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ATCC human cervical cancer hela cells

Dynamic TIRF microscopy measurements of IGF1- and EGF-induced PIP 3 /PI(3,4)P 2 levels in live <t>HeLa,</t> MEF, or A549 cells expressing WT or loss-of-function PIK3CA as indicated. The cells were serum-starved for 3 h prior to stimulation with either saturating doses (100 nM) of IGF1 or EGF and treatment with the PI3Kα inhibitor BYL719 (500 nM). The traces represent the mean PH AKT2 reporter fold change relative to baseline (the median signal of the first four time points). The shading signifies bootstrapped 95% confidence intervals of the mean. The number ( n ) of single cells for each genotype is indicated on the plots. For wild-type (WT) PIK3CA HeLa cells, two <t>independent</t> <t>CRISPR/Cas9</t> clones were used, with and without silent mutations. The 3xFS HeLa cells originate from a single CRISPR/Cas9 clone, engineered with a frameshift (FS) mutation in all three PIK3CA alleles (see also Appendix Fig. ). The MEFs were from polyclonal cultures established from mice with WT or CRE-deleted (KO) PIK3CA , followed by immortalization in vitro (Foukas et al, ). The A549 cells were from a single CRISPR/Cas9 clone per genotype, with knock-out (KO) of PIK3CA caused by a frameshift mutation in exon 3 (Gong et al, ). HeLa datasets for IGF1 and EGF are from six and seven independent experiments, respectively. MEF and A549 IGF1 and EGF data are from 3 independent experiments each. Non-PI3Kα activity refers to the PI3K activity that remains following pharmacological inhibition of PI3Kα.

Human Cervical Cancer Hela Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 945 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human cervical cancer hela cells - by Bioz Stars, 2026-08

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1) Product Images from "Oncogenic PIK3CA corrupts growth factor signaling specificity"

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

Journal: Molecular Systems Biology

doi: 10.1038/s44320-024-00078-x

Dynamic TIRF microscopy measurements of IGF1- and EGF-induced PIP 3 /PI(3,4)P 2 levels in live HeLa, MEF, or A549 cells expressing WT or loss-of-function PIK3CA as indicated. The cells were serum-starved for 3 h prior to stimulation with either saturating doses (100 nM) of IGF1 or EGF and treatment with the PI3Kα inhibitor BYL719 (500 nM). The traces represent the mean PH AKT2 reporter fold change relative to baseline (the median signal of the first four time points). The shading signifies bootstrapped 95% confidence intervals of the mean. The number ( n ) of single cells for each genotype is indicated on the plots. For wild-type (WT) PIK3CA HeLa cells, two independent CRISPR/Cas9 clones were used, with and without silent mutations. The 3xFS HeLa cells originate from a single CRISPR/Cas9 clone, engineered with a frameshift (FS) mutation in all three PIK3CA alleles (see also Appendix Fig. ). The MEFs were from polyclonal cultures established from mice with WT or CRE-deleted (KO) PIK3CA , followed by immortalization in vitro (Foukas et al, ). The A549 cells were from a single CRISPR/Cas9 clone per genotype, with knock-out (KO) of PIK3CA caused by a frameshift mutation in exon 3 (Gong et al, ). HeLa datasets for IGF1 and EGF are from six and seven independent experiments, respectively. MEF and A549 IGF1 and EGF data are from 3 independent experiments each. Non-PI3Kα activity refers to the PI3K activity that remains following pharmacological inhibition of PI3Kα.

Figure Legend Snippet: Dynamic TIRF microscopy measurements of IGF1- and EGF-induced PIP 3 /PI(3,4)P 2 levels in live HeLa, MEF, or A549 cells expressing WT or loss-of-function PIK3CA as indicated. The cells were serum-starved for 3 h prior to stimulation with either saturating doses (100 nM) of IGF1 or EGF and treatment with the PI3Kα inhibitor BYL719 (500 nM). The traces represent the mean PH AKT2 reporter fold change relative to baseline (the median signal of the first four time points). The shading signifies bootstrapped 95% confidence intervals of the mean. The number ( n ) of single cells for each genotype is indicated on the plots. For wild-type (WT) PIK3CA HeLa cells, two independent CRISPR/Cas9 clones were used, with and without silent mutations. The 3xFS HeLa cells originate from a single CRISPR/Cas9 clone, engineered with a frameshift (FS) mutation in all three PIK3CA alleles (see also Appendix Fig. ). The MEFs were from polyclonal cultures established from mice with WT or CRE-deleted (KO) PIK3CA , followed by immortalization in vitro (Foukas et al, ). The A549 cells were from a single CRISPR/Cas9 clone per genotype, with knock-out (KO) of PIK3CA caused by a frameshift mutation in exon 3 (Gong et al, ). HeLa datasets for IGF1 and EGF are from six and seven independent experiments, respectively. MEF and A549 IGF1 and EGF data are from 3 independent experiments each. Non-PI3Kα activity refers to the PI3K activity that remains following pharmacological inhibition of PI3Kα.

Techniques Used: Microscopy, Expressing, CRISPR, Clone Assay, Mutagenesis, In Vitro, Knock-Out, Activity Assay, Inhibition

( A ) TIRF microscopy measurements of IGF1- and EGF-induced PIP 3 /PI(3,4)P 2 kinetics in live HeLa cells with endogenous, dose-controlled expression of PIK3CA H1047R (see also Appendix Fig. ). The cells were serum-starved for 3 h prior to stimulation with the indicated growth factors and treatment with the PI3Kα inhibitor BYL719 (500 nM). Measurements were obtained every 70 s for a total of 60 min. The traces represent the mean PH AKT2 reporter fold change relative to the median signal for the 40–60 min time window, used here to capture the baseline signaling elevation in PIK3CA H1047R mutant cells. The shaded areas represent bootstrapped 95% confidence intervals of the mean. The shading signifies the 95% confidence intervals of the mean. The number ( n ) of single cells for each genotype is indicated on the plots. For WT HeLa cells, two independent CRISPR/Cas9 clones were used, with and without silent mutations. The data are from two independent WT, two independent 1xH1047R, and three independent 2xH1047R CRISPR/Cas9 clones. The data are from the following number ( n ) of independent experiments: n = 6 for 100 nM IGF1; n = 7 for 100 nM EGF; n = 2 for 10 nM IGF1 and 10 nM EGF; n = 3 for 1 nM IGF1; n = 4 for 1 nM EGF. ( B ) Median information capacity in bits (log2) for IGF1 and EGF calculated from the trajectory responses from all independent experiments and individual cells specified in ( A ). Capacity is a measure of the maximum amount of information that flows from the pathway input to its output. The theoretical maximum for three inputs (doses) is 1.5 bits if all the information is captured by the PIP 3 /PI(3,4)P 2 dynamics. The height of each bar specifies the median capacity value, with error bars corresponding to the interquartile range (the distance between the first and the third quartiles). These were estimated following 40 bootstrap repetitions using 80% of the initial observations (default diagnostic settings in the SLEMI package (Jetka et al, )). ( C ) Median information capacity in bits (log2) calculated from snapshot measurements at the indicated time points from the datasets in ( A ).

Figure Legend Snippet: ( A ) TIRF microscopy measurements of IGF1- and EGF-induced PIP 3 /PI(3,4)P 2 kinetics in live HeLa cells with endogenous, dose-controlled expression of PIK3CA H1047R (see also Appendix Fig. ). The cells were serum-starved for 3 h prior to stimulation with the indicated growth factors and treatment with the PI3Kα inhibitor BYL719 (500 nM). Measurements were obtained every 70 s for a total of 60 min. The traces represent the mean PH AKT2 reporter fold change relative to the median signal for the 40–60 min time window, used here to capture the baseline signaling elevation in PIK3CA H1047R mutant cells. The shaded areas represent bootstrapped 95% confidence intervals of the mean. The shading signifies the 95% confidence intervals of the mean. The number ( n ) of single cells for each genotype is indicated on the plots. For WT HeLa cells, two independent CRISPR/Cas9 clones were used, with and without silent mutations. The data are from two independent WT, two independent 1xH1047R, and three independent 2xH1047R CRISPR/Cas9 clones. The data are from the following number ( n ) of independent experiments: n = 6 for 100 nM IGF1; n = 7 for 100 nM EGF; n = 2 for 10 nM IGF1 and 10 nM EGF; n = 3 for 1 nM IGF1; n = 4 for 1 nM EGF. ( B ) Median information capacity in bits (log2) for IGF1 and EGF calculated from the trajectory responses from all independent experiments and individual cells specified in ( A ). Capacity is a measure of the maximum amount of information that flows from the pathway input to its output. The theoretical maximum for three inputs (doses) is 1.5 bits if all the information is captured by the PIP 3 /PI(3,4)P 2 dynamics. The height of each bar specifies the median capacity value, with error bars corresponding to the interquartile range (the distance between the first and the third quartiles). These were estimated following 40 bootstrap repetitions using 80% of the initial observations (default diagnostic settings in the SLEMI package (Jetka et al, )). ( C ) Median information capacity in bits (log2) calculated from snapshot measurements at the indicated time points from the datasets in ( A ).

Techniques Used: Microscopy, Expressing, Mutagenesis, CRISPR, Clone Assay, Diagnostic Assay

( A ) Live-cell fluorescence-based measurements of a miniFOXO-based AKT kinase translocation reporter (KTR) (Gross et al, ), stably expressed in HeLa clones with the indicated PIK3CA genotypes. The total duration of the time course was 300 min, with measurements obtained every 6 min. For each time point, the traces correspond to the mean proportion of cytoplasmic KTR signal, with shaded areas representing bootstrapped 95% confidence intervals of the mean (note that these may be too small to be seen on the figure). Y axis represents an approximation of AKT activity: C, cytoplasmic; N, nuclear. Single-cell numbers ( n ) are shown in the plots. For each condition, WT PIK3CA -expressing cells were also seeded at low density to confirm intra-experimental consistency irrespective of cell crowding. The data are representative of a minimum of two independent experiments per condition, performed in two independent CRISPR/Cas9 clones per genotype. Plots from all independent experiments are shown in Fig. and include control experiments with the 3xFS PIK3CA LOF mutant line. ( B ) Mutual information (MI) in bits (log2) for IGF1 versus each one of the indicated growth factors (EGF, epigen, insulin), calculated using the corresponding KTR trajectory responses ( A ) prior to inhibitor addition. MI values from individual experimental replicates are indicated as dots overlaid on barplots which correspond to the respective mean of each set of measurements. Because IGF1 gave highly robust KTR dynamics, associated with relatively low single-cell noise as reflected in consistently high MI values, it was chosen as the control stimulus in all experimental replicates. ( C ) A graphic summarizing the biochemical signal blurring caused by oncogenic PIK3CA H1047R .

Figure Legend Snippet: ( A ) Live-cell fluorescence-based measurements of a miniFOXO-based AKT kinase translocation reporter (KTR) (Gross et al, ), stably expressed in HeLa clones with the indicated PIK3CA genotypes. The total duration of the time course was 300 min, with measurements obtained every 6 min. For each time point, the traces correspond to the mean proportion of cytoplasmic KTR signal, with shaded areas representing bootstrapped 95% confidence intervals of the mean (note that these may be too small to be seen on the figure). Y axis represents an approximation of AKT activity: C, cytoplasmic; N, nuclear. Single-cell numbers ( n ) are shown in the plots. For each condition, WT PIK3CA -expressing cells were also seeded at low density to confirm intra-experimental consistency irrespective of cell crowding. The data are representative of a minimum of two independent experiments per condition, performed in two independent CRISPR/Cas9 clones per genotype. Plots from all independent experiments are shown in Fig. and include control experiments with the 3xFS PIK3CA LOF mutant line. ( B ) Mutual information (MI) in bits (log2) for IGF1 versus each one of the indicated growth factors (EGF, epigen, insulin), calculated using the corresponding KTR trajectory responses ( A ) prior to inhibitor addition. MI values from individual experimental replicates are indicated as dots overlaid on barplots which correspond to the respective mean of each set of measurements. Because IGF1 gave highly robust KTR dynamics, associated with relatively low single-cell noise as reflected in consistently high MI values, it was chosen as the control stimulus in all experimental replicates. ( C ) A graphic summarizing the biochemical signal blurring caused by oncogenic PIK3CA H1047R .

Techniques Used: Fluorescence, Translocation Assay, Stable Transfection, Clone Assay, Activity Assay, Expressing, CRISPR, Control, Mutagenesis

( A ) The pNDRG1 and pSMAD2/3 signaling responses captured as part of the dataset shown in Fig. . The phosphorylation of NDRG1 on Thr 346 is a marker of mTORC2 activation (García-Martínez and Alessi, ). The phosphorylation of SMAD2/3 (Ser 465/423 ; Ser 467/425 ) is a marker for activated TGFβ signaling which is associated with PIK3CA H1047R phenotypes in human iPSCs (Madsen et al, ). ( B ) CyTOF data from an independent repeat of the experiment in Fig. , using independent CRISPR/Cas9-engineered, 3D-cultured HeLa clones, including the PIK3CA loss-of-function 3xFS clone as an additional control. The spheroids were serum-starved for 4 h prior to the indicated perturbations. The signaling data are from cycling, non-apoptotic cells. The stippled line indicates the position of the peak in WT spheroids treated with vehicle (H 2 O). The gray shading highlights the response region not shown by WT PIK3CA -expressing cells in the absence of stimulation.

Figure Legend Snippet: ( A ) The pNDRG1 and pSMAD2/3 signaling responses captured as part of the dataset shown in Fig. . The phosphorylation of NDRG1 on Thr 346 is a marker of mTORC2 activation (García-Martínez and Alessi, ). The phosphorylation of SMAD2/3 (Ser 465/423 ; Ser 467/425 ) is a marker for activated TGFβ signaling which is associated with PIK3CA H1047R phenotypes in human iPSCs (Madsen et al, ). ( B ) CyTOF data from an independent repeat of the experiment in Fig. , using independent CRISPR/Cas9-engineered, 3D-cultured HeLa clones, including the PIK3CA loss-of-function 3xFS clone as an additional control. The spheroids were serum-starved for 4 h prior to the indicated perturbations. The signaling data are from cycling, non-apoptotic cells. The stippled line indicates the position of the peak in WT spheroids treated with vehicle (H 2 O). The gray shading highlights the response region not shown by WT PIK3CA -expressing cells in the absence of stimulation.

Techniques Used: Phospho-proteomics, Marker, Activation Assay, CRISPR, Cell Culture, Clone Assay, Control, Expressing

( A , B ) The plots in ( A , B ) are from two independent CyTOF datasets using independent CRISPR/Cas9-engineered, 3D-cultured HeLa clones stimulated with 1, 10 or 100 nM of IGF1 or EGF as a function of time. The spheroids were serum-starved for 4 h prior to the indicated perturbations. The signaling data are from cycling, non-apoptotic cells. The stippled line indicates the position of the peak in WT spheroids treated with vehicle (H 2 O). The gray shading highlights the response region not shown by WT PIK3CA -expressing cells in the absence of stimulation. ( C ) Graphical summary of the key observations in the datasets in ( A , B ). A positive response is indicated with (+), the size of which indicates the response magnitude.

Figure Legend Snippet: ( A , B ) The plots in ( A , B ) are from two independent CyTOF datasets using independent CRISPR/Cas9-engineered, 3D-cultured HeLa clones stimulated with 1, 10 or 100 nM of IGF1 or EGF as a function of time. The spheroids were serum-starved for 4 h prior to the indicated perturbations. The signaling data are from cycling, non-apoptotic cells. The stippled line indicates the position of the peak in WT spheroids treated with vehicle (H 2 O). The gray shading highlights the response region not shown by WT PIK3CA -expressing cells in the absence of stimulation. ( C ) Graphical summary of the key observations in the datasets in ( A , B ). A positive response is indicated with (+), the size of which indicates the response magnitude.

Techniques Used: CRISPR, Cell Culture, Clone Assay, Expressing

( A ) Bulk transcriptional profiling of EGF-dependent immediate early and delayed early gene expression in HeLa spheroids with endogenous expression of either WT PIK3CA or one or two copies of PIK3CA H1047R (1-2xH1047R). Expression values are relative and represented as log2 fold-changes, normalized internally to each genotype’s control (H 2 O) response after 30 min of stimulation. All data were further normalized to the expression values of TBP (housekeeping gene). The data are representative of two independent experiments (indicated with solid and stippled lines) with one CRISPR-derived clone per genotype. IGF1 and EGF were used at 100 nM, either alone or in combination as indicated. Note the log2 scale of the y axis. ( B ) Representative fluorescence images of HeLa cells with the indicated genotypes during normal maintenance culture. The cells express a nuclear mCherry marker and were stained with CellMaskBlue and Phalloidin to demarcate their cytoplasm and actin cytoskeleton, respectively. The cells are representative of images from three independent wells per clone and one CRISPR/Cas9 clone per genotype (see also Appendix Fig. for brightfield images of independent HeLa clones for each genotype). The scale bar in ( B ) corresponds to 100 µm. ( C , D ) The cytoplasmic images from all replicates were used for deep learning-based segmentation with Cellpose (Stringer et al, ). Cell shape solidity ( C ) and area ( D ) were quantified for n > 900 single cells per genotype. The P values in ( C , D ) were calculated according to a one-way ANOVA with Tukey’s Honest Significant Difference to correct for multiple comparisons. The exact P value in ( C ) is 0.00066. The P value in ( D ) is <0.0000001. The boxplots display five summary statistics. The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the upper hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range, or distance between the first and third quartiles). The lower whisker extends from the lower hinge to the smallest value at most 1.5 * IQR of the hinge. Data beyond the end of the whiskers are individually plotted outliers.

Figure Legend Snippet: ( A ) Bulk transcriptional profiling of EGF-dependent immediate early and delayed early gene expression in HeLa spheroids with endogenous expression of either WT PIK3CA or one or two copies of PIK3CA H1047R (1-2xH1047R). Expression values are relative and represented as log2 fold-changes, normalized internally to each genotype’s control (H 2 O) response after 30 min of stimulation. All data were further normalized to the expression values of TBP (housekeeping gene). The data are representative of two independent experiments (indicated with solid and stippled lines) with one CRISPR-derived clone per genotype. IGF1 and EGF were used at 100 nM, either alone or in combination as indicated. Note the log2 scale of the y axis. ( B ) Representative fluorescence images of HeLa cells with the indicated genotypes during normal maintenance culture. The cells express a nuclear mCherry marker and were stained with CellMaskBlue and Phalloidin to demarcate their cytoplasm and actin cytoskeleton, respectively. The cells are representative of images from three independent wells per clone and one CRISPR/Cas9 clone per genotype (see also Appendix Fig. for brightfield images of independent HeLa clones for each genotype). The scale bar in ( B ) corresponds to 100 µm. ( C , D ) The cytoplasmic images from all replicates were used for deep learning-based segmentation with Cellpose (Stringer et al, ). Cell shape solidity ( C ) and area ( D ) were quantified for n > 900 single cells per genotype. The P values in ( C , D ) were calculated according to a one-way ANOVA with Tukey’s Honest Significant Difference to correct for multiple comparisons. The exact P value in ( C ) is 0.00066. The P value in ( D ) is <0.0000001. The boxplots display five summary statistics. The lower and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the upper hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range, or distance between the first and third quartiles). The lower whisker extends from the lower hinge to the smallest value at most 1.5 * IQR of the hinge. Data beyond the end of the whiskers are individually plotted outliers.

Techniques Used: Gene Expression, Expressing, Control, CRISPR, Derivative Assay, Fluorescence, Marker, Staining, Clone Assay, Whisker Assay

Figure Legend Snippet: Reagents and tools table

Techniques Used: Knock-Out, Recombinant, Western Blot, Mass Cytometry, Sequencing, Cloning, Mutagenesis, Cell Culture, Membrane, Transfection, Lysis, Extraction, Protease Inhibitor, Sterility, Gel Extraction, Reverse Transcription, SYBR Green Assay, Blocking Assay, Staining, Software, Cytometry, Real-time Polymerase Chain Reaction, Microscopy

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human cervical cancer hela cells (ATCC)

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