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