hek293 atcc crl 1573 human  (ATCC)

Journal: Cell reports

Article Title: Liprin-α1 enhances PHLDB3 oncogenic function in colorectal cancer via activation of mTORC2-AKT1 pathway

doi: 10.1016/j.celrep.2025.116722

Figure Lengend Snippet: (A) HEK293T cells were transfected with either FLAG-PHLDB3 or FLAG-vector. Proteins were extracted and subjected to co-immunoprecipitation (co-IP). The resulting protein complexes were separated by SDS-PAGE, visualized using Coomassie blue staining, and the excised gel bands were analyzed by LC-MS/MS to identify potential PHLDB3-interacting proteins. (B) Co-localization of exogenous PHLDB3 and RICTOR in HCT116 cells. Cells were transfected with FLAG-PHLDB3 and HA-RICTOR, immunostained with the specified antibodies, and visualized using confocal microscopy. Scale bars, 20 μm. (C) Co-IP and WB analysis showing that endogenous PHLDB3 can be pulled down by endogenous RICTOR in HCT116 cells. (D and E) Co-IP and WB analysis demonstrating that endogenous (D) or exogenous (E) RICTOR can be pulled down by exogenous PHLDB3 in HCT116 cells. (F) Co-IP and WB analysis with indicated antibodies showed that exogenous PHLDB3 was inversely pulled down by exogenous RICTOR in HCT116 cells. (G and H) Mapping the RICTOR-binding domain of PHLDB3 through co-IP and WB analysis. HCT116 cells were co-transfected with FLAG-PHLDB3 fragment plasmids and HA-RICTOR plasmids. Co-IP was performed using FLAG beads, and bands were detected with specified antibodies. (I) WB analysis of protein lysates from stable cells transfected with PLKO or PHLDB3 shRNA using the indicated antibodies. (J and K) WB analysis of HCT116 cells transfected with either FLAG-vector or FLAG-PHLDB3 for 48 h, with or without co-treatment with (J) LY294002 or (K) Torin-1. (L) WB analysis of HCT116 cells transfected with either RICTOR siRNA or control siRNA for 24 h, followed by transfection with either FLAG-control vector or FLAG-PHLDB3 for an additional 48 h.

Article Snippet: Human: HEK293 , ATCC , CRL-1573.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Binding Assay, shRNA, Control

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A ) FRET signal as a function of time of a native lysate of HEK293 cells containing an ECFP/YPET FRET sensor of protease activity that was incubated with PK, PK exposed to electroporation conditions, or water ( n = 4 technical replicates, error bars indicate the standard deviation). ( B ) FRET signal from HEK293 cells expressing an ECFP/YPET FRET sensor that were electroporated with PK, electroporated without protease, or that were incubated with PK without electroporation ( n = 3 technical replicates, error bars indicate the standard deviation). ( C ) PK-Cy5 signal after background subtraction in cells with and without electroporation from three independent technical replicates. p -value between mean of electroporated and control replicates <0.001, two-tailed t-test (replicate 1: p -value = 2.045E-07; replicate 2: p -value = 0.0006261; replicate 3: p -value = 2.032E-07). ( D ) Distribution of the average PK-Cy5 signal intensity from ( C ). ( E ) Example image of PK-Cy5 with and without electroporation. To generate technical replicates, cells were split into the indicated number of aliquots before treatment. .

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques: Activity Assay, Incubation, Electroporation, Standard Deviation, Expressing, Control, Two Tailed Test

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A ) Changes in in-cell LiP-MS peptide intensities in HEK293 cells treated with 20 μM rapamycin compared to DMSO control. Each data point represents a single peptide; half-tryptic peptides of FKBP1A are shown in orange, fully-tryptic peptides of FKBP1A are shown in blue. The lines marks significance levels (FC > 1.5, two-sample unpaired t-test, p -value < 0.01, n = 6 technical replicates). ( B ) Changes in standard LiP-MS peptide intensities in a native lysate of HEK293 cells treated with 10 nM rapamycin compared to DMSO control (6 technical replicates). Each data point represents a single peptide; half-tryptic peptides of FKBP1A are shown in orange, fully-tryptic peptides of FKBP1A are shown in blue. The shaded gray region marks significance levels (FC > 1.5, two-sample unpaired t-test, p -value < 0.01, n = 4 technical replicates). The four FKBP1A peptides with the lowest p -value are highlighted. ( C ) Overall sequence coverage of experiments in ( A , B ). The plot shows the indicated quantities across both conditions (in-cell LiP-MS n = 12 technical replicates; standard LiP-MS n = 8 technical replicates). Error bars are shown in black. ( D ) Number of peptides with missing values per treatment in ( A , B ). The plots report the number of replicates per condition, in which a specific peptide was not quantified. A missing value of 0 indicates that the peptide was quantified in all six replicates, 1 indicates that the peptide was not quantified in 1 out of 6 replicates, and so on. ( E ) Coefficient of variation (CV) of peptide intensities per treatment in ( A , B ). ( F ) Fraction of half-tryptic peptides relative to total peptide intensity for the experiments in ( A , B ). ( G , H ) Plots show the indicated quantities across both conditions (in-cell LiP-MS n = 12; standard LiP-MS n = 8). ( G ) Fraction of peptides with missed cleavages after tryptic digestion relative to total peptide intensity for the experiments in ( A , B ). ( H ) Fraction of peptides with indicated length relative to total peptide intensity in ( A , B ). Only peptides with up to 50 amino acids are shown. To generate technical replicates, cells were split into the indicated number of aliquots before treatment.

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques: Control, Sequencing

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A – E ) In-cell LiP-MS of HEK293 cells treated with 20 μM rapamycin compared to DMSO control; standard LiP-MS in a native lysate of HEK293 cells treated with 10 nM rapamycin compared to DMSO control. Plots ( B – E ) show indicated quantities across both conditions (in-cell LiP-MS n = 12; standard LiP-MS n = 8 technical replicates). Error bars are shown in black. ( A ) Heatmap of peptide intensities (stripped sequence level). Colors above the heatmap correspond to the indicated conditions. ( B ) Number of detected proteins. ( C ) Number of detected peptides. ( D ) Overall sequence coverage considering peptides in both rapamycin treated and control samples (in-cell LiP-MS n = 12 replicates; standard LiP-MS n = 8 technical replicates). ( E ) Number of peptides with indicated length relative to total peptide intensity. Only peptides with up to 50 amino acids are shown. To generate technical replicates, cells were split into the indicated number of aliquots before treatment. ( F ) HEK293T cells were treated with rapamycin under the indicated conditions and probed for mTORC1 or mTORC2 activity using Western blots against the indicated phospho-proteins. The plots (right) show quantification of the Western blots on the left. Statistics were performed with two-way ANOVA on three biological replicates (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). Each replicate was biologically independent, consisting of cells grown separately prior to treatment.

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques: Control, Sequencing, Activity Assay, Western Blot

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A ) Number of peptides with missing values per treatment detected by in-cell LiP-MS. The plots report the number of replicates per condition, in which a specific peptide was not quantified. A missing value of 0 indicates that the peptide was quantified in all six replicates, 1 indicates that the peptide was not quantified in 1 out of 6 replicates. The bars show the total number of peptides detected by in-cell LiP-MS and peptides unique to in-cell LiP-MS in HEK293 cells between in-cell LiP-MS replicates after 5 min of DMSO treatment (in-cell LiP n = 6 technical replicates; lysate LiP n = 4 technical replicates). ( B ) Ratio of average peptide intensities across replicates in lysate and in-cell LiP-MS. ( C , D ) For proteins with >3 peptides detected in at least 3 replicates of both datasets, a protein score was calculated (protein score: mean ratio of log 10 -transformed peptide intensities between in-cell LiP and lysate LiP across peptides). Gene ontology enrichment of cellular compartments was performed for ( C ) proteins with protein scores <0.9 or >1.1, and ( D ) protein scores >0.9 and <1.1 ( p -value < 0.001, Fisher’s exact test using the elim-algorithm in topGO ). ( E ) Ratio of log 10 -transformed peptide intensities between in-cell LiP and lysate LiP, for all peptides and for peptides mapping to proteins located in the mitochondrion based on gene ontology annotation. ( F ) Gene ontology enrichment of protein domains was performed for peptides with a ratio >1.1 of log 10 -transformed peptide intensities between in-cell LiP and lysate LiP (excluding mitochondrial peptides). Domain annotations are from Interpro database ( p -value < 0.001, one-sided Fisher’s exact test). To generate technical replicates, cells were split into the indicated number of aliquots before treatment.

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques: Transformation Assay

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A ) Correlation of average peptide intensities in indicated organelles in HEK293 cells between LiP-MS in lysate and in cells after 5 min of DMSO treatment (in-cell LiP-MS n = 6 replicates; lysate LiP-MS n = 4 technical replicates); ρ indicates the Pearson correlation coefficient. Peptides in blue are mapping to proteins located at the indicated organelle based on gene ontology annotation in Spectronaut. Peptides from other organelles are colored gray. ( B ) Ratio of average peptide intensities in lysate and in-cell LiP-MS for indicated organelles. To generate technical replicates, cells were split into the indicated number of aliquots before treatment.

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques:

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A ) Representative micrographs of HEK293 cells stained with anti-G3BP1 antibody (green) and Hoechst (blue) at the indicated time points of sodium arsenite treatment. Scale bar, 30 µm. ( B ) Peptide intensities from HEK293 cells treated with sodium arsenite compared to untreated cells at 10, 20, and 90 min. Each data point represents a single peptide. The shaded gray region marks significance levels (FC > 1.5, two-sample unpaired t-test with Benjamini–Hochberg adjustment, q-value < 0.05, n = 6 biological replicates). ( C ) Gene ontology enrichment analysis (cellular component) of proteins showing structural changes upon arsenite treatment ( p -value < 0.01, Fisher’s exact test using the elim-algorithm in topGO ). Each replicate was biologically independent, consisting of cells grown separately prior to treatment. .

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques: Staining

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A ) Comparison of HEK293 cells treated with sodium arsenite to untreated cells. Each data point represents a single protein. The shaded gray region marks significance levels (FC > 1.5, p -value < 0.05, n = 6 biological replicates). ( B , C ) Gene ontology enrichment analysis of proteins with peptide-level structural changes upon arsenite treatment (q-value < 0.01). ( B ) Molecular function. ( C ) Biological process. Each replicate was biologically independent, consisting of cells grown separately prior to treatment.

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques: Comparison

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A , B ) SERBP1 is associated with polysome disassembly after 10 min of arsenite treatment. ( A ) Polysome profiling of HEK293 with or without 10 min arsenite treatment (2 biological replicates). ( B ) Western blots for SERBP1 and the ribosomal protein Rpl7 in selected fractions. Left blot, control; right blot, after 10 min of arsenite treatment. ( C , D ) Nuclear speckles become rounder upon sodium arsenite treatment. ( C ) Representative images of HEK293 cells stained with anti-SC35 antibody and Hoechst after 90 min treatment with sodium arsenite. Scale bars, 10 µm. ( D ) Circularity of nuclear speckles with and without sodium arsenite treatment measured for three biological replicates. Colors correspond to replicates. The mean of all replicates (horizontal lines) was compared (mean of control: 0.604; mean of sodium arsenite treatment: 0.741; error bars indicate the standard deviation; two-tailed t test, *** p -value = 0.0007). Each replicate was biologically independent, consisting of cells grown separately prior to treatment. .

Article Snippet: HEK293 cells , ATCC , CRL-1573.

Techniques: Western Blot, Control, Staining, Standard Deviation, Two Tailed Test