Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Kefir peptides mitigate bleomycin-induced pulmonary fibrosis in mice through modulating oxidative stress, inflammation and gut microbiota.

doi: 10.1016/j.biopha.2024.116431

Figure Lengend Snippet: Fig. 5. Pretreatment KPs attenuates the expression of fibrotic-related genes in the bleomycin-induced pulmonary fibrosis mouse model. Mice pre-treated with water or KPs for 4 days following intratracheal injection of bleomycin for 21 days. Lung tissues were harvested to detect fibrotic-related mRNA, including (A) Fn1, (B) Col3a1, (C) Col1a1, (D) Timp1, and (E) Ctgf expression by quantitative RT-PCR (n=5 mice per group). Values are normalized to the β-actin gene and are expressed relative to the control group. (F) Western blot analysis was performed to analyze the expression of Collagen I and fibronectin in the lung tissues from each group (n=4–5 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001. All the statistical information and details of tests for each figure are available in the supple mentary files.

Article Snippet: After transferring to PVDF membranes, the membranes were blocked with 5% nonfat dry milk in TBS-T (0.1% Tween-20 in TBS buffer) for 1 h. Proteins were then probed overnight at 4 ◦C with the following antibodies at appropriate dilutions: 1:2000 dilution of Fibronectin and Collagen type I (Proteintech) and 1:25,000 dilution of β-actin (Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Injection, Quantitative RT-PCR, Control, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: Analysis of ROS intracellular amounts and FHC antioxidant protein levels in OVCAR3 and OVCAR8 cells. (a) Immunofluorescence analysis of ROS levels in OVCAR3 and OVCAR8 cells by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported. (b) Immunofluorescence analysis of superoxide radical levels in OVCAR3 and OVCAR8 cells by staining with MitoSOX™ Red Indicator (red). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported. (c) Representative western blot of antioxidant protein FHC in OVCAR3 and OVCAR8 cells. γ -Tubulin was used as internal control. WB has been quantified by using ImageJ software and optical densitometry is reported. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Western Blot, Control, Software

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: OVCAR3 cells exhibit increased growth potential compared to OVCAR8 cells. (a) Cell cycle FACS analysis of OVCAR3 and OVCAR8 cells stained with PI. The experiments were performed in triplicate. Representative plots of a single experiment (left); histograms showing the mean ± SD of three independent experiments (right). ∗ p value < 0.05, OVCAR3 vs . OVCAR8. (b) MTT analysis of OVCAR3 and OVCAR8 cell growth at 12 h, 24 h, 48 h, and 72 h. Data are reported as absorbance measured at 595 nm and shown as mean ± SD of three independent replicates ( ∗ p < 0.05, OVCAR3 vs . OVCAR8); (° p < 0.01, OVCAR3 vs . OVCAR8); N.S.: not significant. (c) Representative WB of c-Myc, cyclin E1 (CCNE1), pChk2 (Thr68), pERK1/2, and pAKT in OVCAR3 and OVCAR8 cells. γ -Tubulin was used as internal control. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Staining, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: OVCAR8 cells are characterized by ROS accumulation and FHC downregulation upon 6 μ M cisplatin treatment. (a) Cell viability assay performed by MTT analysis in OVCAR3 and OVCAR8 cells treated with 6 μ M, 12 μ M, 24 μ M and 48 μ M of cisplatin for 24 h. Cisplatin concentrations are reported as log [cisplatin ( μ M)]. Cell viability is expressed as percentage (%). Treatments were performed at least three times on independent biological replicates and the mean concentration of the drug that gives half-maximal response (log EC 50 ) was used to compare cytotoxicity. (b) Quantification of ROS amounts through DCFDA staining in OVCAR3 and OVCAR8 untreated (NT) and upon treatment with 6 μ M, 12 μ M, 24 μ M and 48 μ M cisplatin for 24 h. Data represent the mean ± SD of three biological replicates. ∗ p value < 0.01 OVCAR3 NT vs . OVCAR8 NT; ° p value < 0.01 OVCAR3 6 μ M cisplatin vs . OVCAR8 6 μ M cisplatin; ∗∗ p value < 0.05 OVCAR3 12 μ M cisplatin vs . OVCAR8 12 μ M cisplatin; °° p value < 0.05 OVCAR3 24 μ M cisplatin vs . OVCAR8 24 μ M cisplatin; N.S.: not significant: OVCAR3 48 μ M cisplatin vs . OVCAR8 48 μ M cisplatin. (c) Representative western blot of FHC, cleaved caspase 3, and caspase 3 in OVCAR3 and OVCAR8 untreated (NT) and upon treatment with 6 μ M cisplatin for 24h. γ -Tub was used as internal control. WB analysis was performed three times and results were reproducible. (d) Immunofluorescence analysis of ROS levels in untreated (NT) and treated with 6 μ M cisplatin OVCAR3 and OVCAR8 cells by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported. (e) Immunofluorescence analysis of superoxide radical levels untreated (NT) and treated with 6 μ M cisplatin OVCAR3 and OVCAR8 cells by staining with MitoSOX™ Red Indicator (red). Nuclei were stained with DAPI (blue). Analysis was performed in duplicate and representative images are reported.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Viability Assay, Concentration Assay, Staining, Western Blot, Control, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: FHC knockdown improves OVCAR3 response to 6 μ M cisplatin by increasing ROS production. (a) Representative plots of Annexin V/7-AAD apoptosis assays in OVCAR3 Neg Control , OVCAR3 Neg Control (6 μ M) cisplatin, OVCAR3 Neg Control 10 mM NAC, OVCAR3 Neg Control (6 μ M) cisplatin/10 mM NAC, OVCAR3 siFHC , and OVCAR3 siFHC (6 μ M) cisplatin. Cisplatin treatment was performed for 24 h while NAC treatment was performed for 2 h. FACS plots are representative of single experiments. Values are expressed as mean ± SD of three biological replicates. (b) Immunofluorescence analysis of ROS levels in OVCAR3 Neg Control , OVCAR3 Neg Control (6 μ M) cisplatin, OVCAR3 Neg Control 10 mM NAC OVCAR3 Neg Control (6 μ M) cisplatin/10 mM NAC, OVCAR3 siFHC , and OVCAR3 siFHC (6 μ M) cisplatin, by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). (c) Representative western blot of FHC in OVCAR3 Neg Control , OVCAR3 Neg Control (6 μ M) cisplatin, OVCAR3 Neg Control 10 mM NAC, OVCAR3 Neg Control (6 μ M) cisplatin/10 mM NAC, OVCAR3 siFHC , and OVCAR3 siFHC (6 μ M) cisplatin. γ -Tubulin was used as internal control. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Knockdown, Control, Immunofluorescence, Staining, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS

doi: 10.1155/2019/3461251

Figure Lengend Snippet: FHC overexpression or NAC treatment reduces OVCAR8 response to cisplatin. (a) Representative plots of Annexin V/7-AAD apoptosis assays in OVCAR8 pc3DNA , OVCAR8 pc3DNA (6 μ M) cisplatin, OVCAR8 pc3DNA (6 μ M) cisplatin/10 mM NAC, and OVCAR8 pc3FHC (6 μ M) cisplatin. Cisplatin treatment was performed for 24 h while NAC treatment was performed for 2 h. FACS plots are representative of single experiments. Values are expressed as mean ± SD of three biological replicates. (b) Immunofluorescence analysis of ROS levels in OVCAR8 pc3DNA , OVCAR8 pc3DNA (6 μ M) cisplatin, OVCAR8 pc3DNA (6 μ M) cisplatin/10 mM NAC, and OVCAR8 pc3FHC (6 μ M) cisplatin, by staining with CellROX® Green Reagent (green). Nuclei were stained with DAPI (blue). (c) Representative western blot of FHC in OVCAR8 pc3DNA , OVCAR8 pc3DNA (6 μ M) cisplatin, OVCAR8 pc3DNA (6 μ M) cisplatin/10 mM NAC, and OVCAR8 pc3FHC (6 μ M) cisplatin. γ -Tubulin was used as internal control. WB analysis was performed three times and results were reproducible.

Article Snippet: To ensure equal loading of proteins, we used goat polyclonal anti- γ -tubulin antibody (C-20) (1 : 2000, sc-7396, Santa Cruz Biotechnology).

Techniques: Over Expression, Immunofluorescence, Staining, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Hepatocyte-specific Wtap deficiency promotes hepatocellular carcinoma by activating GRB2–ERK depending on downregulation of proteasome-related genes

doi: 10.1016/j.jbc.2023.105301

Figure Lengend Snippet: Wtap deletion in hepatocytes promotes hepatocellular carcinoma (HCC) progression in young mice. At 2 weeks old, male Wtap -HKO mice and Wtap flox/flox mice received a single intraperitoneal dose of DEN (50 mg/kg). Mice were fed either a normal choe (NC) diet or a high-fat diet (HFD) for 11 weeks. A , representative pictures of mouse livers from the indicated genotype are shown. B , tumor number was counted (n = 10–12 per group; NC: p = 0.0026; HFD: p < 0.0001). C , maximal tumor size was measured (n = 10–12 per group; NC: p < 0.0001; HFD: p < 0.0001). D , representative Ki67 staining photographs were shown. E , the number of Ki67-positive cells was counted (n = 6 per group; NC: p = 0.0002; HFD: p < 0.0001). F and G , triglyceride (TAG) levels in the livers of Wtap -HKO mice and Wtap flox/flox mice fed an NC diet for 35 weeks or an HFD for 11 weeks were assessed (n = 10 per group; F : p < 0.0001; G : p = 0.0012). H , serum-free fatty acid levels in Wtap -HKO mice and Wtap flox/flox mice fed with an NC diet for 11 weeks (n = 6–8 per group; p = 0.0022). I and J , immunoblotting of IGFBP1, CD36, CCL2, WTAP, and Tubulin in the livers of Wtap -HKO and Wtap flox/flox mice fed NC or HFD for 11 weeks following DEN treatment (n = 7 per group; n = 4 for representative images; NC: IGFBP1, p = 0.0033; CD36, p = 0.0013; and CCL2, p = 0.019; HFD: IGFBP1, p = 0.0115; CD36, p = 0.0264; and CCL2, p = 0.0011). K , heatmap of interleukin 6 (IL6) cytokine family ( Clcf1 , Cntf , Ctf1 , Il6 , IL11 , Il27 , Lif , and Osm ) and Tnfa in Wtap -HKO and Wtap flox/flox livers (n = 3 for each group, RNA-Seq data GSE168850). L , immunoblotting analysis of p-STAT3, STAT3, p-p65, p65, p-IκBα, IκBα, WTAP, and Tubulin in the livers of NC-fed Wtap -HKO and Wtap flox/flox mice pretreated with or without DEN (n = 7 for each group; n = 3 for representative images). ∗ p < 0.05; ∗∗ p < 0.01. Data represent the mean ± SD. CCL2, chemokine (C–C motif) ligand 2; DEN, diethylnitrosamine; IGFBP1, insulin-like growth factor–binding protein 1; Wtap -HKO, hepatocyte-specific Wtap knockout.

Article Snippet: The following antibodies were displayed: FLAG (catalog no.: F1804, 1:5000 dilution; Sigma); WTAP (catalog no.: 10200-1-AP, 1:2000 dilution; Proteintech); PSMB4 (catalog no.: 11029-1-AP, 1:5000 dilution; Proteintech); PSMB6 (catalog no.: 11684-2-AP, 1:5000 dilution; Proteintech); p-STAT3 (catalog no.: 9145, 1:3000 dilution; Cell Signaling Technology); STAT3 (catalog no.: 10253-2-AP, 1:3000 dilution; Proteintech); p-IκBα (Ser32) (catalog no.: 2859, 1:3000 dilution; Cell Signaling Technology); IκBα (catalog no.: 10268-1-AP, 1:2000 dilution; Proteintech); Tubulin (catalog no.: sc-5286, 1:5000 dilution; Santa Cruz); CD36 (catalog no.: 18836-1-AP, 1:2500 dilution; Proteintech); CCL2 (catalog no.: 66272-1-Ig, 1:2000 dilution; Proteintech); p-p65 (catalog no.: 3033, 1:2500 dilution; Cell Signaling Technology); p65 (catalog no.: 8242, 1:2500 dilution; Cell Signaling Technology); p-ERK1/2 (catalog no.: 4377, 1:5000 dilution; Cell Signaling Technology); ERK1/2 (catalog no.: 4695, 1:5000 dilution; Cell Signaling Technology); GRB2 (catalog no.: sc-8034, 1:1000 dilution; Santa Cruz); p-Met (catalog no.: 3077, 1:4000 dilution; Cell Signaling Technology); Met (catalog no.: 3127, 1:4000 dilution; Cell Signaling Technology); p-EGFR (catalog no.: 3777, 1:4000 dilution; Cell Signaling Technology); EGFR (catalog no.: 4267, 1:5000 dilution; Cell Signaling Technology); p-MEK1/2 (catalog no.: 9154, 1:4000 dilution; Cell Signaling Technology); MEK1/2 (catalog no.: 8727, 1:4000 dilution; Cell Signaling Technology); and IGFBP1 (catalog no.: A11672, 1:3000 dilution; ABclonal).

Techniques: Staining, Western Blot, RNA Sequencing, Binding Assay, Knock-Out

Journal: Scientific Reports

Article Title: Resolvin D1 combined with exercise rehabilitation alleviates neurological injury in mice with intracranial hemorrhage via the BDNF/TrkB/PI3K/AKT pathway

doi: 10.1038/s41598-024-83019-w

Figure Lengend Snippet: RvD1 combined with exercise rehabilitation training can activate BDNF/TrκB/p-Akt/PI3K related pathway and increase its protein and mRNA expression. ( a ) Western blot analysis was used to detect the expression levels of BDNF, TrκB, PI3K proteins, and both phosphorylated and total Akt. ( b - d ) Quantitative analysis of BDNF, TrκB, and PI3K protein expressions in brain tissue adjacent to the injection site on the ipsilateral side after the experiment. ( e ) Semiquantitative measurements of the ratio of phosphorylated to total Akt. ( f - i ) Expression levels of BDNF ( f ), TrκB ( g ), PI3K ( h ), and Akt ( i ) mRNAs at the injection site were determined. Western blot and RT-qPCR analyses revealed that the expression levels of BDNF, TrκB, p-Akt, and PI3K in the ICH + RvD1 + exercise training group were significantly higher compared to those in the ICH group. (*Significant vs. ICH group, p < 0.05).

Article Snippet: Equal amounts of extracted proteins was added to 5X loading buffer, heated to 95 ° C for denaturation, and the proteins were separated by electrophoresis in 10% polyacrylamide gel.After transferring the proteins to PVDF membrane by wet transfer method, they were blocked in 5% bovine serum protein for 1 h, then the following primary antibodies were added and incubated overnight at 4 ° C: BDNF (1:4000, 25699-1-AP, Proteintech), AKT (1:4000, 10176-2-AP, Proteintech), phospho-AKT (1:2000, 66444-1-Ig, Proteintech), PI3K (1:1000, 20584-1-AP, Proteintech), TrkB (1:4000, 13129-1-AP, Proteintech), GAPDH (1:4000, GB12002-100, Servicebio).The following day, the primary antibodies were removed, and the PVDF membranes were washed three times with TBST, each wash lasting 5 min.

Techniques: Expressing, Western Blot, Injection, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Resolvin D1 combined with exercise rehabilitation alleviates neurological injury in mice with intracranial hemorrhage via the BDNF/TrkB/PI3K/AKT pathway

doi: 10.1038/s41598-024-83019-w

Figure Lengend Snippet: RvD1 combined with exercise rehabilitation training activates BDNF/TrkB/PI3K/AKT signaling pathway to reduce neuroinflammation after intracerebral hemorrhage and improve neurological prognosis.

Article Snippet: Equal amounts of extracted proteins was added to 5X loading buffer, heated to 95 ° C for denaturation, and the proteins were separated by electrophoresis in 10% polyacrylamide gel.After transferring the proteins to PVDF membrane by wet transfer method, they were blocked in 5% bovine serum protein for 1 h, then the following primary antibodies were added and incubated overnight at 4 ° C: BDNF (1:4000, 25699-1-AP, Proteintech), AKT (1:4000, 10176-2-AP, Proteintech), phospho-AKT (1:2000, 66444-1-Ig, Proteintech), PI3K (1:1000, 20584-1-AP, Proteintech), TrkB (1:4000, 13129-1-AP, Proteintech), GAPDH (1:4000, GB12002-100, Servicebio).The following day, the primary antibodies were removed, and the PVDF membranes were washed three times with TBST, each wash lasting 5 min.

Techniques:

Journal: Scientific Reports

Article Title: Resolvin D1 combined with exercise rehabilitation alleviates neurological injury in mice with intracranial hemorrhage via the BDNF/TrkB/PI3K/AKT pathway

doi: 10.1038/s41598-024-83019-w

Figure Lengend Snippet: The primer sequences for BDNF, TrkB, PI3K, AKT, IL-1β, IL-10, TNF-α, CD206 and GAPDH.

Article Snippet: Equal amounts of extracted proteins was added to 5X loading buffer, heated to 95 ° C for denaturation, and the proteins were separated by electrophoresis in 10% polyacrylamide gel.After transferring the proteins to PVDF membrane by wet transfer method, they were blocked in 5% bovine serum protein for 1 h, then the following primary antibodies were added and incubated overnight at 4 ° C: BDNF (1:4000, 25699-1-AP, Proteintech), AKT (1:4000, 10176-2-AP, Proteintech), phospho-AKT (1:2000, 66444-1-Ig, Proteintech), PI3K (1:1000, 20584-1-AP, Proteintech), TrkB (1:4000, 13129-1-AP, Proteintech), GAPDH (1:4000, GB12002-100, Servicebio).The following day, the primary antibodies were removed, and the PVDF membranes were washed three times with TBST, each wash lasting 5 min.

Techniques: Sequencing