hek293 kidney 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

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Comparison of stable polyclonal HEK cells expressing UNC13A-TS, CFTR-TS, or CUTS following treatment with siRNA control (siControl) (20nM) or TDP-43 (siTDP-43) (0.6nM - 20nM). Cells were reverse transfected with siRNA treatment in complete media supplemented with doxycycline (1000 ng/mL). After 72 h, cells were analyzed by live imagining and protein lysate was harvested for western blot analysis. (A) Schematic of the TDP-43 loss of function Sensor (TS) system design. (B) Overview of the UNC13A-TS, CFTR-TS, and CUTS cassette design. (C) Representative live imaging of TS comparison (10X). (D) Mean intensity quantification of GFP signal intensity as shown in (C). (E-G) Western blot analysis of (E) UNC13A-TS, (F) CFTR-TS, and (G) CUTS developing again GFP and TDP-43 proteins. (H-J) Relative pixel quantification of GFP and TDP-43 band normalized to total protein (Ponceau S) for the indicated TS from E-G. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). Green = GFP signal; red = mCherry signal. Scale bar = 100 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Comparison, Expressing, Control, Transfection, Western Blot, Imaging

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Comparison of stable polyclonal HEK cells expressing UNC13A-TS, CFTR-TS, or CUTS following treatment with siRNA control (siControl) (20nM) or TDP-43 (siTDP-43) (0.6nM - 20nM). Cells were reverse transfected with siRNA treatment in complete media supplemented with doxycycline (1000 ng/mL). After 72 h, cells were analyzed by live imagining and protein lysate was harvested for western blot analysis. ( A) Mean intensity quantification of GFP signal intensity from live imaging, presented as fold change from mock. (B) Relative pixel quantification of GFP normalized to total protein (Ponceau S), presented as fold change from mock. Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparison test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Comparison, Expressing, Control, Transfection, Western Blot, Imaging

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Low-dose siRNA TDP-43 (siTDP-43) treatment was performed in stable polyclonal HEK cells expressing CUTS. CUTS-expressing cells were reverse transfected with siRNA control (siControl) or siTDP43 in a dose-response curve (38 to 1200pM) in doxycycline supplemented media (1000ng/ml) for 72hr. (A) Representative immunofluorescence images of CUTS-expressing HEK cells under low doses of siRNA TDP-43 treatment. (60X). (B) Mean intensity quantification of GFP signal from (A) with normalization to the number mCherry positive cells. (C) Western blot of GFP and TDP-43 proteins from HEK cell lysate expressing CUTS under low doses of siRNA TDP-43. Ponceau S is shown as a loading control. (D) Pixel intensity quantification of the GFP and TDP-43 bands shown in (C), presented as fold-change from the mock-treated sample. (E) Schematic showing the position of qPCR primers, developed to detect CUTS cryptic exon inclusion (referred to as ’CUTS-CE’ and ’CUTS-J’). (F) Representative agarose gel showing qPCR product from melting curve detecting CUTS cryptic exon inclusion using the primers shown in (E). (G) qPCR quantification of the siTDP-43 dose curve presented as fold-change from the mock-treated sample. Purple text indicates the GFP fold change from the mock-treated sample. Red text indicates the percentage of total detectable TDP-43 knockdown. Linear regression analysis shown in (D) and (G) was performed on Log values. Fitting method = least squares regression. Green = GFP; red = mCherry. Scale bar = 50 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Expressing, Transfection, Control, Immunofluorescence, Western Blot, Agarose Gel Electrophoresis, Knockdown

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: Stable HEK cells expressing CUTS were induced with doxycycline (1000 ng/mL) for 24 hours before transfection with the following plasmids: pCMV backbone, TDP-43 WT , TDP-43 ΔNLS , TDP-43 5FL , TDP-43 ΔNLS 5FL , or non-transfected. Following transfection, plasmids were expressed for 72 h, followed by live imaging and protein analysis. (A) Live-imaging of CUTS HEK cells expressing WT or mutant TDP-43 gene cassettes. (B) Representative western blot of exogenous and endogenous GFP and TDP-43. Ponceau S is shown as a loading control. (C) Relative GFP pixel intensity quantification of the band is shown in (B). Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). Green = GFP; red = mCherry. Scale bar = 100 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Expressing, Transfection, Imaging, Mutagenesis, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function

doi: 10.1101/2024.07.12.603231

Figure Lengend Snippet: (A) Schematic of CUTS as an autoregulatory controller of TDP-43 expression (CUTS-TDP43). (B-C) TDP-43 siRNA (siTDP43) dose-response curve in stable polyclonal HEK cells expressing CUTS, CUTS-TDP43, or CUTS-TDP43 (codon optimized). The codon-optimized variation allows for continued expression during siTDP-43 treatment. HEK cells expressing the CUTS, CUTS-TDP-43 and CUTS-TDP-43 codon optimize system were reverse transfected with control siRNA (siControl) or siTDP43 in a dose-response curve (0.6nM-20nM) in a doxycycline (1000ng/ml) supplement media for 72hr. Cells were then used for live imaging or protein analysis. (B) Live imaging of the CUTS variants. (C) Immunoblot assay of GFP and TDP-43. Ponceau S is shown as a loading control. (D-E) CFTR minigene assay in stable CUTS or CUTS-TDP43 (codon optimized) expressing HEK cells. Cells were induced with doxycycline (1000 ng/mL) for 24 h before transfection with the CFTR minigene. Following an additional 24h of expression, cells were transfected with 20nM siControl or siTDP-43. Cells were harvested 48 h following siRNA transfection for RNA extraction and RT-PCR analysis. (D) PCR agarose gel of CFTR minigene. (E) PCR analysis of the ratio between the CFTR cryptic exon inclusion and the correctly spliced product from CFTR as shown in (D). Statistical significance was determined by student t-test (* = P < 0.03; ** = P < 0.002; *** = P < 0.0002; **** = P < 0.0001). Green = GFP; red = mCherry. Scale bar = 100 µm. N=3 biological replicates.

Article Snippet: Human Embryonic Kidney 293 (HEK293) cells (female genotype, acquired from the American Type Culture Collection (ATCC)) and Hela TDP-43 knock-out (KO) cells (a kind gift from Dr. Shawn M Ferguson) ( ) were cultivated in Dulbecco’s Modified Eagle Medium high glucose, pyruvate (DMEM, Thermo Fisher Scientific, 10-313-039) supplemented with 10% HyClone Bovine Growth Serum (Cytiva HyClon, SH3054103HI) and 1X GlutaMAX (Thermo Fisher Scientific, 10-313-039).

Techniques: Expressing, Transfection, Control, Imaging, Western Blot, Mini Gene Assay, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis