/KG vs CaSki CB-839 100 mg/KG. For SiHa xenograft model Ctrl vs CB-839 50 mg/KG, p<0.05; Ctrl vs CB-839 100 mg/KG, p>0.05; CB-839 50 mg/KG vs CB-839 100 mg/KG, p>0.05.

Article Snippet: For tumor generation, 3.5×10 6 SiHa and or CaSki cells were injected subcutaneously into the left flank of 6–8 week old, female nude mice (Crl:NU(NCr)-Foxn1 nu, Charles River Laboratories, Wilmington, MA) in a half matrigel, half serum-free IMDM mixture.

Techniques: Activity Assay, Incubation, Two Tailed Test, In Vivo