antioxidant response element are luciferase reporter hepg2 hepatic cell line  (BPS Bioscience)

Journal: bioRxiv

Article Title: Discovery and Preclinical Validation of a Clinically Optimized Mitochondrial Complex I Modulator for Alzheimer’s Disease

doi: 10.64898/2026.04.10.717554

Figure Lengend Snippet: a A drug discovery funnel used for the identification of C273. b C273 protects MC65 Tet-Off cells from Aβ toxicity (red line). # indicates cell death in Tet-Off cells treated with vehicle. No toxicity was observed in MC65 Tet-On cells (Aβ not expressed, black line). EC 50 values were calculated using nonlinear regression in GraphPad Prism 10. c Treatment with C273 for 24 h does not induce cytotoxicity in HepG2 human hepatic cells at concentrations up to 100 μM. d C273 increases NADH levels in MC65 Tet-On cells, consistent with inhibition of mtCI. NADH levels were measured over 24 h. e Inhibition of mtCI activity in isolated mouse brain mitochondria by C273 measured using an NADH oxidation assay. f Activities of mitochondrial ETC complexes I–IV were assessed in permeabilized MC65 cells using an electron flow assay in the Seahorse XFe96 Extracellular Flux Analyzer in the presence of C273 (25 and 50 μM). g Injection of rotenone or C273 to monitor OCR kinetics in MC65 Tet-On cells. h Pretreatment with rotenone blocks the protective effect of C273 in MC65 Tet-Off cells (Aβ expressed). Open orange squares: Tet-On cells, no Aβ expression, treated with vehicle (positive control, 100% viability). Blue triangles: Tet-Off cells (Aβ expressed), which undergo extensive cell death after 3 days. Tet-Off cells treated with C273 (2 nM - 5 μM; blue line) show complete protection from Aβ toxicity. Solid red triangles: Tet-On cells treated with 32 nM rotenone, which show no toxicity. Open red triangles: Tet-Off cells treated with 32 nM rotenone alone, resulting in complete cell death. Pretreatment with 32 nM rotenone blocks the protective effect of C273 in Tet-Off cells (red line, grey box), supporting a competitive interaction at mtCI. Partial rescue at higher C273 concentrations reflects incomplete occupancy of the mtCI binding site by rotenone, which inhibits ∼50% of mtCI activity at this dose. The grey box indicates the EC 50 concentration range for C273. P values were calculated using unpaired two-tailed Student’s t -tests; ** P < 0.01. Data are presented as mean ± SD ( n = 4 technical replicates). All experiments were replicated at least twice.

Article Snippet: NF-κB Reporter (Luc) HEK293 cell line and the antioxidant response element (ARE) Luciferase Reporter HepG2 hepatic cell line were purchased from BPS Bioscience (CA, USA) and were cultured in accordance of manufacturer instructions.

Techniques: Drug discovery, Inhibition, Activity Assay, Isolation, Oxidation Assay, Injection, Expressing, Positive Control, Binding Assay, Concentration Assay, Two Tailed Test

Journal: Hepatology (Baltimore, Md.)

Article Title: HAF prevents hepatocyte apoptosis and progression to MASH and HCC through transcriptional regulation of the NF-κB pathway

doi: 10.1097/HEP.0000000000001070

Figure Lengend Snippet: HAF depletion in vivo and in vitro is associated with decreased NF-κB activity. (A, B) Effect of HAF depletion on phosphorylation of NF-κB p65 (P-p65) and NF-κB p50 (P-p50) through western blotting in whole-liver lysates from 6-month-old male LPC-S +/− (A) or female hepS −/ − mice (B); and in pooled isolated hepatocytes from two 3-month-old male LPC-S +/− or control mice after ex vivo treatment with indicated durations of TNF (C), with quantitation shown above plots. (D, E) Effect of transient transfection with 2 independent HAF siRNA versus nontargeting control siRNA on (D), protein levels of P-p65/GAPDH and P-p50/T-p50, or (E), NF-κB response element luciferase (NF-κB-RE Luc) reporter activity in HepG2 cells (data shown as mean ± SEM is representative of 3 independent experiments). (F) Western blot showing the effect of FLAG-HAF overexpression on indicated NF-κB pathway components after treatment with indicated durations of 20 ng/mL TNF in HepG2 cells. (G) Effect of FLAG-HAF overexpression on NF-κB-RE Luc in the absence or presence of TNF. Data are representative of 3 independent experiments and are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, not significant. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAF, hypoxia-associated factor; LPC, liver parenchymal cell; siRNA, small interfering RNA.

Article Snippet: The NF-κB Luciferase Reporter HepG2 stable cell line was purchased from Signosis.

Techniques: In Vivo, In Vitro, Activity Assay, Phospho-proteomics, Western Blot, Isolation, Control, Ex Vivo, Quantitation Assay, Transfection, Luciferase, Over Expression, Small Interfering RNA

Journal: Hepatology (Baltimore, Md.)

Article Title: HAF prevents hepatocyte apoptosis and progression to MASH and HCC through transcriptional regulation of the NF-κB pathway

doi: 10.1097/HEP.0000000000001070

Figure Lengend Snippet: HAF regulates the transcription of TRADD and RIPK1 . (A) Western blot showing the effect of HAF siRNA on major components of the canonical NF-κB pathway in HepG2 cells. (B) Impact of 20 µM MG132 (6 h) on major NF-κB pathway components impacted by HAF siRNA. (C) Quantitation of relative levels of TRADD, RIPK1, TAK1, and NEMO protein normalized to GAPDH after treatment with 25 µg/mL of CHX added 24 hours after HAF siRNA transfection in HepG2 cells and lysed 30, 48, or 72 hours later, based on average values from western blots in Supplemental Figure S4B, http://links.lww.com/HEP/I648 . Data are the mean of 3 independent experiments ± SEM. (D, E) Quantitative qRT-PCR analysis of SART1 , RIPK1 , and TRADD mRNA levels after transfection with HAF siRNA (D), or stable HAF overexpression (E), in HepG2 cells. Data are representative of 3 independent experiments and shown as the mean ± SEM. (F) (i) Construction of luciferase reporters containing 3 tandem repeats of the HAF HBS and flanking regions (SART1 site) or those bearing only the flanking regions without the HBS (SART1 del). F(ii), (iii) Relative luciferase activity of TRADD site 2 (ii), and RIPK1 site (iii), in EV and FLAG-HAF overexpressing ACHN cells. Data are representative of 3 independent experiments (mean ± SEM). (G, H) Western blot showing the impact of TRADD and RIPK1 siRNA (72 h, G), or TRADD/RIPK1 overexpression in cells transfected with HAF siRNA (H), on components of the NF-κB pathway in HepG2 cells. Data are representative of 2 independent experiments. Abbreviations: CHX, cycloheximide; EV, empty vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAF, hypoxia-associated factor; HBS, HAF binding site; qRT-PCR, quantitative reverse transcription polymerase chain reaction; siRNA, small interfering RNA.

Article Snippet: The NF-κB Luciferase Reporter HepG2 stable cell line was purchased from Signosis.

Techniques: Western Blot, Quantitation Assay, Transfection, Quantitative RT-PCR, Over Expression, Luciferase, Activity Assay, Plasmid Preparation, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Small Interfering RNA

Journal: Hepatology (Baltimore, Md.)

Article Title: HAF prevents hepatocyte apoptosis and progression to MASH and HCC through transcriptional regulation of the NF-κB pathway

doi: 10.1097/HEP.0000000000001070

Figure Lengend Snippet: HAF depletion triggers spontaneous cell death through apoptosis. (A) Impact of HAF siRNA on cell viability (resazurin assay) in indicated cell lines. Data are representative of 3 independent experiments and are shown as mean ± SEM. (B) Western blot showing the impact of HAF siRNA transfection on cell death markers (B) and splicing of Bcl-X L and Bcl-X s (C) in indicated cell lines. Densitometric ratios of Bcl-X L and Bcl-X s are shown within blots. (D) Effect of HAF overexpression on Bcl-X L /Bcl-X s after treatment with TNF. (E) Effect of HAF siRNA ± the pan-caspase inhibitor ZVAD-FMK (20 µM), the necroptosis/RIPK1 inhibitor, necrostatin-1 (50 µM), or both combined, on annexin V staining in HepG2 cells. Inhibitors were added 48 hours after siRNA transfection. Data are representative of 3 independent experiments and are shown as mean ± SEM. (F, G) Western blots showing effects of HAF siRNA ± ZVAD-FMK (20 µM) for 72 hours (F), or overexpression of a HAF siRNA-resistant construct (FLAG-HAF-r) in the context of HAF siRNA in HEPG2 cells (G). (H) Impact of FLAG-HAF-(r) overexpression in the context of HAF siRNA on annexin V staining in HEPG2 cells. (I, J) Quantitation of immunohistochemical staining for cleaved caspase 3 in livers of 6-month-old male and female hep S −/ − (I) and LPC -S +/− mice (J) with representative images shown inset. Each data point represents an individual mouse. Data are the mean ± SEM with significance determined using Student t tests between control and experimental mice. Abbreviations: HAF, hypoxia-associated factor; LPC, liver parenchymal cell; siRNA, small interfering RNA.

Article Snippet: The NF-κB Luciferase Reporter HepG2 stable cell line was purchased from Signosis.

Techniques: Resazurin Assay, Western Blot, Transfection, Over Expression, Staining, Construct, Quantitation Assay, Immunohistochemical staining, Control, Small Interfering RNA