cancer derived cell line helas3  (ATCC)

Journal: bioRxiv

Article Title: The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response

doi: 10.1101/2023.09.13.556486

Figure Lengend Snippet: A- Graphical representation of NBirA*-FLAG-TRIP12 fusion protein (top left). IDR: Intrinsically Disordered Region, ARM: armadillo repeats, WWE: tryptophan-tryptophan-glutamate rich domain and HECT: Homologous to E6-AP Carboxyl Terminus. Black rectangles represent Nuclear Localization Signals (NLS). Representative image of NBirA*-FLAG-TRIP12 and biotinylated protein localization in NBirA*-FLAG-TRIP12 expressing HelaS3 cells in interphase (bottom left) or in mitosis (bottom right) treated or not with doxycycline (1 µg/ml) and biotin (50 µM) for 24 h by immunofluorescence using an anti-FLAG antibody and streptavidin-FITC conjugate. Nuclei were counterstained with DAPI. B- Nuclear TRIP12 expression and biotinylation profile in response to doxycycline treatment in NBirA*-FLAG-TRIP12 expressing HelaS3 cells was determined by Western blot using TRIP12 antibody and streptavidin-HRP conjugate. Cells were treated with increasing doses of doxycycline (100, 350, 600 and 1000 ng/ml), biotin (50 µM) for 24 h and with MG-132 (1.25 µM) for 15 h. PCNA level was used as loading control. The black and orange arrows indicate endogenous and exogenous TRIP12 protein, respectively. C- Volcano plot of identified proteins at proximity (∼15 nm) of TRIP12 determined by BioID and label-free mass spectrometry-based quantitative proteomics. The volcano plot represents the statistical significance distribution against the log2-transformed fold change between doxycycline addition (NBirA*-FLAG-TRIP12 expression) and no doxycycline addition (Control). Proteins (n=328) were considered significantly enriched (red on the right) when displaying a corrected p value ≤ 0.05 and an absolute fold change ≥ 2. calculated from four independent experiments. Biotinylated TRIP12 protein is indicated in green on the volcano plot. D- Gene ontology analysis of TRIP12 interactors identified by BioID approach using Metascape software. GO terms having the highest significance are shown. E- Protein networks analysis using Metascape software of TRIP12 protein partners identified by BioID (left). Statistic values are expressed as log10(p-value) (right). F- Protein functional network using String software of TRIP12 protein partners identified by BioID. Coloured circles correspond to proteins that belong to SAGA (yellow), MLL1 (orange), PRC (violet), NURD (blue), SWI/SNF (green) complexes. Corresponding False Discovery Rate (FDR) are indicated.

Article Snippet: VHL-NbGFP4-FLAG HelaS3 cell line was obtained after selection with 1 μg/mL of puromycin (Invivogen).

Techniques: Expressing, Immunofluorescence, Western Blot, Control, Mass Spectrometry, Quantitative Proteomics, Transformation Assay, Software, Functional Assay

Journal: bioRxiv

Article Title: The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response

doi: 10.1101/2023.09.13.556486

Figure Lengend Snippet: A- Representative images of NBirA*-FLAG-TRIP12 and NBirA*-FLAG-EGFP-NLS expressing HelaS3 cells treated or not with doxycycline (1 µg/ml) for 48 h by immunofluorescence using indicated antibodies. Nuclei were counterstained with DAPI. A blue arrow represents the thickening of perinucleolar regions while a red arrow represents chromatin condensates. B- Graphical representation of GFP fusion proteins. The yellow star locates the ubiquitin ligase catalytic site. IDR: Intrinsically Disordered Region, ARM: armadillo repeats, WWE: tryptophan-tryptophan-glutamate rich domain, HECT: Homologous to E6-AP Carboxyl Terminus, GFP: Green Fluorescent Protein, RFTD: Replication Foci Targeting Domain, BAH: Bromo-Adjacent Homology and MTase: MethylTransferase. Black rectangles represent Nuclear Localization Signals (NLS). Numbers indicate the length in amino acids of the proteins. C- Representative images of TRIP12-GFP (left) and GFP-TRIP12 (right) overexpressing HelaS3 cells by immunofluorescence after transient transfection. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Blue arrows represent the thickening of perinuclear and perinucleolar regions. Red arrows represent chromatin condensates. D- Determination of DNA granularity in function to GFP expression level in TRIP12-GFP and GFP-TRIP12 expressing HelaS3 cells. For each cell, the DAPI granularity and GFP expression were determined as described in Materials and Methods on over than 50 cells. Blue and green filled circles correspond to individual non transfected and transfected cells, respectively. The linear regression curve is indicated in black. E- Representative images of ΔHECT-GFP, TRIP12(C1959A)-GFP, H2B-GFP and GFP-DNMT1 expressing HelaS3 cells by immunofluorescence after transient transfection. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. F- Determination of DNA granularity in function of GFP expression level in ΔHECT-GFP, TRIP12(C1959A)-GFP, H2B-GFP and GFP-DNMT1 expressing HelaS3 cells. For each cell, DAPI granularity and GFP expression were determined as described in Materials and Methods on over than 50 cells. Blue and green filled circles correspond to individual non transfected and transfected cells, respectively. The linear regression curve is indicated in black.

Article Snippet: VHL-NbGFP4-FLAG HelaS3 cell line was obtained after selection with 1 μg/mL of puromycin (Invivogen).

Techniques: Expressing, Immunofluorescence, Ubiquitin Proteomics, Transfection

Journal: bioRxiv

Article Title: The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response

doi: 10.1101/2023.09.13.556486

Figure Lengend Snippet: A- Prediction of TRIP12 3D-structure by AlphaFold website. Predicted local distance difference test (pLDDT) indicates the confidence level of the predicted structure. Dark blue, light blue, orange and yellow represent very high, confident, low and very low model confidence, respectively. B- Graphical representation of TRIP12 different domains fused to the GFP reporter protein. Dark blue rectangles locate endogenous or artificial nuclear localization signals (NLS). IDR: Intrinsically Disordered Region, ARM: armadillo domain, WWE: tryptophan-tryptophan-glutamate rich domain, HECT: Homologous to E6-AP Carboxyl Terminus and GFP: Green Fluorescent Protein. C- Representative images of TRIP12-domains fused to GFP expressing HelaS3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Insertion of an artificial NLS in ARM-WWE-GFP, ΔIDR/ARM-WWE/HECT-GFP, HECT-GFP and ΔIDR-GFP constructs allowed a nuclear localization with a substantial leaking in the cytoplasm. D- Determination of DNA granularity in function of GFP expression level in TRIP12-domains fused to GFP expressing HelaS3 cells. For each cell, DAPI granularity and GFP expression were determined as described in Materials and Methods on over than 50 cells. Blue and green filled circles correspond to individual non transfected and transfected cells, respectively. The linear regression curve is indicated in black. E- Representative images of chromatin condensates induced by TRIP12-IDR overexpression in HCT-116, U2OS and hTert-RPE1 cell lines by immunofluorescence. Nuclei were counterstained with DAPI. F- Comparison of isoelectric point (pI) and capacity to form chromatin condensates (slope) of the different TRIP12-GFP constructs. The pI of the different TRIP12 fragments was determined using ProtParam on Expasy website. The capacity to form chromatin condensates is indicated by the slope values obtained in , and . G- Representative images of DNA organization in IDR-GFP deletion constructs in high expressing HelaS3 cells (left). The cytoplasmic leaking of 208-324-GFP and 325-445-GFP constructs is explained by the loss of NLS sequences. Determination of DNA granularity in function of GFP expression level for the different IDR-GFP deletion constructs (right). The DAPI granularity and GFP expression were determined as described in Materials and Methods on over than 50 cells. Blue and green filled circles correspond to individual non transfected and transfected cells, respectively. The linear regression curve is indicated in black. H- Comparison of isoelectric point (pI), capacity to form chromatin condensates (slope) and the length (in aa) of the different IDR-GFP deletion constructs. The pI and the length of the different TRIP12 fragments were determined using ProtParam on Expasy website. The capacity to form chromatin condensates was determined by slope values obtained in . I- Graphical representation of MED1-IDR fused to GFP protein. The dark blue rectangle locates an artificial nuclear localization signals (NLS). Representative images of MED1-IDR-GFP expressing HelaS3 cells obtained by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. For each cell, DAPI granularity and GFP expression were determined as described in Materials and Methods on over than 50 cells. Blue and green filled circles correspond to individual non transfected and transfected cells, respectively. The linear regression curve is indicated in black.

Article Snippet: VHL-NbGFP4-FLAG HelaS3 cell line was obtained after selection with 1 μg/mL of puromycin (Invivogen).

Techniques: Expressing, Immunofluorescence, Construct, Transfection, Over Expression, Comparison

Journal: bioRxiv

Article Title: The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response

doi: 10.1101/2023.09.13.556486

Figure Lengend Snippet: A- Representative images of HelaS3 cells transfected with IDR-GFP construct and localization of TRIP12-IDR mediated condensates and HP1α, H3K9me3, EZH2, H3K27me3 and H2AK119Ub heterochromatin marks by immunofluorescence. Nuclei were counterstained with DAPI. B- Representative images of localization of TRIP12-IDR mediated condensates and phosphorylated RNA polymerase II on serine 2 and serine 5. White arrows show the localization of RNA polymerase II staining at the periphery of the chromatin condensates. Nuclei were counterstained with DAPI.

Article Snippet: VHL-NbGFP4-FLAG HelaS3 cell line was obtained after selection with 1 μg/mL of puromycin (Invivogen).

Techniques: Transfection, Construct, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response

doi: 10.1101/2023.09.13.556486

Figure Lengend Snippet: A- Visualization of CREST (top) and LAMIN B1 (bottom) and chromatin condensates in TRIP12-IDR expressing HelaS3 cells by immunofluorescence. Nuclei were counterstained with DAPI. B- Representative images of chromatin condensate formation and GFP expression in IDR-GFP expressing HelaS3 cells by live cell microscopy. Images were acquired every hour for 24 h after transfection. For the clarity of the figure, representative image of a cell 8 h after transfection and every two hours are represented. Nuclei were counterstained with SiR-DNA-647 nm. C- Quantification of GFP expression and DNA granularity in IDR-GFP expressing HelaS3 cells obtained by live cell microscopy. Images were acquired every hour for 24 hours by live cell confocal microscopy. GFP intensity and DNA granularity were determined using Columbus and FIJI software. Results are expressed as mean ± SEM of at least five independent cells. D- Expression of IDR-GFP, FLAG-tagged anti-GFP degrader in VHL-NbGFP4-FLAG expressing HelaS3 cells determined by Western blot 24 h after increasing doses of doxycycline (0.25, 0.5 and 1 µg/ml). The immunoblots are representative of three independent experiments. HSP90 protein level was used as loading control. E- Representative images of VHL-NbGFP4-FLAG expressing in HelaS3 cells transfected with IDR-GFP construct in the presence or not of doxycycline (1 µg/ml) for 24 h obtained by immunofluorescence. Nuclei were counterstained with DAPI. F- Determination of DNA granularity in function of IDR-GFP expression level in VHL-NbGFP4-FLAG expressing in Hela cells transfected with IDR-GFP construct in the presence or not of doxycycline (1 µg/ml) for 24 h or vehicle. For each cell, DAPI granularity and GFP expression were determined as described in Materials and Methods on over than 50 cells. Blue and green filled circles correspond to individual non transfected and transfected cells, respectively. The linear regression curve is indicated in black. G- Representative images of DNA condensates and IDR-GFP expression in VHL-NbGFP4-FLAG expressing HelaS3 cells transfected with IDR-GFP construct every hour (for 13 h) after addition of doxycyclin (1 µg/ml) by cell live microscopy. DNA is stained with SiR-DNA-647 nm. H- Quantification of DNA granularity and GFP intensity in VHL-NbGFP4-FLAG expressing HelaS3 cells transfected with IDR-GFP construct every hour (for 13 h) after addition of doxycyclin (1 µg/ml) by cell live microscopy. ± SEM of at least five independent cells. I- Representative images of DAPI organization in IDR-GFP expressing HelaS3 cells in the presence of 1,6-hexanediol 0,5% or vehicle for 18 h. Cells with three different GFP intensities are represented. Nuclei were counterstained by DAPI. J- Determination of DAPI granularity in IDR-GFP high expressing HelaS3 cells in the presence of 1,6-hexanediol 0,5% or vehicle for 18 h as described in Materials and Methods section. K- IDR-GFP mobility in DNA condensates by FRAP analysis. H2B-dsRed expressing HelaS3 cells were transfected with IDR-GFP construct or H2B-GFP used as control. After 24 h, GFP fluorescence was photo-bleached using a FRAP module of confocal microscope. The recovery of fluorescence was measured every second for 35 sec. Representative images of IDR-GFP and H2B-GFP expressing cells with bleached and non-bleached areas at indicated times are represented (left). The graph represents the mean ± SD of GFP fluorescence intensity obtained from four independent cells. L- IDR-GFP mobility in DNA condensates by half-bleached FRAP analysis. HelaS3 cells were transfected with IDR-GFP construct. After 24 h, GFP fluorescence in a half of a condensate was photo-bleached using a FRAP module of confocal microscope. The recovery of GFP fluorescence was measured every second for 50 sec. Representative images of an IDR-GFP expressing cell with bleached and non-bleached areas before and just after bleaching are represented (left). The graph represents the fluorescence intensity in a bleached and a none bleached condensate in one cell.

Article Snippet: VHL-NbGFP4-FLAG HelaS3 cell line was obtained after selection with 1 μg/mL of puromycin (Invivogen).

Techniques: Expressing, Immunofluorescence, Microscopy, Transfection, Confocal Microscopy, Software, Western Blot, Control, Construct, Staining, Fluorescence

Journal: bioRxiv

Article Title: The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response

doi: 10.1101/2023.09.13.556486

Figure Lengend Snippet: A- Cellular fate of IDR-GFP (left) and H2B-GFP (right) transfected HelaS3 cells. Twenty-four hours after transfection, fluorescence of IDR-GFP transfected cells was determined using Incucyte apparatus to classify cells in three categories: low, moderate and high. Eleven to twelve cells per category were monitored for 30 h every 2 h. Mitotic divisions were annotated with a M and nuclear fusion with a NF. Green bars represent living cells whereas black bars represent dead cells. B- Distribution in the cell cycle of IDR-GFP (left) and H2B-GFP (right) transfected HelaS3 cells. Twenty-four hours after transfection, the position in the cell cycle was determined by immunofluorescence using CYCLIN A and EdU staining as described in Materials and Methods. The graph represents the percentage of cells in the different phases of the cell cycle expressed as mean (± SEM) of at least three independent experiments. C- Representative image of EdU incorporation in IDR-GFP-mediated chromatin condensates (left). HelaS3 cells were transfected with IDR-GFP construct. Twenty-four hours after transfection, cells were incubated with EdU (1 µM) for 20 min. Incorporation of EdU was visualized by Click-It™ followed by confocal microscopy. Nuclei were counterstained with DAPI. The graph represents the intensity of fluorescence along the arrow (right). D- Determination of genomic accessible regions in IDR-GFP expressing cells by ATAC-seq experiments. Only merged regions with a Max tags > 50 were considered. Results were obtained from two independent experiments and expressed as a percentage ±SEM of genomic accessible regions compared to non-transfected cells (set as 100%). E- Percentage of accessible regions in IDR-GFP expressing HelaS3 cells with a lower (Down, fold change<2), higher (Up, fold change>2) and unchanged accessibility compared to non-transfected cells (set as 100%). Results were obtained from two independent experiments. F- Percentage of down (fold change<2) and up-regulated (fold change>2) mRNA in IDR-GFP expressing HelaS3 cells compared to non-transfected cells by RNA-seq approach. Results were obtained from two independent experiments.

Article Snippet: VHL-NbGFP4-FLAG HelaS3 cell line was obtained after selection with 1 μg/mL of puromycin (Invivogen).

Techniques: Transfection, Fluorescence, Immunofluorescence, Staining, Construct, Incubation, Confocal Microscopy, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: The intrinsically disordered region of the E3 ubiquitin ligase TRIP12 induces the formation of chromatin condensates and interferes with DNA damage response

doi: 10.1101/2023.09.13.556486

Figure Lengend Snippet: A- Representative images of MDC1 foci, EdU and FLAG in irradiated NBirA*-FLAG-TRIP12 and NBirA*-FLAG-EGFP-NLS cell lines treated or not with doxycycline (1 µg/ml for 48 h) during cell cycle phases by immunofluorescence. Cells were X ray-irradiated (1 Gy) and treated with EdU 1 h and 20 min, respectively, before the end of 48 h-doxycycline treatment. The determination of the cell cycle phase is described in Materials and Methods. Nuclei were counterstained with DAPI. B- Quantification of MDC1 foci in irradiated NBirA*-FLAG-TRIP12 and NBirA*-FLAG-EGFP-NLS cells treated or not with doxycycline during the different phases of the cell cycle. Each dot corresponds to one cell. Results are expressed as mean ±SEM obtained from independent experiments determined by confocal microscopy and FIJI macro command. C- Representative images of MDC1 foci and GFP staining in irradiated HelaS3 cells transfected or not indicated GFP constructs by immunofluorescence. Nuclei were counterstained with DAPI. D- Quantification of MDC1 foci in irradiated HelaS3 cells transfected or not indicated GFP constructs. Each dot corresponds to one cell. Results are expressed as mean ± SEM obtained from independent experiments determined by confocal microscopy and FIJI macro command. E- Representative images of MDC1 foci and GFP by immunofluorescence in X ray-irradiated HelaS3 cells expressing IDR-GFP or H2B-GFP. Twenty-three hours after transfection, cells were X-ray irradiated (1 Gy). A non-transfected cell and cells with three different GFP intensities are represented. Nuclei were counterstained with DAPI. F- Quantification of MDC1 foci in irradiated IDR-GFP and H2B-GFP expressing HelaS3 cells depending on GFP expression. IDR-GFP expressing cells were classified in low, moderate and high depending on the intensity of GFP expression. Each dot corresponds to one cell. Results are expressed as mean ± SEM obtained from independent experiments determined by confocal microscopy and FIJI macro command. G- Representative images of GFP and RFP fluorescence in HelaS3 cells expressing the indicated constructs and UV-laser (405 nm) micro-irradiated (time 0) at the indicated times in min by live cell microscopy. For clarity, not all the time points performed in the experiments are represented on the figure. H- Quantification of GFP-MDC1 recruitment kinetics in UV-laser micro-irradiated HelaS3 cells transfected with the indicated constructs at the indicated times. The results are expressed as mean ±SD obtained from 10 cells of two independent experiments. The dotted line represents 100% of recruitment.

Article Snippet: VHL-NbGFP4-FLAG HelaS3 cell line was obtained after selection with 1 μg/mL of puromycin (Invivogen).

Techniques: Irradiation, Immunofluorescence, Confocal Microscopy, Staining, Transfection, Construct, Expressing, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Streamlined rAAV HeLaS3 producer cell line generation via GS selection

doi: 10.1038/s41598-025-34826-2

Figure Lengend Snippet: Generation and validation of GLUL -/- HeLaS3 cell lines.( A ) Schematic representation of the GLUL gene structure, showing three transcript variants with exons represented as boxes and coding sequences highlighted in yellow. Exon 5, where the protein catalytic site is located, is also highlighted in orange. ( B ) T7 endonuclease I assay of PCR-amplified genomic DNA from nucleofected cells. Original gel is presented in Supplementary Figure . ( C ) Phase-contrast microscopy images of GLUL -/- clones GKO1, GKO2, and GKO3 at Day 0, Day 3, and Day 10 post single-cell seeding. White arrows indicate clonal outgrowth. ( D ) Western blot analysis of GLUL protein expression in parental HeLaS3 (GLUL +/+ ) and GLUL -/- clones. β-actin was used as a loading control. Original blots are presented in Supplementary Figure .

Article Snippet: HeLaS3 cell line was acquired from ATCC (CCL-2.2) and maintained in adherent culture at 37 oC and in a 5 % CO 2 atmosphere, in Dulbecco’s Modified Eagle’s Medium (DMEM) (10‐013‐CV, Corning) supplemented with 10 % Fetal Bovine Serum (FBS) (1027016, Gibco).

Techniques: Biomarker Discovery, T7EI Assay, Amplification, Microscopy, Clone Assay, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Streamlined rAAV HeLaS3 producer cell line generation via GS selection

doi: 10.1038/s41598-025-34826-2

Figure Lengend Snippet: Viability of HeLaS3 wild-type and GLUL -/- clones under Gln-supplemented and Gln-depleted conditions. Cells were cultured in standard conditions in serum-containing medium supplemented with Gln ( A ) or depleted of Gln ( B ). Five cell viability measurements were taken by Trypan blue exclusion method over a 9 day period.

Article Snippet: HeLaS3 cell line was acquired from ATCC (CCL-2.2) and maintained in adherent culture at 37 oC and in a 5 % CO 2 atmosphere, in Dulbecco’s Modified Eagle’s Medium (DMEM) (10‐013‐CV, Corning) supplemented with 10 % Fetal Bovine Serum (FBS) (1027016, Gibco).

Techniques: Clone Assay, Cell Culture

Journal: Scientific Reports

Article Title: Streamlined rAAV HeLaS3 producer cell line generation via GS selection

doi: 10.1038/s41598-025-34826-2

Figure Lengend Snippet: Establishment and characterization of rAAV producer clones using GLUL -based selection in suspension-adapted HeLaS3 cells. ( A ) Schematic overview of the streamlined 2-month workflow for rAAV producer cell line generation. The process includes plasmid transfection, 3-week selection in Gln-depleted suspension culture, 4-week single-cell cloning, and expansion of selected producer clones. ( B ) Cell viability and viable cell density (VCD) of HeLaS3 cells non-transfected (in black) or transfected with the rAAV- GLUL producer plasmid (in pink) cultured in Gln-depleted EX-CELL medium over time. The selected pool underwent single-cell cloning, and 120 wells with clonal outgrowth were screened for productivity. rAAV5 vector genome titers (vg/mL) ( C ) and qP ( D ) were measured in culture cell lysates of selected clones by qPCR.

Article Snippet: HeLaS3 cell line was acquired from ATCC (CCL-2.2) and maintained in adherent culture at 37 oC and in a 5 % CO 2 atmosphere, in Dulbecco’s Modified Eagle’s Medium (DMEM) (10‐013‐CV, Corning) supplemented with 10 % Fetal Bovine Serum (FBS) (1027016, Gibco).

Techniques: Clone Assay, Selection, Suspension, Plasmid Preparation, Transfection, Cloning, Cell Culture

Journal: Scientific Reports

Article Title: Streamlined rAAV HeLaS3 producer cell line generation via GS selection

doi: 10.1038/s41598-025-34826-2

Figure Lengend Snippet: Characterization of rAAV5 production and GLUL expression in HeLaS3-derived cell lines. ( A ) Quantification of rAAV5 vector genome (vg) titers expressed as vg/mL for Clone 1, Clone 2, and Clone 3 across different amplification stages: primary screen, secondary screen (static culture), and suspension culture production. Data shown as mean ± SD (n=2) is presented for suspension culture conditions. ( B ) Western blot analysis of GLUL protein levels with β-actin as loading control across HeLaS3 GLUL -/- , transfected Pool, Clone 1, Clone 2, and Clone 3. Original blots are presented in Supplementary Figure . ( C ) Digital Droplet PCR analysis of rAAV5 plasmid copy number per cell for the same cell lines. Data shown as mean ± SD (n=2). ( D ) Mass photometry analysis of rAAV5 particles purified from Clone 1 production, showing empty capsids (~3.7 MDa) and full capsids (~4.7 MDa).

Article Snippet: HeLaS3 cell line was acquired from ATCC (CCL-2.2) and maintained in adherent culture at 37 oC and in a 5 % CO 2 atmosphere, in Dulbecco’s Modified Eagle’s Medium (DMEM) (10‐013‐CV, Corning) supplemented with 10 % Fetal Bovine Serum (FBS) (1027016, Gibco).

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Amplification, Suspension, Western Blot, Control, Transfection, Purification

Journal: Scientific Reports

Article Title: Streamlined rAAV HeLaS3 producer cell line generation via GS selection

doi: 10.1038/s41598-025-34826-2

Figure Lengend Snippet: Optimization of rAAV5 production in HeLaS3-derived cell lines. ( A ) Population doubling time (PDT) and viability of rAAV5 producer Clone 1 grown in the presence (black square) or absence (pink circle) of Gln in growth media. Cells were cultured in standard serum-free conditions and subcultured every 3-4 days. ( B ) rAAV5 vector genome titers (vg/mL; black bars) and cell specific production yield (qP, vg/cell; pink bars) measured in culture cell lysates by qPCR of Clone 1 with production media either supplemented or depleted of Gln. Data shown as mean ± SD (n=4). Statistical analysis was performed using unpaired t-test with Welch’s correction (*: p < 0.05; ***: p < 0.001). ( C ) Representation of ammonia (NH 3 ) concentration (mM) up to 66 h post wtAd5 infection, either in Gln-supplemented medium (black circles) or Gln-depleted (pink squares). Data shown as mean ± SD (n=4). ( D ) Gln and NH 3 metabolic rates (mM/10 6 cells h -1 ) with production media either supplemented (black bars) or depleted (pink bars) in Gln. Data shown as mean ± SD (n=3). Statistical analysis was performed using unpaired t-test with Welch’s correction (*: p < 0.05; ***: p < 0.001).

Article Snippet: HeLaS3 cell line was acquired from ATCC (CCL-2.2) and maintained in adherent culture at 37 oC and in a 5 % CO 2 atmosphere, in Dulbecco’s Modified Eagle’s Medium (DMEM) (10‐013‐CV, Corning) supplemented with 10 % Fetal Bovine Serum (FBS) (1027016, Gibco).

Techniques: Derivative Assay, Cell Culture, Plasmid Preparation, Concentration Assay, Infection

Journal: The Journal of Cell Biology

Article Title: Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins

doi: 10.1083/jcb.202406097

Figure Lengend Snippet: hnRNP A1 downregulation promotes MP20 expression. (A) WB analysis confirming the expression of FLAG-MPs in HEK293T cells. The anti–TDP-43 antibody detected endogenous TDP-FL (FL-endo) and FLAG-MPs. (B) Subcellular localization of FLAG-MPs in HeLaS3 cells. FLAG (green) and Hoechst (blue). Magnified images of FLAG are shown in the right panels. Scale bars: 20 μm (left) and 10 μm (right). (C) WB analysis of TDP-43 protein levels in HEK293T with TDP-FL KD (FL KD). Data are normalized to β-actin. Statistical analysis was performed using Welch’s t test. (D) RT-qPCR analysis of TDP-FL, GPSM2 CE , and ATG4B CE mRNA levels in HEK293T with FL KD. Data are normalized to ACTB . Statistical analysis was performed using Welch’s t test ( n = 3 for each group). (E) WB analysis of FLAG-MPs with Rmt’s (see ). (F) Schematic representation of the sMP13 isoform of TARDBP , induced by hnRNP A1 and E1. (G) Percentage of cytoplasmic leakage in FLAG-hnRNPs–positive cells. Data present 564 cells per group. (H) RT-qPCR analysis of hnRNP A1 , TDP-FL, and MP20 (127) mRNA levels in HEK293T cells with hnRNP A1 KD. Data are normalized to ACTB . Statistical analyses were performed using Welch’s t test ( n = 3 for each group). (I) WB analysis of TDP-43 protein levels in HEK293T cells with hnRNP A1 KD. Quantification of FL-endo, SVs-endo, and MP20-endo protein levels is normalized to β-actin. Statistical analyses were performed using Welch’s t test. (J) Immunocytochemistry of HeLaS3 cells with hnRNP A1 KD. hnRNP A1 (magenta), MP20 Ab (green), and Hoechst (blue). Scale bar: 50 μm. (K) Quantification of nuclear hnRNP A1 or MP20 intensity. Data present 100 cells per group. Statistical analyses were performed using Welch’s t test. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***<0.001, and ****P < 0.0001. Ab, antibody. Source data are available for this figure: .

Article Snippet: HEK293T and HeLaS3 cells were obtained from ATCC, and SH-SY5Y (EC94030304-F0) cells were purchased from KAC Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR, Immunocytochemistry

Journal: The Journal of Cell Biology

Article Title: Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins

doi: 10.1083/jcb.202406097

Figure Lengend Snippet: Quality evaluation of a polyclonal antibody against endogenous TDP-MP20. (A) Schematic representation of MP20 antibody (MP20 Ab) recognizing a unique C-terminal peptide sequence of MP20. (B) Immunocytochemistry results showing FLAG-MP20 overexpression in HeLaS3 cells. MP20 Ab (green), FLAG (magenta), and Hoechst (blue). Scale bar: 20 μm. (C) Immunocytochemistry showing MP20-endo staining in HeLaS3 cells, using a peptide-absorbed MP20 Ab (green) and Hoechst (blue). MP18 peptide served as a negative control. Scale bar: 20 μm. (D) IP analysis of FLAG-MP20–overexpressing HEK293T cells using FLAG or MP20 Ab. An anti-IgG Ab was used as a negative control. Endogenous TDP-43 SVs (SVs-endo) include cleaved FL-endo and variants derived from short isoforms. (E) RT-PCR results comparing TARDBP splicing patterns in human cell lines (HEK293T and SH-SY5Y), adult mouse (11 mo old, male) brain cortex, and spinal cord. (F) WB analysis of MP20 expression in adult mouse (22 mo old, male) brain cortex and spinal cord. (G) Fluorescent immunostaining of adult mouse (22 mo old, male) brain cortex, stained with MP20 (green), MAP2 (magenta), and Hoechst (blue). Arrowheads and arrows denote MAP2-positive and MAP2-negative cells, respectively. Scale bar: 50 μm (upper panel), 10 μm (lower panel). Source data are available for this figure: .

Article Snippet: HEK293T and HeLaS3 cells were obtained from ATCC, and SH-SY5Y (EC94030304-F0) cells were purchased from KAC Co., Ltd.

Techniques: Sequencing, Immunocytochemistry, Over Expression, Staining, Negative Control, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins

doi: 10.1083/jcb.202406097

Figure Lengend Snippet: hnRNP K induces the protein expression of endogenous TDP-MP20. (A) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-hnRNPs. MP20-endo (green), FLAG (magenta), and Hoechst (blue). Yellow boxes indicate magnified images (scale bar: 10 μm). Arrowheads and arrows denote FLAG-positive and FLAG-negative cells, respectively. Scale bar: 50 μm. (B) Quantification of nuclear MP20 fluorescence intensity in FLAG-hnRNPs–positive cells. A total of 100 FLAG-positive and 100 FLAG-negative cells per group were analyzed. Fluorescence signals were normalized to the average intensity of 400 FLAG-negative cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. (C) Comparison of nuclear MP20 signals in cells with FLAG-hnRNP K localized in the nucleus only (Nuc) versus in both the cytoplasm and nucleus (Cyto+Nuc). Nuc: 123 cells; Cyto+Nuc: 177 cells. Welch’s t test was used for statistical analysis. (D and E) WB analysis of HEK293T cells overexpressing FLAG-hnRNPs (D) or with hnRNP K KD (E). Quantification of the relative expression of FL-endo, SVs-endo, and MP20-endo ( n = 3 for each group). Data are normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test (D) or Welch’s t test (E). (F) IP of FLAG-hnRNP K overexpressed in HEK293T cells using MP20 or FLAG Ab, with or without peptide absorption by MP20- or MP18-immunizing peptides. An anti-IgG Ab was used as a negative control. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001. Ab, antibody. Source data are available for this figure: .

Article Snippet: HEK293T and HeLaS3 cells were obtained from ATCC, and SH-SY5Y (EC94030304-F0) cells were purchased from KAC Co., Ltd.

Techniques: Expressing, Immunocytochemistry, Fluorescence, Comparison, Negative Control

Journal: The Journal of Cell Biology

Article Title: Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins

doi: 10.1083/jcb.202406097

Figure Lengend Snippet: hnRNP K enhances hnRNP A1 expression. (A) WB analysis of HEK293T cells overexpressing FLAG-hnRNP K. Quantification of relative expression levels of hnRNP A1 and hnRNP A2/B1 is shown ( n = 3 for each group). (B) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-hnRNP K. FLAG (magenta), hnRNP A1 or hnRNP A2/B1 (green), and Hoechst (blue). Arrowheads indicate FLAG-positive cells, while arrows indicate FLAG-negative cells. Scale bar: 10 μm. (C) Quantification of nuclear hnRNP A1 or hnRNP A2/B1 fluorescence intensity in FLAG-hnRNP K–positive and –negative cells. A total of 100 FLAG-positive and 100 FLAG-negative cells per group were analyzed. Statistical analyses were performed using Welch’s t test. (D) Comparison of nuclear hnRNP A1 or hnRNP A2/B1 fluorescence intensity in cells with FLAG-hnRNP K localized only in the nucleus (Nuc) versus in both the cytoplasm and nucleus (Cyto+Nuc). For hnRNP A1, Nuc: 52 cells, Cyto+Nuc: 48 cells; for hnRNP A2/B1, Nuc: 64 cells, Cyto+Nuc: 35 cells. Statistical analyses were performed using Welch’s t test. (E) WB analysis of HEK293T cells with hnRNP K KD. Quantification of relative expression levels of hnRNP K or hnRNP A1 is shown ( n = 3 for each group). In A and E, data are normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test (A) or Welch’s t test (E). All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are available for this figure: .

Article Snippet: HEK293T and HeLaS3 cells were obtained from ATCC, and SH-SY5Y (EC94030304-F0) cells were purchased from KAC Co., Ltd.

Techniques: Expressing, Immunocytochemistry, Fluorescence, Comparison

Journal: The Journal of Cell Biology

Article Title: Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins

doi: 10.1083/jcb.202406097

Figure Lengend Snippet: hnRNP K levels are not influenced by hnRNP A1 expression. (A–C) RT-qPCR analysis of hnRNP A1 (A and B) and hnRNP K (C) mRNA levels in HEK293T cells overexpressing FLAG-hnRNP K (A) or with hnRNP K KD (B) or overexpressing hnRNP A1 (C). Data are normalized to ACTB . Statistical analyses were performed using one-way ANOVA followed by Tukey’s test (A and C) or Welch’s t test (B), respectively ( n = 3 for each group). (D) WB analysis of HEK293T cells overexpressing FLAG-hnRNP A1. Quantification of hnRNP K is normalized to β-actin. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test ( n = 3 for each group). (E) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-hnRNP K. FLAG (magenta), hnRNP A1 (green), and Hoechst (blue). Arrowheads and arrows indicate FLAG-positive and FLAG-negative cells, respectively. Scale bar: 10 μm. (F) WB analysis of HEK293T cells with hnRNP A1 KD. Quantification of hnRNP A1 and hnRNP K is normalized to β-actin. Statistical analyses were performed using Welch’s t test ( n = 3 for each group). (G) RT-qPCR analysis of hnRNP K mRNA levels in HEK293T cells overexpressing FLAG-hnRNP A1. Data are normalized to ACTB . Statistical analysis was performed using Welch’s t test ( n = 3 for each group). (H) IP of FLAG-hnRNP A1–overexpressing HEK293T cells. FLAG antibody was used for IP, and endogenous hnRNP K was detected by WB. An anti-IgG antibody was used as a negative control for IP. (I–K) Schematic diagram showing conditions with excess hnRNP K (I), hnRNP A1 (J), or hnRNP K coexpressed with hnRNP A1 (K). All graphs show the mean ± SEM. *P < 0.05, **P < 0.01. Source data are available for this figure: .

Article Snippet: HEK293T and HeLaS3 cells were obtained from ATCC, and SH-SY5Y (EC94030304-F0) cells were purchased from KAC Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR, Immunocytochemistry, Negative Control

Journal: The Journal of Cell Biology

Article Title: Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins

doi: 10.1083/jcb.202406097

Figure Lengend Snippet: ALS-associated mutant FUS exhibits a restricted capacity to suppress the expression of hnRNP K and MP20. (A) Comparison of sequences downstream of the FL-stop in exon 6 529–585 in TARDBP between humans and mice. Annotated with FL-stop (magenta), GU-rich (light blue), and AU-rich region (purple), respectively. Magenta lines and square box indicate the FUS-binding consensus reported previously. (B) Schematic representation of the FUS with P525L mutation. (C) RT-qPCR analysis of relative mRNA expression of MP20 (127), TDP-FL, and hnRNP K in HEK293T cells overexpressing FLAG-FUS. Data are normalized to ACTB . Statistical analyses were performed using one-way ANOVA followed by Tukey’s test ( n = 3 for each group). (D) WB of HEK293T cells overexpressing FLAG-FUS. Quantification of relative expression of hnRNP K ( n = 3 for each group). (E) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-FUS. HnRNP K (green), FLAG (magenta), and Hoechst (blue). Arrowheads or arrows indicate FLAG-positive or FLAG-negative cells, respectively. Scale bar: 10 μm. Quantification of the fluorescence intensity of nuclear hnRNP K in FLAG-FUS–positive cells (right). (F) WB of HEK293T cells overexpressing FLAG-FUS. Quantification of relative expression of MP20-endo ( n = 3 for each group). In D and F, data are normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Tukey’s test. (G) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-FUS. MP20 (green), FLAG (magenta), and Hoechst (blue). Arrowheads or arrows indicate FLAG-positive or FLAG-negative cells, respectively. Scale bar: 10 μm. Quantification of the fluorescence intensity of nuclear MP20 in FLAG-FUS–positive cells (right). In E and G, a total of 100 FLAG-positive and 100 FLAG-negative cells per group were analyzed. Fluorescence signals were normalized to the average intensity of 200 FLAG-negative cells. Statistical analysis was performed using Welch’s t test. (H) Comparison of sequences downstream of the MP20 stop codon in exon 6 1,198–1,246 in TARDBP between humans and mice. Annotated with MP20 stop codon (magenta) and GU-rich (light blue), respectively. The magenta lines and square box indicate the FUS-binding consensus reported previously. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Ab, antibody; RMM, RNA recognition motif; ZnF, zinc finger; NES, nuclear export signal; NLS, nuclear localization signal; QSYG, glutamine-glycine-serine-tyrosine-rich domain. Source data are available for this figure: .

Article Snippet: HEK293T and HeLaS3 cells were obtained from ATCC, and SH-SY5Y (EC94030304-F0) cells were purchased from KAC Co., Ltd.

Techniques: Mutagenesis, Expressing, Comparison, Binding Assay, Quantitative RT-PCR, Immunocytochemistry, Fluorescence