Journal: Oncotarget

Article Title: Endoplasmic reticulum chaperone prolyl 4-hydroxylase, beta polypeptide (P4HB) promotes malignant phenotypes in glioma via MAPK signaling.

doi: 10.18632/oncotarget.18026

Figure Lengend Snippet: Figure 5: Transient over-expression of P4HB promoted glioma cell proliferation, migration, invasion and tube formation ability in vitro. (A) Western blot analysis showed upregulated expression of P4HB post-transfection. (B) MTT assay was performed on cells with transient P4HB over-expression (D54 P4HB, U87 P4HB and U251 P4HB) and empty vector controls (D54 Vec, U87 Vec and U251 Vec). After 5 days incubation, cells over-expressing P4HB showed higher proliferative rates than controls. (C) Migration assay showed greater motility of U87 P4HB and U251 P4HB than their respective vector controls (U87 Vec and U251 Vec). (D) Matrigel cell invasion assay similarly showed greater invasiveness in U87 and U251 cells with P4HB over-expression (** p <0.05). (E) Angiogenesis, as measured by tube formation ability, was again higher in P4HB overexpressing cells (U87 and U251). (F) Western blot analysis revealed that P4HB over-expression was associated with increased MAPK phosphorylation. (G) Suppression of MAPK activities by U0126 reduced VEGF expression in P4HB over-expressing cells, while P4HB expression level was unaffected. (H) Representative pictures from three independent assays showed decreased cell invasion abilities of U87 P4HB and U251 P4HB cells after treatment with U0126. (I) U0126 pretreatment also inhibited tube formation in these cells after 24 h (Magnification: ×200).

Article Snippet: Briefly, after blocking with 5% non-fat milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6), the membrane was probed with one of the following primary antibodies (at 1:1000 dilution) at 4oC overnight: rabbit monoclonal antibodies against P4HB, total p44/42 MAP kinase (Erk 1/2) (total MAPK), phosphor-p44/42 MAP kinase (Erk1/2) (Thr202/Tyr204) (pMAPK), and VEGF (all from Cell Signaling Technology Inc.).

Techniques: Over Expression, Migration, In Vitro, Western Blot, Expressing, Transfection, MTT Assay, Plasmid Preparation, Incubation, Invasion Assay, Phospho-proteomics

Journal: Journal of Advanced Research

Article Title: Exploiting metabolic vulnerabilities through synergistic ferroptosis and disulfidptosis for breast cancer therapy

doi: 10.1016/j.jare.2025.03.052

Figure Lengend Snippet: FCSP@876 MOFs induced disulfidptosis under the synergistic action of BAY876 − mediated glucose starvation and FCSP − induced adaptively high SLC7A11. (A) Non reducing and reducing western blot analysis of the indicated actin cytoskeleton proteins (FLNA, MYH9) in 4 T1 cells cultured in different groups for 24 h. (B) Fluorescent staining of F-actin with phalloidin in 4 T1 cells cultured in different groups for 24 h, scale bar = 25 μm. The treatment dosage: BAY876: 100 nmol, FCSP: 125 μg/mL, FCSP@876:125 μg/mL.

Article Snippet: The primary antibodies for the following proteins were used at the specified concentrations for western blotting: FLNA (1:500, 4762S, Cell Signaling Technology), myosin IIa (MYH9, 1:500, 3403S, Cell Signaling Technology), GPX4 antibody (ab125066, 1:200, Abcam), SLC7A11 antibody (ab307601, 1:200, Abcam), PTGS2 antibody (66351-1-IG, 1:200, Proteintech), secondary antibody IgG/HRP (bs-0295G-HRP, 1:10000, Beijing Biosynthesis biotechnology Co., Ltd), beta-Actin (AF7018, 1:10000, Affinity).

Techniques: Western Blot, Cell Culture, Staining

Journal: Journal of Advanced Research

Article Title: Exploiting metabolic vulnerabilities through synergistic ferroptosis and disulfidptosis for breast cancer therapy

doi: 10.1016/j.jare.2025.03.052

Figure Lengend Snippet: FCSP@876 MOFs achieved combined ferroptosis/disulfidptosis strategy for enhancing breast cancer therapy. (A) Representative photographs of 4 T1 xenograft tumors from mice after different treatments (B) The tumor growth curves and (C) mice body weights change after the different treatments. (D) Representative images of immunohistochemical staining (Ki67, SLC7A11, GPX4 and PTGS2) and hematoxylin-eosin staining (H&E) of 4 T1 xenograft tumors with the indicated treatments. Data are presented as mean ± SD (n = 5). Scale bars, 200 μm. (E) Western blot analysis of GPX4 and PTGS2 in tumor treated in different groups, and (F) (G) relative quantification of GPX4 and PTGS2. (H) Western blot analysis of SLC7A11 in tumor treated in different groups, and (I) relative quantification of SLC7A11. (J) Non reducing and reducing western blot analysis of the indicated actin cytoskeleton proteins (FLNA, MYH9) of tumor treated in different groups. The treatment dosage; BAY876: 3 mg/kg, FCSP: 60 mg/kg, FCSP@876: 60 mg/kg, ns: P＞0.05, *:P＜0.05, **:P＜0.01, ***:P＜0.001, ****:P＜0.0001.

Article Snippet: The primary antibodies for the following proteins were used at the specified concentrations for western blotting: FLNA (1:500, 4762S, Cell Signaling Technology), myosin IIa (MYH9, 1:500, 3403S, Cell Signaling Technology), GPX4 antibody (ab125066, 1:200, Abcam), SLC7A11 antibody (ab307601, 1:200, Abcam), PTGS2 antibody (66351-1-IG, 1:200, Proteintech), secondary antibody IgG/HRP (bs-0295G-HRP, 1:10000, Beijing Biosynthesis biotechnology Co., Ltd), beta-Actin (AF7018, 1:10000, Affinity).

Techniques: Immunohistochemical staining, Staining, Western Blot, Quantitative Proteomics

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deferasirox Targeting Ferroptosis Synergistically Ameliorates Myocardial Ischemia Reperfusion Injury in Conjunction With Cyclosporine A

doi: 10.1161/JAHA.123.031219

Figure Lengend Snippet: A, Experimental protocol for inducing ischemia reperfusion (I/R) in mice. B, Measurement of total myocardial iron levels 24 hours after DFX administration (200 mg/kg, per os [p.o.]; n=15, each group). C, Measurement of iron levels in the ER fraction of I/R‐injured myocardium 24 hours after I/R (n=4, each group). D, Measurement of iron levels in the ER fraction of I/R‐injured myocardium treated with vehicle or DFX (200 mg/kg, per os [p.o.]; n=8 in vehicle group and 7 in DFX group). Data are presented as the mean±SEM. Statistical significance was determined using unpaired t ‐test. * P <0.05, ** P <0.01. 4‐HNE indicates 4‐hydroxynonenal; CCM, cleaved caspase substrate motif; HO‐1, heme oxygenase‐1; Hx, hypoxia; LAD, left anterior descending artery; Nx, normoxia; TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling; and Veh, vehicle.

Article Snippet: Western blotting was performed as described previously , with the following primary antibodies against: 4‐hydroxy‐2‐nonenal (4‐HNE; MHN‐020P, JaICA; Shizuoka), acrolein (MAR‐020n, JaICA), cyclooxygenase‐2 (COX2; #12282, Cell Signaling Technology [CST]), heme oxygenase‐1 (HO‐1; #43966, CST), cleaved caspase substrate motif (#8698, CST), BAX (#14796, CST), and BCL2 (ab182858, Abcam); the antibody against GAPDH (internal standard; Santa Cruz Biotechnology, Inc., sc‐32 233) was used as an internal control to standardize the signal intensities.

Techniques: TUNEL Assay, End Labeling