Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Cells were transiently transfected with a plasmid encoding the red fluorescence protein and LC3 fusion protein (RFP-LC3). After 48 h post-transfection, cells were counterstained with Hoechst 33342 (blue). RFP-LC3 (red) shows a homogeneous staining in the cytoplasm of MDA-MB-231 cells, indicating the absence or low level of autophagosomes. Both BT474 and AU565 cells show punctate or dotted staining patterns of RFP-LC3 which is a typical marker of autophagosome structures. Scale bar, 10 μm. (B) Numbers of RFP-LC3 dots (Puncta) in the transfected cells as in A. Shown are the average numbers of puncta per cell estimated by examining at least ten images per cell line (mean ± SD).

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Staining, Marker

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Electron microscopy (EM) images show the ultrastructural features of untreated cells or cells treated with 3-methyladenine (3-MA) at 10 mM for 24 h (Bar =1 μm). Arrows denote the autophagosome ultrastructures in cytoplasm. Lower panel shows the average number of autophagosome structures per view (371 μm 2 ) obtained by examining at least 50 images per testing sample. *p<0.0001. (B) EM images of parental cells and cells transfected with siRNA specific to the autophagy regulatory genes ATG7, Beclin 1, and LC3, respectively. Images are representatives of at least 50 captures. Lower panel shows the average number of autophagosome structures as determined in (B) for individual samples. *p<0.0001. (C) Nude mice were injected s.c. with BT474 or MDA-MB-231 cells per the protocol described in the Materials and Methods. When tumors reached 0.6 cm 3 in size, tumor tissues were harvested and analyzed by EM imaging. Bar =1 μm. Lower panel shows the quantification of autophagosome numbers in the respective tissues.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Electron Microscopy, Transfection, Injection, Imaging

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A & B) The indicated cell lines were transiently transfected with a scramble siRNA (siCtrl) as a negative control or siRNA against ATG7 for 72 h, followed by incubation with rhTRAIL (100 ng/ml) for an additional 24 h. The resultant cells were analyzed by flow cytometry for apoptosis (A) or immunoblotting for caspase cleavage (B). (C & D) BT474 cells were transiently transfected with a control siRNA (siCtrl) or siRNA specific to Beclin 1, LC3 or in combination. After 48 h post-transfection, cells were analyzed for apoptosis and caspase activation. *p<0.0001.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Transfection, Negative Control, Incubation, Flow Cytometry, Western Blot, Control, Activation Assay

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Flow cytometry analysis of death receptor expressions on cell surface. Cells were transiently transfected with a control siRNA (siCtrl) or siATG7. After 72 h post transfection, cells were incubated with PE-conjugated antibodies to DR4 (IgG1) or DR5 (IgG2b) or corresponding control IgG isotypes. Shown are representative histograms of siCtrl cells stained with control PE-IgG1 or PE-IgG2b (purple), PE-anti-DR4 or PE-anti-DR5 (green), and siATG7 transfected cells stained with PE-anti-DR4 or PE-anti-DR5 (yellow). The right-shift of a histogram peak indicates the increase in surface expression of the receptors. (B) Western blots of DR4 and DR5 total proteins. Knockdown of ATG7 is indicated by the decrease in ATG7 protein and the simultaneous loss of LC3-II. (C) Confocal images show the redistribution of DR4 from cytosol to plasma membrane upon inhibition of autophagy. Stable BT474/RFP-LC3 cells were left untreated or treated with 3-MA (10 mM) for 24 h. Alternatively, cells were transiently transfected with siCtrl or siATG7 for 48 h. Resultant cells were transfected with plasmids for GFP-DR4 or GFP-DR5 for 18 h. (D) Functional DISC formation assay. The indicated cells were transiently transfected with siATG7 for 72 h and incubated with (His) 6 -TRAIL (1 μg/ml) for 1 h. Affinity isolated DISC complexes were analyzed by western blotting. As a positive control, MDA-MB-231 cells (expressing both DR4 and DR5 on cell surface and are sensitive to TRAIL) recruited adaptor protein FADD and pro-caspase-8 into the DISC complexes upon TRAIL treatment. Only little DISC components were detected in parental BT474 and AU565 cells which are deficient in surface DR4/DR5 (Fig. ). Knockdown of ATG7 enhanced FADD and caspase 8, particularly the cleaved forms p43/41 and p21/18, in the DISC complexes, while had no effect on their total protein expressions.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Flow Cytometry, Transfection, Control, Incubation, Staining, Expressing, Western Blot, Knockdown, Clinical Proteomics, Membrane, Inhibition, Functional Assay, Tube Formation Assay, Isolation, Positive Control

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: (A) Cells were treated with bafilomycin A1 (BafA1) at 100 nM for 8 or 24 h, and the resultant whole cell extracts were analyzed by immunoblotting for LC3, p62, DR4 and DR5. Actin was used as a loading control. (B) Relative protein levels were estimated by densitometry analysis of the blots in A, and normalized to the corresponding actin intensity. The level of each protein in the untreated cells (time 0) was arbitrarily set as 1. Shown are representatives of two independent experiments. (C) MDA-MB-231/RFP-LC3 cells and BT474/RFP-LC3, both stably express RFP-LC3 protein, were treated with BafA1 (100nM) for the indicated times, countered stained with Hoechst 33342 (blue), and analyzed by confocal microscopy. Scale bar, 10 μm. MDA-MB-231 cells accumulated punctate structures at a much higher rate compared to BT474 cells.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Western Blot, Control, Stable Transfection, Staining, Confocal Microscopy

Journal: Oncotarget

Article Title: Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5

doi:

Figure Lengend Snippet: In MDA-MB-231 cells, TRAIL binds DR4 and/or DR5 expressed on cell surface, thereby recruiting adaptor protein Fas-associated death domain (FADD) and pro-caspase 8 into a death inducing signaling complex (DISC). Within the DISC, caspase 8 undergoes self-cleavage and activation which triggers the caspase cascade, cleavage of structural proteins, and eventually apoptosis. Both BT474 and AU565 cells are characterized by high basal level of autophagosomes that sequester DR4 and DR5, which may contribute to their deficiency on cell surface. Disruption of autophagosome structures (e.g. by 3-MA or siATG7) restores the surface expression of DR4 and DR5 which make the cells susceptible to TRAIL induced apoptosis.

Article Snippet: The human breast cancer cell lines AU565, BT474, HCC1428, MCF-7, MDA-MB-453, BT549, HCC38, HCC1954, MDA-MB-157, MDA-MB-231 and Hs578T were purchased from the American Type Culture Collection (ATCC), where the cell lines were tested and authenticated by growth rate, morphology, isoenzymology, short tandem repeat profiling, and Mycoplasma testing ( www.ATCC.org ).

Techniques: Activation Assay, Disruption, Expressing

Journal: bioRxiv

Article Title: Tumor cell-specific loss of GPX4 reprograms triacylglycerol metabolism to escape ferroptosis and impair antitumor immunity in NSCLC

doi: 10.1101/2025.11.12.687999

Figure Lengend Snippet: Inhibition of TAG synthesis re-sensitizes Gpx4 -deficient tumor cells to ferroptosis. (A) A workflow scheme of RNA-seq and CUT&Tag (H3K4me3 and H3K27ac) of KL and KLG4 m/m CD45 - CD31 - EpCAM + tumor cells. (B) The principal component analysis (PCA) plot showing the differentially expressed genes (DEGs) in RNA-Seq and the differential histone modifications in CUT&Tag assays of KL and KLG4 m/m CD45 - CD31 - EpCAM + tumor cells (left). Dotplot showing the upregulated DEGs and the histone modifications in CUT&Tag assays of KL and KLG4 m/m CD45 - CD31 - EpCAM + tumor cells (middle). Scheme of TAG and phospholipids synthesis pathway (right). GPD, glycerol-3-phosphate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase; AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase; PLPP, phospholipid phosphatase; DGAT, diacylglycerol acyltransferase; PLD, Phospholipase D; PLA2, Phospholipase A2; FA, fatty acid. (C) Heatmap showing the expression levels, H3K4me3 and H3K27ac modifications of the 10 TAG synthesis-related DEGs. (D) Representative Integrative Genomics Viewer (IGV) displaying CUT&Tag sequencing and RNA-sequencing signal profiles across Dgat2 , Gpd1l and Apoe loci in CD45 - CD31 - EpCAM + tumor cells derived from lung tumors of KL and KLG4 m/m mice. (E) Schematic illustration depicts tumor induction and iDGAT1/2 (composed of T863 and PF06424439) treatment in KL and KLG4 m/m mice that were intranasally injected with Ad-Cre (2×10 6 PFU per mouse) for 5 weeks followed by intraperitoneal injection of tamoxifen every other day for 2 weeks. One week after Tam treatment, the mice were injected with iDGAT1/2 (composed of T863 and PF06424439, 20 mg and 40 mg per kg body weight, respectively) every other day by gavage for 5 weeks. The mice were rested for one week followed by various analyses. (F) Representative images from transmission electron microscopy showing the morphology of lipid droplets (red arrowheads, in middle panel images) and mitochondria (bottom panel images, mitochondria were indicated by magenta arrowheads and ruptured mitochondria were highlighted with orange asterisks) along with statistics of the numbers and the sizes of lipid droplets (LDs) and the percentage of ruptured mitochondria in CD45 - CD31 - EpCAM + tumor cells from lung tumors of KL and KLG4 m/m mice treated as in (E). The orange and blue boxed areas are shown at higher magnifications in the middle (orange box) and the bottom (blue box), respectively. (G) The levels of C11-bodipy staining (upper panels) and SYTOX staining (lower panels) of CD45 - CD31 - EpCAM + tumor cells from lung tumors of KL and KLG4 m/m mice treated as in (E). N=6 for each group. (H, I) Representative images of HE staining (H, left), statistics of tumor burdens and individual tumor sizes (H, right) and representative images of micro-CT (I) of tumor-burdened lungs from the KL (n = 4 for control and n = 5 for iDGAT1/2, respectively) and KLG4 m/m (n = 6 for control and n = 6 for iDGAT1/2) mice treated as in (E). N=2 mice per group (B-D). Graphs show mean ± SEM (F-H). Statistical analyses were performed with two-way ANOVA (F-H). Scale bars represent 2 μm (white, F), 1 μm (magenta, F), 500 nm (blue, F), and 5 mm (H and I). Data are representative of three (F-I) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P <0.0001. ns, not significant.

Article Snippet: The iDGAT1/2, consisting of T863 (a DGAT1 inhibitor , , 20mg per kg of body weight, HY-32219, MedChemExpress) and PF-06424439 (a DGAT2 inhibitor , 40mg per kg of body weight, HY-108341, MedChemExpress), were solubilized in a solvent containing 5% DMSO, 30%PEG300, 5% Tween-80 and 60% H 2 O.

Techniques: Inhibition, RNA Sequencing, Expressing, Sequencing, Derivative Assay, Injection, Transmission Assay, Electron Microscopy, Staining, Micro-CT, Control

Journal: bioRxiv

Article Title: Tumor cell-specific loss of GPX4 reprograms triacylglycerol metabolism to escape ferroptosis and impair antitumor immunity in NSCLC

doi: 10.1101/2025.11.12.687999

Figure Lengend Snippet: Inhibition of DGAT1/2 restores CD8 + T cell function in the TME of KLG4 m/m tumors. (A) Schematic illustration depicts tumor induction and iDGAT1/2 (composed of T863 and PF06424439) treatment in KL and KLG4 m/m mice that were intranasally injected with Ad-Cre (2×10 6 PFU per mouse) for 5 weeks followed by intraperitoneal injection of tamoxifen every other day for 2 weeks. One week after Tam treatment, the mice were injected with iDGAT1/2 (composed of T863 and PF06424439, 20 mg and 40 mg per kg body weight, respectively) every other day by gavage for 5 weeks. The mice were rested for one week followed by various analyses. (B-C) Representative flow cytometry images (B) and quantification analysis (C) of tumor-infiltrated lymphocytes (TILs) from lung tumors of KL (n = 6 for iDGAT1/2 or control dissolvent) and KLG4 m/m (n = 6 for iDGAT1/2 or control dissolvent) mice treated as in (A) under indicated treatment. Graphs show mean ± SEM (C). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P <0.0001. ns: not significant (two-way ANOVA for C). Data are representative results of two independent experiments (B, C).

Article Snippet: The iDGAT1/2, consisting of T863 (a DGAT1 inhibitor , , 20mg per kg of body weight, HY-32219, MedChemExpress) and PF-06424439 (a DGAT2 inhibitor , 40mg per kg of body weight, HY-108341, MedChemExpress), were solubilized in a solvent containing 5% DMSO, 30%PEG300, 5% Tween-80 and 60% H 2 O.

Techniques: Inhibition, Cell Function Assay, Injection, Flow Cytometry, Control

Journal: ACS nano

Article Title: Fluorinated Gold Nanoparticles for Nanostructure Imaging Mass Spectrometry

doi: 10.1021/acsnano.8b02376

Figure Lengend Snippet: (A) Schematic of the perfluorinated monolayer on the surface of the f-AuNP. When the laser hits the nanoparticle, the energy is transferred to the surface causing the thermal release of the fluorinated chains; (B) phase separation between the f-AuNPs dissolved in perfluorohexane and the surface of the sample.

Article Snippet: 36 Briefly, 1 mL of 2% (v/v) octanethiol functionalized gold nanoparticles (2–4 nm diameter, 20 mg/mL in toluene, Sigma, Milwaukee, WI) were washed with 20 mL neat ethanol, followed by 20 mL acetone, sonicated for 1 minute and centrifuged for 20 minutes at 3000 rpm between each wash.

Techniques:

Journal: Frontiers in Immunology

Article Title: Optimized peptide nanofibrils as efficient transduction enhancers for in vitro and ex vivo gene transfer

doi: 10.3389/fimmu.2023.1270243

Figure Lengend Snippet: Biophysical and functional characterization of RM-8 PNFs. (A) TEM image of RM-8 PNFs. Scale bar indicates 500 nm. (B) RM-8 PNFs are chemically stable for 10 days, as analyzed by HPLC. (C) ATR-FTIR spectrum of RM-8, showing spectral maxima in the amide I region indicating β-sheet structure. (D) RM-8 PNFs efficiently enhance transduction rates of GFP-expressing GALV-RV in Jurkat cells. Two days after transduction GFP+ cells were determined by flow cytometry. Shown are average values ( ± SD) of three independent experiments. (E) RM-8 PNFs efficiently enhance transduction rates of GFP-expressing RD114/TR-RV in HEK293T cells. Three days after transduction GFP+ cells were determined by flow cytometry. Shown are average values ( ± SD) of three independent experiments. (F) RM-8 PNFs efficiently enhance transduction rates of Luciferase-expressing VSV-G-LV in HEK293T cells. Two days after transduction infection rates were determined by measuring Luciferase signal. Shown are average values of triplicates (± SEM) of three independent experiments. (G) RM-8 PNFs efficiently enhance transduction rates of Luciferase-expressing RD114/TR-LV in HEK293T cells. Three days after transduction infection rates were determined by measuring Luciferase signal. Shown are average values of triplicates (± SEM) of three independent experiments. In D-F the numbers above the bars show the n-fold enhancement of infection relative to the average of controls without peptide. (H) Transduction protocol for using RetroNectin and RM-8 PNFs as transduction enhancer in an ex vivo gene transfer. Created with BioRender.com. (I) Viability of T cells was determined using trypan blue staining after 7 days. Shown are average values (± SD) of two donors. (J) RM-8 PNFs enhance retroviral transduction of T cells similar to Vectofusin-1 and RetroNectin. GALV-RV was incubated with indicated transduction enhancers before transducing activated T cells according to protocol of (H) GFP+ cells were determined by flow cytometry after 7 days. Shown are values of two donors. (K) RM-8 forms aggregates 1 day after transduction. Scale bar indicates 1000 µm and for the inset 200 µm. ATR-FTIR, Attenuated total reflection Fourier transform infrared spectroscopy; A.u., arbitrary units; centrif., centrifugation step; GALV, glycoprotein of gibbon ape leukemia virus; HPLC, high-performance liquid chromatography; LV, lentiviral vector; RD114/TR, chimeric envelope glycoprotein derived from feline leukemia virus with the cytoplasmic tail derived from the murine leukemia virus glycoprotein; RV; γ-retroviral vector; SEM, standard error of mean; TEM, Transmission electron microscopy; VSV-G, glycoprotein of vesicular stomatitis virus.

Article Snippet: After one day 5×10 5 T cells were transduced with GALV-RV (1:4 dilution on cells) or VSV-G-LV (1:40 dilution on cells) in absence and presence of RM-8 (30 or 50 µg/ml), D4 (30 µg/ml), EF-C (30 or 50 µg/ml), 10 µg/ml Vectofusin-1 (Miltenyi Biotec, Cat: 130-111-163), or 20 µg/ml RetroNectin (Takara Bio, Cat: T100B).

Techniques: Functional Assay, Transduction, Expressing, Flow Cytometry, Luciferase, Infection, Ex Vivo, Staining, Retroviral, Incubation, Fourier Transform Infrared Spectroscopy, Spectroscopy, Centrifugation, Virus, High Performance Liquid Chromatography, Plasmid Preparation, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Cell Proliferation

Article Title: Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β‐cell function in NAFPD by inhibiting MST1 ‐Foxo3a signalling pathway

doi: 10.1111/cpr.13764

Figure Lengend Snippet: Terazosin selectively activated GPR119, leading to increased insulin secretion in MIN6 cells. (A) Flowchart illustrating the screening of drugs based on structural compatibility with GPR119 and their docking score ranking (Top‐60). (B) Structures of candidate drugs and the positive control MBX‐2982 were depicted. (C) Comparative analysis of CRE promoter transcriptional activity following 24 h treatment of MIN6 cells with candidate drugs ( n = 6), * p <0.05 versus control group. (D) Comparative assessment of cAMP levels in MIN6 cells after 24 h treatment with candidate drugs ( n = 6–8), * p <0.05 versus control group. (E), (F) PLIP analysis of terazosin binding to GPR119, with MBX‐2982 as a positive control. (G) Confocal imaging validated the nuclear exclusion of MDM2 in MIN6 cells induced by terazosin. Scale bar = 5 μm. (H) Terazosin decreased the nuclear localization of the MDM2 protein in MIN6 cells, using Lamin‐B1 and β ‐actin as biomarkers for nuclear and cytosolic fractions ( n = 5), * p < 0.05 versus control groups of nuclear and cytosolic fractions. (I), (J) Silencing of GPR119 suppressed the terazosin‐induced elevation of cAMP and ATP levels ( n = 5–6), * p <0.05 versus scramble groups of terazosin or MBX‐2982 induced. (K) Silencing of GPR119 impeded terazosin‐induced glucose‐stimulated insulin secretion, with GLP‐1 and MBX‐2982 serving as positive controls ( n = 4–6), * p <0.05 versus scramble groups of terazosin or MBX‐2982 induced. Statistical analysis was performed using Student's t ‐test, Dunnett's t ‐test, and Mann–Whitney U test.

Article Snippet: MIN6 cells were treated with terazosin (20 μmol/L, MCE, HY‐B0371A) and a set of screened drugs (ZINC‐1872 10 μmol/L, ZINC‐6192 5 μmol/L, ZINC‐3652 1 μmol/L, ZINC‐6601 10 μmol/L, ZINC‐4414 1 μmol/L, ZINC‐2427 20 μmol/L, ZINC‐6792 5 μmol/L) for 24 h individually.

Techniques: Positive Control, Activity Assay, Control, Binding Assay, Imaging, MANN-WHITNEY

Journal: Cell Proliferation

Article Title: Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β‐cell function in NAFPD by inhibiting MST1 ‐Foxo3a signalling pathway

doi: 10.1111/cpr.13764

Figure Lengend Snippet: Terazosin inhibited the interaction between MST1 and Foxo3a, leading to the nuclear exclusion of Foxo3a. (A) Terazosin demonstrated dose‐dependent effects on the protein levels of MST1‐Foxo3a and PDX1 in MIN6 cells after a 24‐h treatment with varying terazosin concentrations ( n = 4), * p <0.05 versus control group. (B), (C) CoIP experiments revealed the interaction between MST1 and Foxo3a in MIN6 cells, with terazosin diminishing this interaction. Quantitative data were displayed on the right ( n = 4). (D) Confocal imaging confirmed terazosin's role in promoting the nuclear exclusion of Foxo3a in MIN6 cells. Scale bar = 20 μm. (E) Terazosin decreased the nuclear distribution of Foxo3a protein in MIN6 cells, with Lamin‐B1 and β ‐actin serving as biomarkers for nuclear and cytosolic fractions ( n = 5), * p <0.05 versus control groups of nuclear and cytosolic fractions. Statistical analysis was performed using Dunnett's t ‐test.

Article Snippet: MIN6 cells were treated with terazosin (20 μmol/L, MCE, HY‐B0371A) and a set of screened drugs (ZINC‐1872 10 μmol/L, ZINC‐6192 5 μmol/L, ZINC‐3652 1 μmol/L, ZINC‐6601 10 μmol/L, ZINC‐4414 1 μmol/L, ZINC‐2427 20 μmol/L, ZINC‐6792 5 μmol/L) for 24 h individually.

Techniques: Control, Imaging

Journal: Cell Proliferation

Article Title: Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β‐cell function in NAFPD by inhibiting MST1 ‐Foxo3a signalling pathway

doi: 10.1111/cpr.13764

Figure Lengend Snippet: Terazosin inhibited the MST1‐Foxo3a pathway, improving β‐cell function. (A), (B) Terazosin suppressed the upregulation of MST1‐Foxo3a and mitophagy induced by MST1 overexpression in MIN6 cells, accompanied by a reduction in protein levels associated with β‐cell function. Representative gel images were presented in (A) and quantitative data in (B) ( n = 5), * p <0.05 versus Ad‐GFP groups of various proteins. (C) Real‐time PCR validation of the regulation of gene expression related to MST1‐Foxo3a, mitophagy, and β‐cell function in MIN6 cells overexpressing MST1 ( n = 5–8), * p <0.05 versus Ad‐GFP groups of various genes. (D) Terazosin prevented the decrease in transcriptional activity of the PDX1 gene promoter induced by MST1 overexpression ( n = 8), * p <0.05 versus Ad‐GFP group. (E) Terazosin counteracted the reduction in intracellular ATP levels induced by MST1 overexpression ( n = 8), * p <0.05 versus Ad‐GFP groups of intracellular or extracellular fractions. (F) Terazosin alleviated the reduction in glucose‐stimulated insulin secretion caused by MST1 overexpression (n = 5). (G), (H) Terazosin suppressed the upregulation of Foxo3a and autophagy proteins induced by Foxo3a overexpression in MIN6 cells, concomitant with a reduction in protein levels associated with β‐cell function. Representative gel images were presented in (G) and quantitative data in (H) ( n = 5), * p <0.05 versus Ad‐GFP groups of various proteins. (I) Real‐time PCR validation of the regulation of gene expression related to Foxo3a, mitophagy, and β‐cell function in MIN6 cells overexpressing Foxo3a ( n = 5–9), * p <0.05 versus Ad‐GFP groups of various genes. (J), (K) Silencing of Foxo3a inhibited the upregulation of Foxo3a and mitophagy induced by MST1 overexpression in MIN6 cells, accompanied by a decrease in protein levels associated with β‐cell function. Representative gel images were presented in (J) and quantitative data in (K) ( n = 5), * p <0.05 versus Ad‐GFP + Scramble groups of various proteins. (L), (M) Silencing of Foxo3a suppressed the upregulation of P27, IL‐1β, and Caspase‐1 protein levels induced by MST1 overexpression in MIN6 cells. Representative gel images were presented in (L), and quantitative data in (M) ( n = 5), * p <0.05 versus Ad‐GFP + Scramble groups of various proteins. TZ, terazosin. Statistical analysis was performed using Student's t ‐test, Dunnett's t ‐test, and Mann–Whitney U test.

Article Snippet: MIN6 cells were treated with terazosin (20 μmol/L, MCE, HY‐B0371A) and a set of screened drugs (ZINC‐1872 10 μmol/L, ZINC‐6192 5 μmol/L, ZINC‐3652 1 μmol/L, ZINC‐6601 10 μmol/L, ZINC‐4414 1 μmol/L, ZINC‐2427 20 μmol/L, ZINC‐6792 5 μmol/L) for 24 h individually.

Techniques: Cell Function Assay, Over Expression, Real-time Polymerase Chain Reaction, Gene Expression, Activity Assay, MANN-WHITNEY

Journal: Cell Proliferation

Article Title: Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β‐cell function in NAFPD by inhibiting MST1 ‐Foxo3a signalling pathway

doi: 10.1111/cpr.13764

Figure Lengend Snippet: Terazosin inhibited the MST1‐Foxo3a pathway, alleviating lipid deposition and improving β‐cell dysfunction. (A)–(C) Terazosin prevented the decrease in MIN6 cell viability induced by PA. Ki67 protein confocal imaging was shown in (A), Quantitative fluorescence data were presented in (B) ( n = 5). CCK‐8 data were presented in (C) ( n = 6), * p <0.05 versus PA group. (D), (E) Terazosin reduced PA‐induced triglyceride (TG) deposition. Quantitative TG data were presented in (D), and Oil Red O staining images were shown in (E). Scale bars: 50 and 200 μm ( n = 6), * p <0.05 versus PA group. (F), (G) Terazosin inhibited the upregulation of MST1‐Foxo3a and PA‐induced mitophagy in MIN6 cells, and it prevented the downregulation of protein levels associated with β‐cell function. Representative gel images were displayed in (F), and quantitative data in (G) ( n = 5), * p <0.05 versus PA groups of various proteins. (H) Real‐time PCR confirmed that terazosin inhibited the upregulation of MST1‐Foxo3a and prevented the downregulation of mitophagy and β‐cell function gene expression induced by PA in MIN6 cells ( n = 6–9), * p <0.05 versus PA groups of various genes. (I) Terazosin prevented the downregulation of PDX1 gene promoter transcriptional activity induced by PA ( n = 6), * p <0.05 versus PA group. (J), (M) Confocal imaging confirmed that terazosin prevented the downregulation of PDX1 and Insulin protein levels induced by PA in MIN6 cells. Scale bar = 20 μm. Quantitative fluorescence data were presented in (K) and (M) ( n = 5), * p <0.05 versus PA groups. TZ, terazosin. MBX, MBX‐2982. PA, palmitic acid. Statistical analysis was performed using Dunnett's t ‐test and Mann–Whitney U test.

Article Snippet: MIN6 cells were treated with terazosin (20 μmol/L, MCE, HY‐B0371A) and a set of screened drugs (ZINC‐1872 10 μmol/L, ZINC‐6192 5 μmol/L, ZINC‐3652 1 μmol/L, ZINC‐6601 10 μmol/L, ZINC‐4414 1 μmol/L, ZINC‐2427 20 μmol/L, ZINC‐6792 5 μmol/L) for 24 h individually.

Techniques: Imaging, Fluorescence, CCK-8 Assay, Staining, Cell Function Assay, Real-time Polymerase Chain Reaction, Gene Expression, Activity Assay, MANN-WHITNEY

Journal: Cell Proliferation

Article Title: Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β‐cell function in NAFPD by inhibiting MST1 ‐Foxo3a signalling pathway

doi: 10.1111/cpr.13764

Figure Lengend Snippet: Terazosin prevented mitochondrial damage and abnormalities in autophagic flux induced by PA. (A), (B) Terazosin reversed the decline in mitochondrial membrane potential caused by PA. (A) Shows immunofluorescence staining images: Red fluorescence indicated JC‐1 aggregate under normal membrane potential, and green indicated JC‐1 monomer under membrane potential loss, with CCCP serving as a positive control for membrane potential loss. Scale bar = 100 μm. Quantitative fluorescence data were presented in (B) ( n = 6), * p <0.05 versus PA group. (C) Terazosin prevented the reduction in ATP content caused by PA in MIN6 cells ( n = 6), * p <0.05 versus PA groups of intracellular or extracellular fractions. (D), (E) Terazosin decreased ROS production induced by PA. (D) Displays confocal imaging with green fluorescence indicating ROS generation. Scale bar = 5 μm. Quantitative fluorescence data were presented in (E) ( n = 6), * p <0.05 versus PA group. (F) Transmission electron microscopy images confirmed that terazosin restored the number and structure of mitochondria, leading to an increase in autophagosomes. White arrows indicated normal mitochondria, black arrows indicated swollen mitochondria and red arrows indicated autophagosomes. Scale bar = 1 μm. (G), (H) Confocal imaging confirmed that terazosin enhanced the recruitment of Parkin to mitochondria inhibited by PA. Green fluorescence indicated Parkin protein, red indicated mitochondria, and blue indicated the cell nucleus. Scale bar = 20 μm. Quantitative fluorescence data were presented in (H) ( n = 5), * p <0.05 versus PA groups. (I), (J) Confocal imaging confirmed that terazosin enhanced the fluorescence of autophagosomes and mitochondria inhibited by PA. Green fluorescence indicated autophagosomes and red indicated mitochondria. Scale bar = 20 μm. Quantitative fluorescence data were presented in (J) ( n = 5), * p <0.05 versus PA groups. (K) Terazosin enhanced the inhibited autophagic flux caused by PA. Representative gel images were displayed on the left, and quantitative data on the right ( n = 6), * p <0.05 versus PA group. (L), (M) The tandem fluorescence‐labelled LC3 system confirmed that terazosin effectively restored the inhibited autophagic flux caused by PA. (L) displayed immunofluorescence staining images: Red fluorescence indicated lysosomes, and yellow indicated autophagosomes. Scale bar = 50 μm. Quantitative fluorescence data were presented in (M) ( n = 6), * p <0.05 versus PA groups. Baf, bafilomycin A1; MTR, mitochondria tracker red; MBX, MBX‐2982; PA, palmitic acid; TZ, terazosin. Statistical analysis was performed using Dunnett's t ‐test.

Article Snippet: MIN6 cells were treated with terazosin (20 μmol/L, MCE, HY‐B0371A) and a set of screened drugs (ZINC‐1872 10 μmol/L, ZINC‐6192 5 μmol/L, ZINC‐3652 1 μmol/L, ZINC‐6601 10 μmol/L, ZINC‐4414 1 μmol/L, ZINC‐2427 20 μmol/L, ZINC‐6792 5 μmol/L) for 24 h individually.

Techniques: Membrane, Immunofluorescence, Staining, Fluorescence, Positive Control, Imaging, Transmission Assay, Electron Microscopy

Journal: Cell Proliferation

Article Title: Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β‐cell function in NAFPD by inhibiting MST1 ‐Foxo3a signalling pathway

doi: 10.1111/cpr.13764

Figure Lengend Snippet: Terazosin treatment ameliorated hyperglycemia, obesity, and pancreatic β‐cell dysfunction in NAFPD mice. (A) Glucose tolerance test (GTT) conducted after 6 weeks of terazosin treatment in NAFPD mice. The upper panel displayed GTT data, while the lower panel showed the corresponding area under the curve (AUC) data ( n = 8). (B) Insulin tolerance test (ITT) performed after 6 weeks of terazosin treatment in NAFPD mice. The upper panel displayed ITT data, and the lower panel showed AUC data ( n = 8). (C) A reduction in fasting blood glucose was observed in NAFPD mice following 6 weeks of terazosin treatment ( n = 6). (D) Terazosin treatment lowered serum insulin levels in NAFPD mice ( n = 8). (E) Terazosin treatment alleviated obesity in NAFPD mice ( n = 8). (F) Terazosin treatment reduced serum TG and CHOL levels in NAFPD mice ( n = 8). (G) Terazosin treatment decreased TG content in the pancreas of NAFPD mice ( n = 7). (H), (I) Terazosin treatment improved lipid deposition in hepatocytes and mesenteric adipose tissue. Oil Red O staining of hepatocytes was presented in (H), while HE staining of adipose tissue in (I). Scale bar = 50 μm. (J), (K) Terazosin inhibited the upregulation of MST1‐Foxo3a, mitigated mitophagy downregulation, and modulated β‐cell functional protein levels in the pancreas of NAFPD mice. Representative gel images were displayed in (J), while quantitative data in (K) ( n = 6). (L)–(M) Real‐time PCR verified that terazosin inhibited the upregulation of MST1‐Foxo3a, alleviated mitophagy downregulation, and modulated β‐cell functional gene expression in the pancreas of NAFPD mice ( n = 7–10). (N)–(Q) Confocal imaging verified that terazosin decreased MST1 protein levels in pancreatic β‐cells of NAFPD mice and elevated levels of PDX1 and Insulin proteins. Scale bar = 20 μm. Quantitative fluorescence data were presented in (O) and (Q) ( n = 5). (R) Terazosin treatment elevated ATP levels in the pancreas of NAFPD mice ( n = 8). (S) Transmission electron microscopy images validated that terazosin reinstated mitochondrial number and structure in pancreatic β‐cells of NAFPD mice. White arrows indicated normal mitochondria, black arrows indicated swollen mitochondria and red arrows indicated autophagosomes. Scale bar = 1 μm. ND, normal diet; NC, negative control (mice treated with saline); TZ, terazosin. Statistical analysis was performed using Dunnett's t ‐test and Mann–Whitney U test, * p <0.05 versus HFD mice treated with saline.

Article Snippet: MIN6 cells were treated with terazosin (20 μmol/L, MCE, HY‐B0371A) and a set of screened drugs (ZINC‐1872 10 μmol/L, ZINC‐6192 5 μmol/L, ZINC‐3652 1 μmol/L, ZINC‐6601 10 μmol/L, ZINC‐4414 1 μmol/L, ZINC‐2427 20 μmol/L, ZINC‐6792 5 μmol/L) for 24 h individually.

Techniques: Staining, Functional Assay, Real-time Polymerase Chain Reaction, Gene Expression, Imaging, Fluorescence, Transmission Assay, Electron Microscopy, Negative Control, Saline, MANN-WHITNEY

Journal: Cell Proliferation

Article Title: Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β‐cell function in NAFPD by inhibiting MST1 ‐Foxo3a signalling pathway

doi: 10.1111/cpr.13764

Figure Lengend Snippet: Terazosin treatment failed to ameliorate hyperglycemia, obesity, and pancreatic β‐cell dysfunction in NAFPD mice with GPR119 deficiency. (A), (B) Confocal imaging and immunoblotting validated GPR119 deficiency in mouse islets. (A) displayed confocal images ( n = 5), Scale bar = 20 μm. Representative gel images were presented in (B). (C) Terazosin treatment failed to ameliorate glucose intolerance in GPR119 −/− mice. GTT data were displayed in the left, and AUC data in the right ( n = 5–8). (D) Terazosin treatment failed to alleviate insulin resistance in GPR119 −/− mice. ITT data were displayed in the left, and AUC data in the right ( n = 5–8). (E) Terazosin treatment failed to reduce fasting blood glucose in GPR119 −/− mice ( n = 5–8). (F) Terazosin treatment failed to decrease serum insulin levels in GPR119 −/− mice ( n = 5–8). (G) Terazosin treatment failed to reduce body weight in GPR119 −/− mice ( n = 5–8). (H) Terazosin treatment failed to decrease serum TG and CHOL levels in GPR119 −/− mice ( n = 5–8). (I) Terazosin failed to reduce TG content in the pancreas of GPR119 −/− mice ( n = 5–8). (J), (K) Terazosin failed to inhibit the upregulation of MST1‐Foxo3a, as well as the downregulation of mitophagy and β‐cell functional protein levels in the pancreas of GPR119 −/− mice. Representative gel images were displayed in (J) and quantitative data in (K) ( n = 6). (L), (M) Real‐time PCR confirmed that terazosin failed to inhibit the upregulation of MST1‐Foxo3a, as well as the downregulation of mitophagy and β‐cell functional gene expression in the pancreas of GPR119 −/− mice ( n = 6). (N), (O) Confocal imaging confirmed that terazosin failed to decrease MST1 protein levels and increase Insulin protein levels in the pancreatic β‐cells of GPR119 −/− mice. Scale bar = 20 μm. Quantitative fluorescence data were presented in (O) ( n = 5). (P) Terazosin treatment failed to increase ATP levels in the pancreas of GPR119 −/− mice ( n = 6). (Q) Transmission electron microscopy images confirmed that terazosin failed to restore the number and structure of mitochondria in the pancreatic β‐cells of GPR119 −/− mice. White arrows indicated normal mitochondria, black arrows indicated swollen mitochondria and red arrows indicated autophagosomes. Scale bar = 1 μm. NC, negative control (mice treated with saline); TZ, terazosin. Statistical analysis was performed using Dunnett's t ‐test and Mann–Whitney U test, * p <0.05 versus GPR119 −/− HFD mice treated with saline.

Article Snippet: MIN6 cells were treated with terazosin (20 μmol/L, MCE, HY‐B0371A) and a set of screened drugs (ZINC‐1872 10 μmol/L, ZINC‐6192 5 μmol/L, ZINC‐3652 1 μmol/L, ZINC‐6601 10 μmol/L, ZINC‐4414 1 μmol/L, ZINC‐2427 20 μmol/L, ZINC‐6792 5 μmol/L) for 24 h individually.

Techniques: Imaging, Western Blot, Functional Assay, Real-time Polymerase Chain Reaction, Gene Expression, Fluorescence, Transmission Assay, Electron Microscopy, Negative Control, Saline, MANN-WHITNEY