ciap2 817 p2  (R&D Systems)

Journal: Antioxidants

Article Title: Targeting SQLE-Mediated Cholesterol Metabolism to Promote Oxidative Stress and Attenuate Drug Resistance in Osteosarcoma

doi: 10.3390/antiox15091122

Figure Lengend Snippet: Enhancement of doxorubicin-induced SaOS-2 cell apoptosis by SQLE inhibition ( A ) Representative flow cytometry scatter plots of SaOS-2 cells following Annexin V-FITC/PI dual staining. Cells were treated with doxorubicin (150 nM), terbinafine (50 and 75 μM), or their combinations for 48 h. ( B ) Quantitative analysis showing the corresponding percentages of total apoptotic cells derived from ( A ). ( C ) Representative flow cytometry histograms of mitochondrial membrane potential (MMP) disruption in SaOS-2 cells using MitoView™ 633 staining. Cells were treated with doxorubicin (150 nM), terbinafine (50 and 75 μM), or their respective combinations for 48 h. ( D ) Quantitative analysis showing the proportion of MMP-disrupted cells normalized to the control group, derived from ( C ). ( E , F ) Western blot analysis of key anti-apoptotic proteins in (BCL-2, BCL-XL, c-IAP2, and c-FLIP) SaOS-2 cells treated with doxorubicin (150 nM), terbinafine (50 and 75 μM), or their respective combinations for 48 h. β-actin was used as the internal control. Representative immunoblots ( E ) and their corresponding relative band intensities calculated relative to the control group ( F ). ( G , H ) Western blot analysis of pro-apoptotic proteins (cleaved PARP-1 and cleaved caspase-3) in SaOS-2 cells treated with doxorubicin (150 nM), terbinafine (50 and 75 μM), or their respective combinations for 48 h. β-actin was used as the internal control. Representative immunoblots ( G ) and quantitative analysis of protein expression levels relative to the control group ( H ). ( I ) Cell viability analysis via MTT assay in SaOS-2 cells treated with 150 nM doxorubicin, 75 μM terbinafine, or their combination for 48 h, evaluated in the absence or presence of apoptosis inhibitor 50 μM Z-VAD-FMK. Data are expressed as the mean ± SD ( n = 3 independent experiments). #, ##, and ### indicate p ≤ 0.05, p ≤ 0.005, and p ≤ 0.001 compared to the control group, respectively. *, **, and *** indicate p ≤ 0.05, p ≤ 0.005, and p ≤ 0.001 for specific comparisons between experimental groups.

Article Snippet: Antibodies specific to c-FLIP, c-IAP2, BCL-2, BCL-XL, p-GSK3, GSK3, p-ERK, ERK, N-cadherin, vimentin, cleaved caspase-3, cyclin-B, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), whereas antibodies specific to MT1MMP, p-mTOR, mTOR, p-AKT, AKT, PARP-1, GPX-4, FSP-1, CDC25C, and p21 were purchased from Abclonal (Woburn, MA, USA).

Techniques: Inhibition, Flow Cytometry, Staining, Analysis, Derivative Assay, Membrane, Disruption, Control, Western Blot, Expressing, MTT Assay