Journal: bioRxiv

Article Title: Neuronal cell line expressing full-length mutant huntingtin exhibits alterations in proteolysis

doi: 10.64898/2026.01.15.699723

Figure Lengend Snippet: Application of the Sleeping Beauty system for the transposition of normal/mutant huntingtin genes into the genome of Neuro-2a cells allows to create HD transgenic cellular model and determine the levels of HttQ15/Q138 genes expression in obtained cell lines. a) The validation of transgenes expression by quantitative PCR (qPCR). SB::Q15/Q138 – pSB tet -Neo vector containing HttQ15 or HttQ138 gene, SB::(-) – pSB tet -Neo vector without huntingtin gene insertion, -SB – Neuro-2a cells without transfection, RT – reverse transcription; b) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression; c) Western blotting of poly/monoclonal transgenic Neuro-2a cells lysates without/with doxycycline induction of HttQ15 or HttQ138 expression. Data for control Neuro-2a lines (SB::(-), with transfection of «empty» vector, and -SB, without transfection, are represented also. Top panel – lysates of four different monoclonal transgenic Neuro-2a lines with HttQ15 expression, middle panel – same as top, but for lysates of HttQ138 monoclonal lines, bottom panel – lysates of polyclonal transgenic Neuro-2a lines; d) The transgene (HttQ15/HttQ138) copy number in the genomes of monoclonal (Q15m/Q138m) and polyclonal (Q15p/Q138p) Neuro-2a lines. The GAPDH-normalized Htt gene copy number in the SB::(-) control line was referred to 1. e) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression, (f) Western blotting of lysates without/with doxycycline induction of HttQ15 or HttQ138 expression after 3 years of storage, cultivation and passaging. Total protein content in each sample was controlled by β-actin immunoreactivity. Data in Tables b) and f) and Fig. d) are represented as mean ± S.D. (n = 3).

Article Snippet: The original pSB tet -Neo vector was from Addgene (#60509).

Techniques: Mutagenesis, Transgenic Assay, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Reverse Transcription, Western Blot, Control, Passaging

Journal: bioRxiv

Article Title: Neuronal cell line expressing full-length mutant huntingtin exhibits alterations in proteolysis

doi: 10.64898/2026.01.15.699723

Figure Lengend Snippet: Application of the Sleeping Beauty system for the transposition of normal/mutant huntingtin genes into the genome of Neuro-2a cells allows to create HD transgenic cellular model and determine the levels of HttQ15/Q138 genes expression in obtained cell lines. a) The validation of transgenes expression by quantitative PCR (qPCR). SB::Q15/Q138 – pSB tet -Neo vector containing HttQ15 or HttQ138 gene, SB::(-) – pSB tet -Neo vector without huntingtin gene insertion, -SB – Neuro-2a cells without transfection, RT – reverse transcription; b) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression; c) Western blotting of poly/monoclonal transgenic Neuro-2a cells lysates without/with doxycycline induction of HttQ15 or HttQ138 expression. Data for control Neuro-2a lines (SB::(-), with transfection of «empty» vector, and -SB, without transfection, are represented also. Top panel – lysates of four different monoclonal transgenic Neuro-2a lines with HttQ15 expression, middle panel – same as top, but for lysates of HttQ138 monoclonal lines, bottom panel – lysates of polyclonal transgenic Neuro-2a lines; d) The transgene (HttQ15/HttQ138) copy number in the genomes of monoclonal (Q15m/Q138m) and polyclonal (Q15p/Q138p) Neuro-2a lines. The GAPDH-normalized Htt gene copy number in the SB::(-) control line was referred to 1. e) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression, (f) Western blotting of lysates without/with doxycycline induction of HttQ15 or HttQ138 expression after 3 years of storage, cultivation and passaging. Total protein content in each sample was controlled by β-actin immunoreactivity. Data in Tables b) and f) and Fig. d) are represented as mean ± S.D. (n = 3).

Article Snippet: After pSB tet -Neo PCR linearization (using Q5® High-Fidelity DNA Polymerase, New England Biolabs, USA) with introduction of MluI and NotI restriction sites (forward primer with NotI site: 5′-AATA GCGGCC GCGCTTCCATCGATAGACATGATAAGATAC-3′, reverse primer with MluI site: 5′-TATT ACGCGT CAGAGGCCTTTCGAGGGTAGG-3′; restriction sites are underlined) as well as restriction of the pCI-HttQ15 and pCI-HttQ138 plasmids at MluI and NotI sites, the resulted fragments were ligated (insertion:vector = 1:3 in molar ratio) and transformation of competent E. coli XL1-Blue cells with the obtained ligase mixture was carried out.

Techniques: Mutagenesis, Transgenic Assay, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Reverse Transcription, Western Blot, Control, Passaging

Journal: Cancers

Article Title: Synergistic Disruption of Survival and Metastatic Potential in Esophageal Adenocarcinoma Cells Through Combined Inhibition of HIF1α and CD73

doi: 10.3390/cancers17244016

Figure Lengend Snippet: EAC cells are sensitive to HIF1α and NT5E inhibition, with enhanced effects under hypoxic conditions. ( A , B ) HIF1α and NT5E expression measured by qPCR ( A ) and cell viability measurements expressed as % growth (cell titer relative to day 0) ( B ) upon NT5E knockdown; qPCR data represent mean ± SEM of n = 4; statistical significance determined by unpaired two-tailed t -test. ( C ) Cell viability in HIF1α knockdown alone and in combination with NT5E knockdown under normoxic or hypoxic conditions at 72 h; data are mean ± SEM ( n = 3); comparisons analyzed by two-way ANOVA with Dunnet’s correction. ( D ) % cell growth (cell titer relative to day 0) comparing acriflavine treatment in normoxic and hypoxic conditions. ( E ) % cell growth for combined treatment with acriflavine and PSB12379 under normoxic and hypoxic conditions, showing synergistic viability reduction. ( F ) NT5E CRISPR dependency scores from DepMap across cancer lineages, indicating NT5E is broadly non-essential. ns = not significant; * p -value < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Cells were treated with acriflavine (Sigma-Aldrich, St. Louis, MO, USA) as a HIF1α inhibitor and PSB12379 (MedChemExpress, Monmouth Junction, NJ, USA) as a CD73/NT5E inhibitor at the indicated concentrations under normoxic or hypoxic conditions for 24–72 h prior to viability, migration, or metabolite assays.

Techniques: Inhibition, Expressing, Knockdown, Two Tailed Test, CRISPR

Journal: Cancers

Article Title: Synergistic Disruption of Survival and Metastatic Potential in Esophageal Adenocarcinoma Cells Through Combined Inhibition of HIF1α and CD73

doi: 10.3390/cancers17244016

Figure Lengend Snippet: HIF1α and NT5E inhibition alter purinergic metabolite levels in esophageal adenocarcinoma cells. ( A ) Schematic illustrating hypoxia-induced NT5E expression and the enzymatic conversion of AMP to adenosine. ( B ) Intracellular and extracellular adenosine levels in FLO-1 cells measured by LC-MS, cultured under normoxia or hypoxia. Data represent mean ± SD ( n = 3 biological replicates); significance determined by unpaired two-tailed t -test. ( C ) Intracellular and extracellular AMP levels under the same conditions. Data represent mean ± SD ( n = 3); significance determined by unpaired two-tailed t -test. ( D ) Dose-dependent changes in intracellular adenosine following treatment with PSB12379 , acriflavine, or their combination under hypoxia. Data represent mean ± SD ( n = 3); analyzed by two-way ANOVA with Tukey’s multiple comparison test. ( E ) Adenosine levels in conditioned media from the same experiment, showing dose-dependent accumulation at low and intermediate PSB12379 concentrations, followed by reduction at the highest dose. Data represent mean ± SD ( n = 3); analyzed by two-way ANOVA with Tukey’s post hoc test. ( F ) Intracellular AMP levels under the same treatment conditions; PSB12379 -induced AMP accumulation further enhanced by acriflavine co-treatment. Data represent mean ± SD ( n = 3); analyzed by two-way ANOVA with Tukey’s multiple comparison test. ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Cells were treated with acriflavine (Sigma-Aldrich, St. Louis, MO, USA) as a HIF1α inhibitor and PSB12379 (MedChemExpress, Monmouth Junction, NJ, USA) as a CD73/NT5E inhibitor at the indicated concentrations under normoxic or hypoxic conditions for 24–72 h prior to viability, migration, or metabolite assays.

Techniques: Inhibition, Expressing, Liquid Chromatography with Mass Spectroscopy, Cell Culture, Two Tailed Test, Comparison