cell lines hela cells atcc ccl 2 cos7 cells atcc n a hek293ft cells atcc n a ss rfp gfp kdel hela stable cells  (ATCC)

Journal: bioRxiv

Article Title: Persistence without turnover: RhoG G12E mutant highlights the role of nucleotide cycling in RhoG signaling

doi: 10.64898/2026.03.25.713116

Figure Lengend Snippet: (A) Coomassie blue stained SDS-PAGE gel from pull down assay with purified RhoG WT and RhoG G12E proteins in controlled GDP or GTP-bound states. The GTP-loaded forms of both proteins interact strongly with GST-ELMO1 coated beads. The results were reproduced in three independent experiments. (B) GST-ELMO1 mediated pull-down of active RhoG in HeLa parental, RhoG WT , and RhoG G12E expressing cells. Lanes: GDP-treated negative control (1–3), GTP-treated positive control (4–6), and cell extract only (7– 9). (C) Input control showing total RhoG expression levels in crude lysates of parental, RhoG WT , and RhoG G12E cells, with GAPDH loading control.

Article Snippet: HeLa (ATCC # CRM-CCL2) cells, stable HeLa cell lines expressing exogenous RhoG WT or RhoG G12E variants and 293T cells (ATCC# CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, 31966-021) supplemented with 10% (v/v) FBS (Eurobio Scientific, CVFSVF00-01) and 1% (v/v) penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2.

Techniques: Staining, SDS Page, Pull Down Assay, Purification, Expressing, Negative Control, Positive Control, Control

Journal: bioRxiv

Article Title: Persistence without turnover: RhoG G12E mutant highlights the role of nucleotide cycling in RhoG signaling

doi: 10.64898/2026.03.25.713116

Figure Lengend Snippet: (A) Scanning electron microscopy of HeLa cells expressing RhoG WT (a) or RhoG G12E (b). Confocal images of phalloidin-stained (red) F-actin and DAPI-stained (blue) nuclei in RhoG WT (c) or RhoG G12E (d) cells. Scale bars, 20 µm. (B) Cell spread area (µm 2 ) quantified from phalloidin-stained boundaries using Fiji (n = 30 cells per condition from 3 independent experiments). Data are mean ± s.e.m. Dots represent individual cells. ****P < 0.0001 (Statistical significance was determine by Mann-Whitney test).

Article Snippet: HeLa (ATCC # CRM-CCL2) cells, stable HeLa cell lines expressing exogenous RhoG WT or RhoG G12E variants and 293T cells (ATCC# CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, 31966-021) supplemented with 10% (v/v) FBS (Eurobio Scientific, CVFSVF00-01) and 1% (v/v) penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2.

Techniques: Electron Microscopy, Expressing, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Persistence without turnover: RhoG G12E mutant highlights the role of nucleotide cycling in RhoG signaling

doi: 10.64898/2026.03.25.713116

Figure Lengend Snippet: (A) Incucyte images of scratch-wound closure by HeLa RhoG WT or RhoG G12E cells at 0 h and 24 h. (B) Trajectories of leading-edge cells over 24 h determined from centroid movement. (C) Migration distance (µm) toward the wound (n = 30 cells per condition from 3 independent experiments). Data are mean ± s.e.m. Dots represent individual cells. ****P < 0.0001 (Statistical significance was determine by Mann-Whitney test).

Article Snippet: HeLa (ATCC # CRM-CCL2) cells, stable HeLa cell lines expressing exogenous RhoG WT or RhoG G12E variants and 293T cells (ATCC# CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, 31966-021) supplemented with 10% (v/v) FBS (Eurobio Scientific, CVFSVF00-01) and 1% (v/v) penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2.

Techniques: Migration, MANN-WHITNEY

Journal: bioRxiv

Article Title: High-throughput imaging of GABA fluorescence as a functional assay for variants in the neurodevelopmental gene, SLC6A1

doi: 10.1101/2025.11.06.687003

Figure Lengend Snippet: ( A ) Schematic of constructs (pCDNA3.1) used to express human GAT-1 ( upper ) and a genetically encoded GABA sensor, iGABA-Snfr ( lower ) with stoichiometrically linked fluorophores. Not to scale. ( B ) Approach to quantifying GAT-1-mediated GABA uptake in HeLa cells. In the presence of exogenous GABA and GAT-1 protein, elevated intracellular concentration of GABA ([GABA] i ) is reflected by increased fluorescence by iGABA-Snfr. ( C ) Workflow for assays to validate the function of GAT-1 variants and the detection of agonist compounds. ( D ) Representative images of iGABA assay and mock-up of automated analysis to measure per-cell average iGABA-Snfr fluorescence. Scale bar = 50 um. ( E ) Relative frequency histograms of iGABA-Snfr fluorescence after incubation with GABA (100uM) versus vehicle (DMSO), as measured by automated analysis at the per-cell level within a single site ( top ) and the per-well averages ( lower ) across a representative 96-well plate (N=48 wells per condition, 4 sites per well).

Article Snippet: A stable HeLa cell line (ATCC, CCL-2) harboring both constructs (V006 and V007) was generated serially by transfection of linearized constructs (PvuI digest) followed by extended antibiotic selection, FACS to select single fluorescent cells for monoclonal culture, followed by FACS reassessment.

Techniques: Construct, Concentration Assay, Fluorescence, Incubation

Journal: bioRxiv

Article Title: High-throughput imaging of GABA fluorescence as a functional assay for variants in the neurodevelopmental gene, SLC6A1

doi: 10.1101/2025.11.06.687003

Figure Lengend Snippet: ( A ) Proposed work-flow for high-throughput screening includes: 1) stable HeLa cell line expressing iGABA-Snfr and hGAT-1 in 384-well format; 2) application of GABA + test compounds using automated liquid handler (Bravo). The GABA concentration for screening is 1uM, given highly specific GAT-1-dependent uptake at this range. As there are no known GAT-1 agonists, a higher concentration of GABA (5uM) is used as a positive control. Incubation duration = 6-8 hrs; 3) measurement of iGABA fluorescence using automated high-content imaging (IXM); 4) plate normalization and quality control; 5) hit detection and prioritization based on robust strictly standardized mean difference (RSSMD). In a primary screen, compounds whose RSSMD versus on-plate negative controls (GABA 1uM) exceed the RSSMD FPR ≤ 5% threshold are considered hits; compounds whose RSSMD exceed the RSSMD TPR ≥ 95% threshold have effect size similar to positive control. ( B ) A linear model (i) estimates the variance in iGABA explained by covariates (ii). Data are barplots of model R 2 or residual R 2 values calculated from ANOVA, and written beside each bar. Statistics are F-tests from ANOVA. ***, p <0.001. ( C ) Assay quality based on RSSMD between positive control (GABA 5uM) and negative control (GABA 1uM) for raw iGABA ( left ) and adjusted iGABA ( right ) following plate normalization. Data are RSSMD of iGABA fluorescence (n=28 wells per replicate x condition) from 2 plates, each with 2 replicas (=4 replicates). ( D-E ) Bootstrap simulations from raw iGABA (D) and adjusted iGABA (E) to identify RSSMD screening thesholds for a primary screen without replicates. Thresholds are shown as functions of the number of on-plate replicate controls versus a single test well. The threshold for hit detection ( blue ) maintains FPR≤5%, while the threshold for detecting compounds with effects as large as positive control (GABA 5uM; red ) maintains TPR≥95%. Vertical dotted lines indicate the RSSMD thresholds @ N=10 replicates. See Method section for further details.

Article Snippet: A stable HeLa cell line (ATCC, CCL-2) harboring both constructs (V006 and V007) was generated serially by transfection of linearized constructs (PvuI digest) followed by extended antibiotic selection, FACS to select single fluorescent cells for monoclonal culture, followed by FACS reassessment.

Techniques: High Throughput Screening Assay, Expressing, Concentration Assay, Positive Control, Incubation, Fluorescence, Imaging, Control, Negative Control

Journal: Biology

Article Title: A Novel Quinoline Inhibitor of the Canonical NF-κB Transcription Factor Pathway

doi: 10.3390/biology13110910

Figure Lengend Snippet: Effect of DMSO on HeLa/NF-κB/luciferase reporter assay. ( A ) Effect of 1% ( v / v ) DMSO (o) on TNF-induced luciferase activity over time: TNF treatment at 20 ng/mL (●). ( B ) Dose effect of DMSO dilutions, tested at 1%, 0.1%, 0.01%, and 0.001% ( v / v ) in medium, on TNF-induced luciferase activity after 3 h compared to unstimulated controls (□). Data are expressed relative to TNF-stimulated cells (100%). One-way ANOVA was performed, followed by Tukey’s post hoc multiple comparisons of treatment means. Significant differences to TNF-induced cells, **** p < 0.0001 ( n = 3). Non-significant, ns. Data are presented as representative of two independent experiments.

Article Snippet: The human NF-κB luciferase reporter HeLa stable cell line (HeLa/NF-κB-Luc; SL-0001—Signosis, Santa Clara, CA, USA) that expresses inducible luciferase under the promoter of the canonical NF-κB pathway was used for the study.

Techniques: Luciferase, Reporter Assay, Activity Assay

Journal: Biology

Article Title: A Novel Quinoline Inhibitor of the Canonical NF-κB Transcription Factor Pathway

doi: 10.3390/biology13110910

Figure Lengend Snippet: Effect of known NF-κB inhibitors on TNF-stimulated HeLa/NF-κB-Luc cell reporter assay. TNF treatment (at 20 ng/mL, for 3 h) of cells pretreated for 20 min with ( A ) proteasome inhibitor MG132, ( B ) macrolide antibiotic clarithromycin, and ( C ) glucocorticoid steroid hydrocortisone ( n = 3). Data are presented as representative of three independent experiments.

Article Snippet: The human NF-κB luciferase reporter HeLa stable cell line (HeLa/NF-κB-Luc; SL-0001—Signosis, Santa Clara, CA, USA) that expresses inducible luciferase under the promoter of the canonical NF-κB pathway was used for the study.

Techniques: Reporter Assay

Journal: Biology

Article Title: A Novel Quinoline Inhibitor of the Canonical NF-κB Transcription Factor Pathway

doi: 10.3390/biology13110910

Figure Lengend Snippet: Effect of quinolines Q1 and Q3 on TNF-induced luciferase activity in HeLa/NF-κB-Luc reporter cell line. ( A ) Serial dilutions of Q1 (▼) and ( B ) Q3 (⬪) and ( C ) the effect of Q1 and Q3 at final concentration of 5 μM on luciferase activity in TNF-stimulated cells (●) compared to unstimulated controls (□); n = 4. ( D ) Cell viability, as assessed by flow cytometry analysis, of HeLa/NF-κB-Luc cells in the absence or presence of 5 μM Q3 for 3 h and 6 h ( n = 3). One-way ANOVA was performed, followed by Tukey’s post hoc multiple comparisons of treatment means. Significant differences, * p < 0.05 and **** p < 0.0001. Non-significant, ns. Data are representative of at least three independent experiments.

Article Snippet: The human NF-κB luciferase reporter HeLa stable cell line (HeLa/NF-κB-Luc; SL-0001—Signosis, Santa Clara, CA, USA) that expresses inducible luciferase under the promoter of the canonical NF-κB pathway was used for the study.

Techniques: Luciferase, Activity Assay, Concentration Assay, Flow Cytometry

Journal: Biology

Article Title: A Novel Quinoline Inhibitor of the Canonical NF-κB Transcription Factor Pathway

doi: 10.3390/biology13110910

Figure Lengend Snippet: IκBα degradation as assessed by immunoblot analysis in TNF-induced HeLa/NF-κB-Luc reporter cells. HeLa/NF-κB-Luc cells were either left unstimulated ( n = 3) or stimulated with 20 ng/mL TNF ( n = 4) for 20 min in the absence or presence of 40 μM MG132 (MG) and 10 μM Q1 or Q3 (all n = 4). ( A ) RIPA cell lysates were analyzed with SDS-PAGE, transferred to a PVDF membrane, and levels of IκBα and β-tubulin were each detected by an immunoblot analysis using specific antibodies. Immunoblots are representative of four experiments. All immunoblot experiments can be reviewed in . ( B ) A quantification analysis of the IκBα bands normalized against β-tubulin levels was performed. One-way ANOVA followed by Tukey’s post hoc multiple comparisons test revealed significant differences. * p < 0.05, **** p < 0.0001. Non-significant, ns.

Article Snippet: The human NF-κB luciferase reporter HeLa stable cell line (HeLa/NF-κB-Luc; SL-0001—Signosis, Santa Clara, CA, USA) that expresses inducible luciferase under the promoter of the canonical NF-κB pathway was used for the study.

Techniques: Western Blot, SDS Page, Membrane

Journal: Biology

Article Title: A Novel Quinoline Inhibitor of the Canonical NF-κB Transcription Factor Pathway

doi: 10.3390/biology13110910

Figure Lengend Snippet: The impact of quinolines Q1 and Q3 on p65/NF-κB nuclear translocation in TNF-induced HeLa/NF-κB-Luc reporter cells. ( A ) Immunocytochemistry images for p65/NF-κB in HeLa/NF-κB-Luc cells left untreated or stimulated with 20 ng/mL TNF for 1 h in the absence or presence of Q1 or Q3. Images are representative of 2 independent experiments, with 3 full field views (100x magnification) per condition for each experiment. Higher-power images can be seen in . ( B , C ) A quantitative analysis to determine TNF-induced p65 nuclear localization (●) compared to unstimulated controls (□) from the immunocytochemistry images and the impact of quinolines Q1 (▼) and Q3 (⬪), performed using ImageJ software 1.54k. Images were analyzed by two approaches. ( B ) Cells showing p65 nuclear staining were counted using the multi-point tool feature in ImageJ; a total of 1000 cells were independently counted per condition by two researchers. Data are illustrated for p65 nuclear (black bars) or cytoplasmic staining (gray bars). ( C ) A digital image analysis was performed using the IHC profiler plugin. Nuclear and cytoplasmic staining, expressed as mean gray value, was measured in cells ( n = 100, randomly selected across 6 full images for each treatment group) and the nuclear/cytoplasmic (N/C) ratio was calculated. The pixel intensity values for any color ranges from 0 to 255, wherein 0 represents the darkest shade and 255 represents the lightest shade of the color. Therefore, a reduction in the N/C ratio reflects higher amounts of p65 localized within the nucleus. Significant differences, **** p < 0.0001, ** p < 0.01, and * p < 0.05 (Kruskal–Wallis test).

Article Snippet: The human NF-κB luciferase reporter HeLa stable cell line (HeLa/NF-κB-Luc; SL-0001—Signosis, Santa Clara, CA, USA) that expresses inducible luciferase under the promoter of the canonical NF-κB pathway was used for the study.

Techniques: Translocation Assay, Immunocytochemistry, Software, Staining

Journal: Biology

Article Title: A Novel Quinoline Inhibitor of the Canonical NF-κB Transcription Factor Pathway

doi: 10.3390/biology13110910

Figure Lengend Snippet: Effect of quinolines Q1 and Q3 on TNF-induced gene expression. Total RNA was isolated from cells either left unstimulated or stimulated with 20 ng/mL TNF for 3 h in the absence or presence of 5 μM Q1 or Q3. Following reverse transcription, a quantification of mRNA abundance was performed by qPCR for ( A ) luciferase and ( B ) TNF in HeLa/NF-κB-Luc reporter cells. ( C – E ) TNF gene expression in colorectal/intestinal cell-lines ( C ) HT-29, ( D ) DLD-1, and ( E ) Int-407 cells following TNF stimulation in the absence or presence of Q3. One-way ANOVA was performed, followed by Tukey’s post hoc multiple comparisons of treatment means. Significant differences, * p < 0.05, **** p < 0.0001. Non-significant, ns. Data are representative of three independent experiments.

Article Snippet: The human NF-κB luciferase reporter HeLa stable cell line (HeLa/NF-κB-Luc; SL-0001—Signosis, Santa Clara, CA, USA) that expresses inducible luciferase under the promoter of the canonical NF-κB pathway was used for the study.

Techniques: Gene Expression, Isolation, Reverse Transcription, Luciferase