Journal: Nutrients

Article Title: Phosphate Metabolic Inhibition Contributes to Irradiation-Induced Myelosuppression through Dampening Hematopoietic Stem Cell Survival

doi: 10.3390/nu14163395

Figure Lengend Snippet: Akt counteracts Pi metabolic inhibition and apoptosis of HSCs post IR. ( A ) Representative flow cytometric analysis and fold change of p-Akt S473 MFI in BM HSCs of mice at 3 dpi ( n = 5). ( B ) Representative flow cytometric analysis and fold change of p-Akt S473 MFI in BM HSCs of WT mice and Slc20a1 +/− mice at 3 dpi ( n = 5). ( C ) Representative flow cytometric analysis and fold change of p-Akt S473 MFI in BM HSCs of mice with or without Klotho treatment at 3 dpi ( n = 5). ( D ) Representative flow cytometric analysis and fold change of p-Akt S473 MFI in BM HSCs of WT and Kl +/− mice at 3 dpi ( n = 5). ( E ) Scheme for MK-2206 treatment and HSC analysis. ( F ) Representative flow cytometric analysis and fold change of SLC20A1 MFI in BM HSCs of mice with or without soluble MK-2206 treatment at 3 dpi ( n = 5). ( G ) Pi content in BM LSK cells of mice with or without MK-2206 treatment post IR ( n = 6). ( H ) Relative HSC numbers in the BM of mice with or without MK-2206 treatment post IR compared to those at 0 dpi. ( n = 6). ( I ) Representative flow cytometric analysis and quantification of apoptotic HSCs in the BM of mice with or without MK-2206 treatment at 3 dpi ( n = 6). ( J – L ) WBC, RBC, and platelet counts in PB of mice with or without MK-2206 treatment post IR ( n = 6). ( M ) Survival rates of mice with or without MK-2206 treatment post IR ( n = 10). Data are mean ± SD. * p < 0.05, ** p < 0.01. Two-tailed unpaired Student’s t -test. ( M ) Log-rank test.

Article Snippet: For the MK-2206 (MedChem Express, Monmouth Junction, NJ, USA) treatment, mice were administrated with a dose of 4 mg/kg intraperitoneally every other day, immediately post IR.

Techniques: Inhibition, Two Tailed Test

Journal: Nutrients

Article Title: Phosphate Metabolic Inhibition Contributes to Irradiation-Induced Myelosuppression through Dampening Hematopoietic Stem Cell Survival

doi: 10.3390/nu14163395

Figure Lengend Snippet: p53 promotes Pi metabolic inhibition and apoptosis of HSCs induced by IR. ( A ) Representative flow cytometric analysis and fold change of p53 MFI in BM HSCs of mice at 3 dpi ( n = 5). ( B ) Representative flow cytometric analysis and fold change of PUMA MFI in BM HSCs of mice at 3 dpi ( n = 5). ( C ) Representative flow cytometric analysis of MMP by TMRE staining and fold change of TMRE MFI in BM HSCs of mice at 3 dpi ( n = 5). ( D ) Fold change of p53 MFI in BM HSCs of WT mice and Slc20a1 +/− mice at 3 dpi ( n = 5). ( E ) Fold change of p53 MFI in BM HSCs of mice with or without Klotho treatment at 3 dpi ( n = 5). ( F ) Fold change of p53 MFI in BM HSCs of mice with or without MK-2206 treatment at 3 dpi ( n = 5). ( G ) Fold change of p53 MFI in BM HSCs of WT and Kl +/− mice at 3 dpi ( n = 5). ( H ) GSEA of p53 pathway gene set in HSCs with enhanced Pi metabolism (PRJNA695319). ( I ) Scheme for the creation of mouse model of irradiation-induced myelosuppression using p53 −/− mice and HSC analysis. ( J ) Relative HSC numbers in the BM of WT and p53 −/− mice post IR compared to those at 0 dpi ( n = 6). ( K ) Representative flow cytometric analysis and quantification of apoptotic HSCs in the BM of WT and p53 −/− mice at 3 dpi ( n = 6). ( L – N ) WBC, RBC, and platelet counts in PB of WT and p53 −/− mice post IR ( n = 6). ( O ) Relative HSC numbers in the BM of p53 −/− mice with or without Klotho or MK-2206 treatment post IR compared to those at 0 dpi ( n = 5). ( P ) Frequency of apoptotic HSCs in the BM of p53 −/− mice with or without Klotho or MK-2206 treatment at 3 dpi ( n = 5). Data are mean ± SD. ns = not significant. * p < 0.05, ** p < 0.01. Two-tailed unpaired Student’s t -test. ( O , P ) One-way ANOVA.

Article Snippet: For the MK-2206 (MedChem Express, Monmouth Junction, NJ, USA) treatment, mice were administrated with a dose of 4 mg/kg intraperitoneally every other day, immediately post IR.

Techniques: Inhibition, Staining, Irradiation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Berberine Protects against NEFA-Induced Impairment of Mitochondrial Respiratory Chain Function and Insulin Signaling in Bovine Hepatocytes

doi: 10.3390/ijms19061691

Figure Lengend Snippet: Activation of PGC-1α increased the beneficial effects of BBR on mitochondrial respiratory chain function and insulin signaling damage induced by NEFA. Hepatocytes were assigned to 3 groups as follows: A 1.2 mmol/L NEFA group, a 1.2 mmol/L NEFA + 20 μmol/L BBR treatment group, and a 1.2 mmol/L NEFA + 10 μmol/L ZLN005 treatment group. ( A , B ) Western blot analysis and quantification of key molecules of the insulin signaling pathway, PGC-1α, and five representative subunits of OXPHOS complexes, and β-Actin served as an internal control; ( C ) TG content in bovine hepatocytes. Quantified data are mean ± SD; ## p < 0.01 versus NEFA group.

Article Snippet: The hepatocytes were treated with NEFA for 9 h, ZLN005 (MedChem Express, NJ, USA) for 24 h, and different levels of berberine (Sigma-Aldrich, St. Louis, MO, USA) for 24h.

Techniques: Activation Assay, Western Blot, Control

Journal: BMC Biology

Article Title: FGF-independent MEK1/2 signalling in the developing foetal testis is essential for male germline differentiation in mice

doi: 10.1186/s12915-023-01777-x

Figure Lengend Snippet: Summary of treatments and doses used in gonad cultures

Article Snippet: Additional inhibitors for FGFR (AZD4547; SelleckChem, S2801 and SU5402; MedChem Express, HY-10407), p38 (Ralimetinib dimesylate; MedChem Express, HY-13241) and PI3K (PF-04691502; MedChem Express, HY-15177) signalling were also used (Table ) to ensure consistency of outcomes with each of the primary inhibitors used.

Techniques: Control, Recombinant

Journal: Journal of Neuroinflammation

Article Title: Transforming growth factor-β1 protects against LPC-induced cognitive deficit by attenuating pyroptosis of microglia via NF-κB/ERK1/2 pathways

doi: 10.1186/s12974-022-02557-0

Figure Lengend Snippet: Activation of ERK1/2 and NF-κB pathways by PMA partially reversed the anti-pyroptotic effect of TGF-β1 in vitro. A The protein expression of Cleaved-GSDMD, NLRP3, and IL-1β in cultured microglia under different treatments was evaluated by Western blot. B Quantitative analysis of Western blot was performed using One-way ANOVA followed by Tukey’s multiple comparisons test. ** P < 0.01, **** P < 0.0001 versus CT group; # P < 0.05, ## P < 0.01 versus LPS group; & P < 0.05, && P < 0.01 versus LPS + TGF-β1 group. N = 6 per group. C Representative confocal images (scale bar = 30 μm) and 3D reconstructed images in high power field (scale bar = 10 μm) of Iba1 and NLRP3 in cultured microglia under different treatments. D Quantitative analysis of the volume of NLRP3 + punctures per cell in immunofluorescent staining. One-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis. **** P < 0.0001 versus CT group; # P < 0.05 versus LPS group, & P < 0.05 versus LPS + TGF-β1 group. N = 9 per group. E The level of IL-1β in microglial CM under different treatments was analyzed by ELISA. Quantitative analysis was performed using One-way ANOVA followed by Tukey’s multiple comparisons test. **** P < 0.0001 versus CT group; ### P < 0.001 versus LPS group; & P < 0.05 versus LPS + TGF-β1 group. N = 6 per group. F The level of TNF-α and IL-6 in microglia-CM exposed to varied stimulations was measured by CBA. Quantitative analysis was performed using One-way ANOVA followed by Tukey’s multiple comparisons test. **** P < 0.0001 versus CT group; ### P < 0.001, #### P < 0.0001 versus LPS group; & P < 0.05, n.s. no significance versus LPS + TGF-β1 group. N = 6 per group. G Representative images of bright field (scale bar = 40 μm) and immunostaining with Iba1 (scale bar = 30 μm) in cultured microglia exposed to varied treatments. H Quantitative analysis of the percent of amoeboid-like microglia in bright-field images. **** P < 0.0001 versus CT group; ## P < 0.01 versus LPS group; & P < 0.05 versus LPS + TGF-β1 group. N = 6 per group

Article Snippet: The applied dose was 1 μg/mL for LPS (sigma, USA), 10 ng/mL for TGF-β1 (Peprotech, USA), 1 μM for PMA (MedChem Express, China).

Techniques: Activation Assay, In Vitro, Expressing, Cell Culture, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways

doi: 10.1590/1414-431X2024e13474

Figure Lengend Snippet: CoQ10 inactivates the PI3K/AKT/mTOR and MAPK signaling pathways in RANKL-treated RAW264.7 cells. After RAW264.7 cells were treated with 10 -3 M CoQ10 with or without 10 -4 M H 2 O 2 in the presence of 50 ng/mL RANKL, PI3K/AKT/mTOR, and MAPK signaling pathway-related proteins were tested. A , The levels of p-PI3K/t-PI3K; B , p-AKT/t-AKT; C , p-mTOR/t-mTOR; D , p-ERK/t-ERK; and E , p-p38/t-p38. CoQ10: Coenzyme Q10; PI3K: phosphatidylinositol 3 kinase; MAPK: mitogen-activated protein kinase; RANKL: receptor activator of NF‐κB ligand. The data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001 compared to the control (RANKL), unless otherwise indicated; ANOVA; ns: not significant.

Article Snippet: These cells were preconditioned with the inhibitors PI3K (LY294002, 30 μM; #L832989; Macklin, China) for 1 h , ERK (PD98059, 10 μM; MedChem Express, USA) for 1 h , p38MAPK (SB203580, 10 μM; Yuanye Bio-Technology Co., Ltd., China) for 1 h , or mitophagy agonist Torin1 (4 nM; #T861013; Macklin) for half an hour.

Techniques: Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways

doi: 10.1590/1414-431X2024e13474

Figure Lengend Snippet: CoQ10 promotes autophagy in RANKL-treated RAW264.7 cells by inactivating the PI3K/AKT/mTOR and MAPK pathways. RAW264.7 cells were pretreated with the PI3K inhibitor LY294002, the ERK inhibitor PD98059, and the p38MAPK inhibitor SB203580, and then, the cells were treated with or without 10 -3 M CoQ10 in the presence of 50 ng/mL RANKL. The levels of autophagy-related proteins were measured. A , Images of autophagy-related proteins determined by western blot; B , Quantitative analysis of FOXO3; C , Quantitative analysis of Beclin1; D , Quantitative analysis of LC3II/LC3I. CoQ10: Coenzyme Q10; RANKL: receptor activator of NF‐κB ligand; FOXO3: forkhead box protein O3. The data are reported as means±SD. ***P<0.001 compared to the corresponding groups; ANOVA.

Article Snippet: These cells were preconditioned with the inhibitors PI3K (LY294002, 30 μM; #L832989; Macklin, China) for 1 h , ERK (PD98059, 10 μM; MedChem Express, USA) for 1 h , p38MAPK (SB203580, 10 μM; Yuanye Bio-Technology Co., Ltd., China) for 1 h , or mitophagy agonist Torin1 (4 nM; #T861013; Macklin) for half an hour.

Techniques: Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways

doi: 10.1590/1414-431X2024e13474

Figure Lengend Snippet: CoQ10 inhibits RANKL-induced osteoclastogenesis by inactivating the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells. RAW264.7 cells were pretreated with the PI3K inhibitor LY294002, the ERK inhibitor PD98059, and the p38MAPK inhibitor SB203580, and then the cells were treated with or without 10 -3 M CoQ10 in the presence of 50 ng/mL RANKL. The protein levels of osteoclast markers were measured. A , Images of osteoclast markers determined by western blot; B , Quantitative analysis of TRAP; C , Quantitative analysis of NFATc1; D , Quantitative analysis of OSCAR. CoQ10: Coenzyme Q10; RANKL: receptor activator of NF‐κB ligand; TRAP: tartrate-resistant acid phosphatase; NFATc1: nuclear factor of activated T cells; OSCAR: osteoclast-associated immunoglobulin-like receptor. The data are reported as means±SD. *P<0.05, ***P<0.001 compared to the corresponding groups; ANOVA.

Article Snippet: These cells were preconditioned with the inhibitors PI3K (LY294002, 30 μM; #L832989; Macklin, China) for 1 h , ERK (PD98059, 10 μM; MedChem Express, USA) for 1 h , p38MAPK (SB203580, 10 μM; Yuanye Bio-Technology Co., Ltd., China) for 1 h , or mitophagy agonist Torin1 (4 nM; #T861013; Macklin) for half an hour.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage.

doi: 10.3390/ijms241310972

Figure Lengend Snippet: Figure 4. Effect of parbendazole on apoptosis, proliferation, microgliosis, and oligodendroglial cell fate of NG2+ cells within the border zone of post-photothrombotic adult coronal organotypic slice cul- tures. (A) Laser scanning micrograph (LSM) image of a photothrombotically lesioned NG2-CreERT2 td-Tomato mice brain after 4 days of cultivation. Immunostaining of cell nuclei using DAPI. The composition of 4 images (1 image measures 2195 × 1949 pixel or 482 × 428 µm) indicates the ROIs. Dashed lines indicate the border zone. Arrows indicate double-positive cells. (F) Quantification of NG2+ cells double positive cells for TUNEL indicating the apoptosis rate after 4 d and 8 d in culture. (G) Percentage of NG2+ and Ki-67+ double positive cells for DMSO control- (B) and parbendazole- (D) treated slice culture after 4 d. (H) Identification of astroglial lineage cells at the border zone of the photothrombotic lesion upon control (C) or parbendazole treatment (E) after 4 d. (I) Identification of Iba1 positive cells at the border zone upon control (J) or parbendazole (K) treatment after 4 d. Double-positive cells for NG2 td-Tomato and GFAP were counted and set in relation to the total amount of NG2+ cells. Dashed lines indicate the border between photothrombotically lesioned tissue and the border zone. Scale bars: (A) 10× magnification, (B,D) 50 µm, 20 µm, (C,E) 100 µm, and 20 µm, (J,K) 100 µm, 20 µm. Data are shown as mean values (±SD) and are derived from (F) n = 3, (G) n = 3, (H) n = 4, and (I) n = 3 experiments. Statistical significance was calculated using the Kruskal–Wallis test with Dunn’s post-test (F), and Mann–Whitney U test (G,H). Data were considered statistically significant (95% confidence interval) at ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: To determine the physiological reaction from OPCs exposed to the substances, OPCs were treated in a dose-dependent manner using concentrations of 10 mM, 20 mM, 40 mM, 50 mM, and 100 mM for sodium azide (NaN3; Sigma-Aldrich, St. Louis, MO, USA Cat# S2002-100G); 4 mM, 8 mM, 16 mM, 20 mM, and 50 mM for 2-Deoxy-D-Glucose (2-DG; Sigma-Aldrich, St. Louis, MO, USA Cat# D8375-5G); and 0.02 μM for parbendazole (MedChem Express, Monmouth Junction, NJ, USA, CAS 14255879).

Techniques: Immunostaining, TUNEL Assay, Control, Derivative Assay, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage.

doi: 10.3390/ijms241310972

Figure Lengend Snippet: Figure 5. Promoting OPC differentiation within the border zone of post-photothrombotic adult coro- nal cultures following stimulation with parbendazole. (A′–G′′) Representative images of oligoden- droglial differentiation-associated protein markers on slices treated with parbendazole (A′′–D′′,G′′) or DMSO, as control (A′–D′,G′). The impact on oligodendroglial cell differentiation and maturation was assessed by the number/mm2 of NG2+ cells and the relative percentage of double-positive cells (NG2+/PDGFR-α+ cells; (A–A′′)), (NG2+/Sox10+ cells; (B–B′′)), (NG2+/CC1+ cells; (C–C′′)), (NG2+/BCAS1+ cells; (D–D′′)), (NG2+/MBP+ cells; (G–G′′)) along the same area. Arrows in detailed

Article Snippet: To determine the physiological reaction from OPCs exposed to the substances, OPCs were treated in a dose-dependent manner using concentrations of 10 mM, 20 mM, 40 mM, 50 mM, and 100 mM for sodium azide (NaN3; Sigma-Aldrich, St. Louis, MO, USA Cat# S2002-100G); 4 mM, 8 mM, 16 mM, 20 mM, and 50 mM for 2-Deoxy-D-Glucose (2-DG; Sigma-Aldrich, St. Louis, MO, USA Cat# D8375-5G); and 0.02 μM for parbendazole (MedChem Express, Monmouth Junction, NJ, USA, CAS 14255879).

Techniques: Control, Cell Differentiation

Journal: International journal of molecular sciences

Article Title: A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage.

doi: 10.3390/ijms241310972

Figure Lengend Snippet: Figure 6. Identification and characterization of cells invading the scratch on myelination co-culture at 17 days in vitro (DIV). Representative bright field images of cells migrating into the scratch 24 and 48 h after stimulation with parbendazole (B–B′′) and DMSO (A–A′′) as control. Cells inside the scratch were counted, and the area of the scratch was calculated (E). Scale bar: 100 µm. After 48 h, the percentage of Olig2+ cells (C,D,F) and GFAP+ cells (C′,D′,G) was calculated in relation to the total number of cells, assessed by the number of DAPI+ cells, as exemplified by merged pictures (C′′, D′′). Scale bar: 100 µm. Data are shown as mean values (±SD) and are derived from n = 7 experiments for bright field images and n = 6 for immunofluorescence staining. Statistical significance was calculated using (E) Mann–Whitney U and Student’s t-test (F,G). Data were considered statistically significant (95% confidence interval) at * p < 0.05, ** p < 0.01, ns, not significant.

Article Snippet: To determine the physiological reaction from OPCs exposed to the substances, OPCs were treated in a dose-dependent manner using concentrations of 10 mM, 20 mM, 40 mM, 50 mM, and 100 mM for sodium azide (NaN3; Sigma-Aldrich, St. Louis, MO, USA Cat# S2002-100G); 4 mM, 8 mM, 16 mM, 20 mM, and 50 mM for 2-Deoxy-D-Glucose (2-DG; Sigma-Aldrich, St. Louis, MO, USA Cat# D8375-5G); and 0.02 μM for parbendazole (MedChem Express, Monmouth Junction, NJ, USA, CAS 14255879).

Techniques: Co-Culture Assay, In Vitro, Control, Derivative Assay, Staining, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage.

doi: 10.3390/ijms241310972

Figure Lengend Snippet: Figure 7. Rescue of oligodendroglial differentiation competence by means of parbendazole treatment following chemical ischemia after 3 d in vitro. Modification of oligodendroglial differentiation competence upon energy restriction removing glucose, blocking oxidative phosphorylation with sodium azide NaN3, and blocking glycolysis with 2-deoxy-D-glucose (2DG). In order to determine functionally relevant cell-specific concentrations, (C) total cell number as well as (D) MBP expression of primary cultured OPCs was investigated in a dose-dependent manner after 3 days in culture (A–A′′′). (E) In response to parbendazole treatment MBP (B–B′′′) as well as (G) CC1 (F–F′′′) expression can be rescued. Scale bar: 50 µm. Data are shown as mean values (±SD) and derive from n = 3 experiments. Statistical significance was calculated using Kruskal–Wallis test with Dunn’s post-test (C–E). Data were considered statistically significant (95% confidence interval) at * p < 0.05, ** p < 0.01, *** p < 0.001 ns, not significant.

Article Snippet: To determine the physiological reaction from OPCs exposed to the substances, OPCs were treated in a dose-dependent manner using concentrations of 10 mM, 20 mM, 40 mM, 50 mM, and 100 mM for sodium azide (NaN3; Sigma-Aldrich, St. Louis, MO, USA Cat# S2002-100G); 4 mM, 8 mM, 16 mM, 20 mM, and 50 mM for 2-Deoxy-D-Glucose (2-DG; Sigma-Aldrich, St. Louis, MO, USA Cat# D8375-5G); and 0.02 μM for parbendazole (MedChem Express, Monmouth Junction, NJ, USA, CAS 14255879).

Techniques: In Vitro, Blocking Assay, Phospho-proteomics, Expressing, Cell Culture

Journal: Circulation Journal

Article Title: Long Non-Coding RNA Nuclear-Enriched Abundant Transcript 1 (<i>NEAT1</i>) Facilitates Foam Cell Formation and Atherosclerosis Progression Through the miR-17-5p/Itchy E3 Ubiquitin Protein Ligase (ITCH)/Liver Kinase B1 (LKB1) Axis

doi: 10.1253/circj.cj-23-0769

Figure Lengend Snippet: Figure 4. Itchy E3 ubiquitin protein ligase (ITCH) promoted the uptake of low-density lipoprotein (LDL) by and lipid accumulation in macrophages by mediating ubiquitination degradation of liver kinase B1 (LKB1). (A) Interaction between ITCH and LKB1 in macrophages formed by phorbol myristate acetate-induction of THP-1 cells (Mφ) and RAW264.7 cells, analyzed using the coimmunoprecipitation assay. (B,C) Western blot analysis of the degradation of LKB1 in ITCH-knocked down or -overexpressing macrophages after MG132 treatment. DMSO, dimethylsulfoxide; NC, negative control; si, short interfering. (D) Western blot analysis of the degradation of LKB1 in ITCH-knocked down macrophages after cycloheximide (CHX) treatment. (E) Ubiquitination analysis of LKB1 ubiquitination in ITCH-knocked down macrophages. (F) Western blot analysis of ITCH and LKB1 protein levels in macrophages following si-ITCH and si-LKB1 co-transfection. (G) Western blot analysis of ITCH and LKB1 protein levels in Dil-oxLDL-treated macrophages were examined by western blot. (H) Uptake of LDL by macrophages observed under a fluorescence microscope. Scale bars, 100 μm. M.I., mean fluorescence intensity. (I) Lipid accumulation in macrophages assessed by Oil red O staining. Scale bars, 100 μm. (J) Lipid droplet formation in macrophages measured by BODIPY staining. Scale bars, 100 μm. DAPI, 4′,6′-diamidino-2-phenylindole. Data in all graphs are presented as the mean ± SD (n=3 replicate experiments). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: When Mφ and RAW264.7 cells reached 70–80% confluence, they were stimulated in DMEM mixed with the fluorescently labeled Dil-oxLDL (10, 20, 30, 40 and 50 mg/L; Yeasen, Shanghai, China) for 48 h. For drug treatment, cells were treated with a concentration of cycloheximide (CHX; 5 μmol/L; MedChem Express, Monmouth Junction, NJ, USA) for 0, 1, 2, and 4 h or proteasome inhibitor MG132 (10 μmol/L; MedChem Express) for 6 h with 40 mg/L Dil-oxLDL for 48 h. As reported in the Supplementary Methods, cells were subsequently used in transfection experiments examining the effects of NEAT1 and ITCH knockdown, as well as ITCH overexpression; coimmunoprecipitation (Co-IP) and RNA immunoprecipitation (RIP) assays; for the analysis of LKB1 ubiquitination; and lipid accumulation using Oil red O and BODIPY staining.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Cotransfection, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of DAPT in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were treated with 20 µM DAPT or the vehicle (DMSO) for 24, 48–72 h and lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Notch1, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 4–6 separate experiments. Bar graphs represent the quantification of protein expression as fold change over the level in vehicle-treated cells and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: To investigate the functional role of Notch1 signaling, cells were treated with 50 μM recombinant Jagged1 protein (Jagged-1 (188–204), Anaspec Inc, Freemont, CA, USA) for 48 h to activate the pathway, and with the γ-secretase inhibitor DAPT (MedChem Express, Monmouth Junction, NJ, USA) to inhibit Notch1 activation for 24 h, 48 h, 72 h or once daily for five consecutive days, depending of experimental procedures.

Techniques: Expressing, SDS Page, Western Blot, Control, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of DAPT in TG-induced Ca 2+ release and entry in non-tumoral breast epithelial cells and breast cancer cells. Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were treated with 20 µM DAPT or the vehicle (DMSO; Control). Fourty-eight hours later cells were loaded with fura-2. Cells were then suspended in a Ca 2+ -free (100 µM EGTA) HBS and stimulated with 2 µM TG followed by reintroduction of external Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ entry. Representative traces are shown in the left panel. Scatter plots represent quantification of TG-evoked Ca 2+ release and Ca 2+ entry determined as described in Materials and Methods. Dots represent single cell data for 2–3 separate experiments. n = 85 and 85 (for Control and DAPT-treated MCF10A cells, respectively), 90 and 90 (for Control and DAPT-treated MDA-MB-231 cells), 99 and 83 (for Control and DAPT-treated BT20 cells), 80 and 90 (for Control and DAPT-treated MCF7 cells) and 80 and 90 (for Control and DAPT-treated T47D cells, respectively). Data are presented as means ± SEM and are statistically analyzed using Student´s t- test. ** p < 0.01.

Article Snippet: To investigate the functional role of Notch1 signaling, cells were treated with 50 μM recombinant Jagged1 protein (Jagged-1 (188–204), Anaspec Inc, Freemont, CA, USA) for 48 h to activate the pathway, and with the γ-secretase inhibitor DAPT (MedChem Express, Monmouth Junction, NJ, USA) to inhibit Notch1 activation for 24 h, 48 h, 72 h or once daily for five consecutive days, depending of experimental procedures.

Techniques: Control, Concentration Assay, Single Cell