Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: SW900 (squamous cell lung carcinoma) , ATCC , ATCC Cat# HTB-59, RRID:CVCL_1731.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: SW620 (Colon adenocarcinoma) , ATCC , Cat# CCL-227, RRID:CVCL_0547.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet: (A) AKTIP mRNA levels between matched normal breast tissues and tumor samples of all subtypes (n = 111 pairs) or luminal subtype only (n = 84 pairs) in TCGA cohort. p values of Wilcoxon signed rank test are shown. (B) Relapse-free survival of patients with ERα-, progesterone receptor (PR)-, and HER2-positive (top) and ERα-, PR-, and HER2-negative (bottom) breast cancer with a lower tertile of AKTIP mRNA level as cutoff. The analysis was generated by KM plotter using expression data obtained from Gene Expression Omnibus datasets. Hazard ratio (HR), 95% confidence interval, and log rank p values are shown. (C and D) Cells were transfected with AKTIP siRNA for 24 h before seeding into 96-well plate or culture insert. (C) Cell viability of ERα-positive MCF7 and MDA-MB-361 cells (left), HER2-enriched SKBR3 and HCC1954 cells (middle), and basal-like MDA-MB-231 and MDA-MB-468 cells (right) was measured over 7 days. Day 0 was the day of cell seeding. Data show mean ± SD of triplicates and one representative of three independent experiments. **p < 0.01; ***p < 0.001; ns, no significant difference by two-way ANOVA with Sidak’s multiple comparison test. (D) Cells were allowed to migrate or invade for 16 h. Mean numbers (top) and representative images (bottom) of migrated or invaded MCF7, MDA-MB-361, SKBR3, or HCC1954 cells of five fields at a magnification of 100× (MCF7 and HCC1954), 200× (MDA-MB-361), or 20× (SKBR3) are shown. Scale bar, 200 μm. Data shown are one of three independent experiments. (E) MCF7 cells stably expressing AKTIP shRNA or vector were subcutaneously injected into nude mice (n = 10) for 6 weeks. Image of tumor nodules extracted (left) and tumor weight and volume (right) are shown. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference compared with mock or vector using t test.

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Generated, Expressing, Gene Expression, Transfection, Comparison, Stable Transfection, shRNA, Plasmid Preparation, Injection

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet: (A) Lysates of MCF7 cells transfected with AKTIP siRNA or mock for 72 h were harvested for reverse-phase protein array. The heatmap shows proteins with >20% change in levels in AKTIP -depleted cells normalized to mock-transfected cells. (B and C) Lysates of MCF7 cells stably expressing AKTIP shRNA and MDA-MB-361 cells transfected with AKTIP siRNA for 72 h were subjected to western blotting for proteins of the (B) AKT and (C) ERα pathways. ERK2 was loading control. (D) Cells were transfected with siRNA targeting the 3′ UTR of AKTIP . Eight h later, lentivirus for AKTIP overexpression (OX) was added to the culture for another 72 h prior to protein harvest for western blotting. ERK2 was loading control. (E) Representative immunohistochemical images of AKTIP -depleted and vector xenograft tumor sections stained with anti- AKTIP , anti-ERα, or anti-Ki67 antibody. Scale bar, 100 μm. (F) Human breast tumor tissue samples were subjected to immunohistochemical staining using anti- AKTIP or anti-ERα antibody. Top, representative immunohistochemical images. The boxes depict magnified areas. Scale bar, 200 μm. Bottom, heatmap illustrating the correlation between ERα and AKTIP staining intensities. p value of Pearson correlation analysis is shown. (G) Lysates of MCF7 cells stably expressing AKTIP shRNA and MDA-MB-361 cells transfected with AKTIP siRNA for 72 h were subjected to western blotting for ERβ levels. The western blots shown are representatives of three independent experiments.

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Transfection, Protein Array, Stable Transfection, Expressing, shRNA, Western Blot, Control, Over Expression, Immunohistochemical staining, Plasmid Preparation, Staining

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet: (A) MCF7 cells stably expressing AKTIP shRNA or vector were treated with 10 μg/mL cycloheximide (CHX) for the indicated hours before being subjected to western blotting. ERK2 was loading control. The graph shows band intensities quantified by densitometry in ImageJ and normalized to that at 0 h (n = 3). *p < 0.05 using two-way ANOVA for comparison between groups with Sidak’s multiple comparison test. (B) Lysates of AKTIP -depleted or vector control cells were subjected to immunoprecipitation (IP) with anti-ERα antibody before western blotting (immunoblotting [IB]). ERα protein levels were normalized prior to IP by using proportionally different amounts of lysates. IP with normal immunoglobulin G (IgG) was control. (C) AKTIP-bound proteins pulled down in MCF7 cells were identified by mass spectrometry. Protein interaction network of the protein partners was generated by STRING. ERα, which was not pulled down in the IP, was included in the STRING analysis to reveal proteins that potentially interact with ERα. Only proteins in the ubiquitin/proteasome degradation pathway are shown in the network map. Red dots: hits known to inhibit protein ubiquitination. Line thickness reflects the degree of confidence of the interaction. (D–G) MCF7 cells stably expressing AKTIP shRNA or vector were transfected with (D) a pool of 4 siRNA of each gene indicated, (E and F) individual siRNA of CAND1 , or (G) OX plasmid of CUL2 or NEDD8 for 72 h. Protein lysates were harvested for western blotting or IP. (H) Lysates of AKTIP -depleted or vector control cells were subjected to IP with anti-CUL2 antibody (top) or anti-CAND1 antibody (bottom) using equal amounts of lysates. (I) Lysates of AKTIP -depleted or vector control cells were subjected to IP with anti-ERα antibody using proportionally different amounts of lysates for equal ERα levels (top) or anti-CUL2 antibody using equal amounts of lysates (bottom). IP with IgG was control. Input was lysate without the IP procedure. Relative densitometric values (after normalization with the immunoprecipitated proteins) are provided below the blots. *p < 0.05; ***p < 0.001; ns, no significant difference compared with mock using t test (n = 3). The western blots shown are representatives of three independent experiments.

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Stable Transfection, Expressing, shRNA, Plasmid Preparation, Western Blot, Control, Comparison, Immunoprecipitation, Mass Spectrometry, Generated, Ubiquitin Proteomics, Transfection

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet: (A) Lysates of MCF7 cells stably expressing AKTIP shRNA or vector were harvested for subcellular fractionation before western blotting. GAPDH and lamin A/C are markers for the cytosolic and nuclear fractions, respectively. (B) MCF7 cells were transfected with AKTIP siRNA for 48 h prior to co-transfection of pRL-TK Renilla luciferase plasmids and 3x-ERE-TATA firefly luciferase or pGL2-Basic (vector backbone without 3x-ERE-TATA) plasmids. Cells were treated with or without 10 nM estradiol (E2) for 24 h. Dual-luciferase reporter assay was performed, and the normalized firefly/Renilla luciferase activities are presented. (C) Volcano plots showing the distribution of DEGs (adjusted p < 5%, fold change > 1.2) identified from the RNA-seq analysis of MCF7 (left) and SKBR3 (right) cellsupon AKTIP loss (n = 2). Upregulated and downregulated DEGs are labeled in red and green, respectively. Labeled genes are DEGs validated by real-time PCR. (D) Venn diagram showing the overlap of DEGs between MCF7 and SKBR3 upon AKTIP knockdown. (E) Diagram showing the percentage of estrogen-responsive genes among the 91 DEGs identified in MCF7 upon AKTIP knockdown. (F) GSEA analysis of hallmark gene sets representing early and late estrogen response using ranked gene expression changes in AKTIP siRNA-transfected cells compared with mock-transfected cells. Normalized enrichment score (NES), p value, and FDR are shown. (G) Total RNA of MCF7 and MDA-MB-361 cells transfected with AKTIP siRNA for 72 h was harvested for real-time PCR. GAPDH was internal control. (H) MCF7 cells stably expressing AKTIP shRNA or vector were treated with 10 nM fulvestrant for 48 h before total RNA was harvested for real-time PCR. (I) Lysates of MCF7 cells stably expressing AKTIP shRNA, MDA-MB-361, SKBR3, and MDA-MB-453 cells transfected with AKTIP siRNA for 72 h were subjected to western blotting. ERK2 was loading control. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference using ordinary one-way ANOVA for analysis within group and two-way ANOVA for comparison between groups with Sidak’s multiple comparison test. Bar graphs are mean ± SD of triplicates and one representative of three independent experiments. The western blots shown are representatives of three independent experiments.

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Stable Transfection, Expressing, shRNA, Plasmid Preparation, Fractionation, Western Blot, Transfection, Cotransfection, Luciferase, Reporter Assay, RNA Sequencing, Labeling, Real-time Polymerase Chain Reaction, Knockdown, Gene Expression, Control, Comparison

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet: (A) MCF7 cells stably expressing AKTIP shRNA or vector were transfected with ESR1 siRNA for 24 h before cells were seeded for cell viability assay, which was measured over 7 days (top), or lysates were harvested for western blotting 8 days after siRNA transfection (bottom). ERK2 was loading control. Day 0 was the day of cell seeding. Bar graphs are mean ± SD of triplicates and one representative of three independent experiments. (B) Lysates of MCF7 cells stably expressing AKTIP shRNA, MDA-MB-361, SKBR3, and MDA-MB-453 cells transfected with AKTIP siRNA for 72 h were subjected to western blotting. (C–F) MCF7 stable cells were (C) treated with 10 nM fulvestrant or 1 μM 4-hydroxytamoxifen (4-OH-Tam) for 48 h; (D) SGK3 siRNA for 72 h; (E) 2 μM AZD1480 for 24 h or STAT3 siRNA for 72 h; or (F) 0.5 μM BBI608 for 2 h before lysates were harvested for western blotting. *p < 0.05; ***p < 0.001; ns, no significant difference compared with vector using t test. The western blots shown are representatives of three independent experiments.

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Stable Transfection, Expressing, shRNA, Plasmid Preparation, Transfection, Viability Assay, Western Blot, Control

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet: (A) MCF7 cells stably expressing AKTIP shRNA or vector were treated with serially diluted concentrations of fulvestrant for 72 h or 4-OH-Tam for 6 days prior to cell viability assay. Lysates of cells treated with the indicated concentration of fulvestrant were harvested for western blotting (right). ERK2 was loading control. (B) Relapse-free survival of patients with ERα-positive breast cancer with high or low AKTIP mRNA levels stratified at the lower quartile. Cohorts of patients with ERα-positive breast cancer treated with adjuvant tamoxifen (n = 158) were selected for the analysis in the KM plotter. Log rank p value is shown. (C) MCF7 cells stably expressing AKTIP shRNA or vector were treated with serial concentrations of AZD1480 for 72 h. (D and E) MCF7 cells stably expressing AKTIP shRNA or vector were treated with serial concentrations of (D, top) fulvestrant (Ful) with or without 2 μM AZD1480 (AZD) for 72 h; (D, bottom) 4-OH-Tam with or without 2 μM AZD; or (E) 4-OH-Tam with or without 2 μM C188–9 for 6 days. The plots show mean ± SD of triplicates and one representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference by two-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Stable Transfection, Expressing, shRNA, Plasmid Preparation, Viability Assay, Concentration Assay, Western Blot, Control, Adjuvant, Comparison

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet: (A) Vector-expressing or AKTIP shRNA-expressing MCF7 tumor-bearing mice (n = 5) were treated with vehicles, 4-OH-TAM (10 mg/kg; once every 2 days), and AZD (20 mg/kg; daily) alone or in combination for 3 weeks. Images of the tumors (top) as well as graphs of tumor weight and tumor volume (bottom) are presented. Error bars represent SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significant difference using two-way ANOVA with Sidak’s multiple comparison test. (B and C) Human breast cancer organoids were transfected with AKTIP siRNA (B) for 9 days prior to harvest for western blotting or immunofluorescence staining or (C) for a period of 14 days, and cell viability (n = 9 of three independent experiments) was measured at 4 and 14 days post-transfection. The western blots and staining shown are representatives of three independent experiments. Scale bar, 20 μm. **p < 0.01; ns, no significant difference by t test. (D) Top, schematic illustration of the experimental design and treatment timeline of the organoids. BME, basement membrane extract. Bottom, viability of the organoids after treatment with 4-OH-Tam in the presence or absence of 2 μM AZD or 0.5 μM BBI608 for 5 days. The plots show mean ± SD of triplicates and one representative of three independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, no significant difference by two-way ANOVA with Sidak’s multiple comparison test. (E) Proposed model of regulation of ERα protein level upon AKTIP loss. When AKTIP is depleted, the binding of CAND1 with cullin 2, and thereby the regulation on cullin 2 by CAND1, is promoted. This may alter the interaction of cullin 2 with substrate receptor (SR) that binds ERα, leading to reduced binding between cullin 2 and ERα. As a result, the ubiquitination and degradation of ERα is decreased. E2, E2 ubiquitin-conjugating enzyme; RBX, RING box protein. The figure was created with BioRender.com .

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Plasmid Preparation, Expressing, shRNA, Comparison, Transfection, Western Blot, Immunofluorescence, Staining, Membrane, Binding Assay, Ubiquitin Proteomics

Journal: Cell reports

Article Title: AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance

doi: 10.1016/j.celrep.2022.111821

Figure Lengend Snippet:

Article Snippet: Human: MCF7 , ATCC , HTB-22.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Membrane, Protease Inhibitor, Extraction, cDNA Synthesis, Reporter Assay, Viability Assay, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: HPAC (pancreatic adenocarcinoma) , ATCC , Cat# CRL-2119, RRID:CVCL_3517.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: HPAF-II (pancreatic adenocarcinoma) , ATCC , Cat# CRL-1997, RRID:CVCL_0313.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend 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.

Article Snippet: Human cervical cancer (HeLa) cells (ATCC #CCL-2 and CRISPR derivatives) and previously established immortalized mouse embryonic fibroblasts (MEFs; generated in Foukas et al, ) were cultured in complete medium consisting of DMEM (with 4 mM l -Glutamine and 1 mM sodium pyruvate; Thermo Fisher Scientific #41966-029) supplemented with an additional 2 mM of l -Glutamine (Sigma #G7513) and 10% fetal bovine serum (FBS; Pan-Biotech #P30-8500).

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

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend 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 ).

Article Snippet: Human cervical cancer (HeLa) cells (ATCC #CCL-2 and CRISPR derivatives) and previously established immortalized mouse embryonic fibroblasts (MEFs; generated in Foukas et al, ) were cultured in complete medium consisting of DMEM (with 4 mM l -Glutamine and 1 mM sodium pyruvate; Thermo Fisher Scientific #41966-029) supplemented with an additional 2 mM of l -Glutamine (Sigma #G7513) and 10% fetal bovine serum (FBS; Pan-Biotech #P30-8500).

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

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend 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 .

Article Snippet: Human cervical cancer (HeLa) cells (ATCC #CCL-2 and CRISPR derivatives) and previously established immortalized mouse embryonic fibroblasts (MEFs; generated in Foukas et al, ) were cultured in complete medium consisting of DMEM (with 4 mM l -Glutamine and 1 mM sodium pyruvate; Thermo Fisher Scientific #41966-029) supplemented with an additional 2 mM of l -Glutamine (Sigma #G7513) and 10% fetal bovine serum (FBS; Pan-Biotech #P30-8500).

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

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend 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.

Article Snippet: Human cervical cancer (HeLa) cells (ATCC #CCL-2 and CRISPR derivatives) and previously established immortalized mouse embryonic fibroblasts (MEFs; generated in Foukas et al, ) were cultured in complete medium consisting of DMEM (with 4 mM l -Glutamine and 1 mM sodium pyruvate; Thermo Fisher Scientific #41966-029) supplemented with an additional 2 mM of l -Glutamine (Sigma #G7513) and 10% fetal bovine serum (FBS; Pan-Biotech #P30-8500).

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

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend 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.

Article Snippet: Human cervical cancer (HeLa) cells (ATCC #CCL-2 and CRISPR derivatives) and previously established immortalized mouse embryonic fibroblasts (MEFs; generated in Foukas et al, ) were cultured in complete medium consisting of DMEM (with 4 mM l -Glutamine and 1 mM sodium pyruvate; Thermo Fisher Scientific #41966-029) supplemented with an additional 2 mM of l -Glutamine (Sigma #G7513) and 10% fetal bovine serum (FBS; Pan-Biotech #P30-8500).

Techniques: CRISPR, Cell Culture, Clone Assay, Expressing

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend 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.

Article Snippet: Human cervical cancer (HeLa) cells (ATCC #CCL-2 and CRISPR derivatives) and previously established immortalized mouse embryonic fibroblasts (MEFs; generated in Foukas et al, ) were cultured in complete medium consisting of DMEM (with 4 mM l -Glutamine and 1 mM sodium pyruvate; Thermo Fisher Scientific #41966-029) supplemented with an additional 2 mM of l -Glutamine (Sigma #G7513) and 10% fetal bovine serum (FBS; Pan-Biotech #P30-8500).

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

Journal: Molecular Systems Biology

Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity

doi: 10.1038/s44320-024-00078-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human cervical cancer (HeLa) cells (ATCC #CCL-2 and CRISPR derivatives) and previously established immortalized mouse embryonic fibroblasts (MEFs; generated in Foukas et al, ) were cultured in complete medium consisting of DMEM (with 4 mM l -Glutamine and 1 mM sodium pyruvate; Thermo Fisher Scientific #41966-029) supplemented with an additional 2 mM of l -Glutamine (Sigma #G7513) and 10% fetal bovine serum (FBS; Pan-Biotech #P30-8500).

Techniques: 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