annexin v apc dapi apoptosis detection kit  (Elabscience Biotechnology)

Journal: International Journal of Oncology

Article Title: Diverse roles of SERPINE1 in regulating cellular proliferation and invasion

doi: 10.3892/ijo.2026.5871

Figure Lengend Snippet: SERPINE1-mediated modulation of the ERK/p38 ratio and anoikis. (A) Upper panel: western blotting showing ERK, p-ERK, p38, and p-p38 levels. Quantitative data showed the p-ERK/ERK and p-p38/p38 ratios in SERPINE1 knockdown cells compared with control cells. Lower panel: western blotting analysis of p-AKT, AKT, p-JNK and JNK (normalized to total protein) levels in the shSE1 and shc groups. The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins. Data represent mean ± SDs of three independent experiments. (B) PLAUR and HSP90AA1 mRNA and protein levels in shSE1 and shc cells analyzed using RNA-seq (left panel) and western blotting (right panel). The numbers beneath the bands represent the ratios of the expression levels of the indicated proteins and represent mean ± SDs of three independent experiments. RNA-seq was performed using one biological replicate per group. (C-D) Upper panel: (C) 231-shSE1 cells were infected with lentivirus containing PLAUR cDNA (ex-PLAUR) or the corresponding empty vector control (vec). (D) H4-shSE1 cells were transfected with siRNA targeting HSP90AA1 (si-HSP90AA1) or siNC. The expression levels of uPAR and HSP90α, as well as the activity of ERK and p38, were detected by western blotting, with GAPDH serving as the loading control. Data represent mean ± SDs of three independent experiments. Lower panel: Cell proliferation was assessed by CCK-8 assay in 231-shSE1 cells infected with ex-PLAUR or vec, and in H4-shSE1 cells transfected with si-HSP90AA1 or siNC. Statistical significance was determined using two-way ANOVA followed by Šídák's multiple comparisons test. The data are presented as the means ± SDs. n=6. (E) Morphology of shSE1 and shc cells cultured under suspension condition. (scale bar, 100 μ m). (F) Apoptotic cells cultured under suspension condition were analyzed by flow cytometry following Annexin V-PE/7AAD staining (72 h of suspension culture). Statistical significance was determined using a two-sided Student's t test. The data are presented as the means ± SDs of three independent experiments. ** P<0.01 *** P<0.001, ns, not significant. SERPINE1, serine protease inhibitor clade e member 1; p-, phosphorylated; shRNA, short hairpin RNA; shSE1, shRNA targeting SERPINE1; shc, shRNA scrambled control; PLAUR, plasminogen activator, urokinase receptor; HSP90AA1, heat shock protein 90 alpha family class a member 1; RNA-seq, RNA sequencing; vec, empty vector control; siRNA, short interfering RNA; siNC, negative control siRNA; uPAR, urokinase-type plasminogen activator receptor; HSP90α, heat shock protein 90-alpha.

Article Snippet: Subsequently, suspended cells were harvested, and anoikis was quantified using an Annexin V-APC/DAPI apoptosis detection kit (cat. no. E-CK-A258; Elabscience Bionovation Inc.) according to the manufacturer's instructions.

Techniques: Western Blot, Knockdown, Control, Expressing, RNA Sequencing, Infection, Plasmid Preparation, Transfection, Activity Assay, CCK-8 Assay, Cell Culture, Suspension, Flow Cytometry, Staining, Protease Inhibitor, shRNA, Small Interfering RNA, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: Cd induces the nuclear expression of FoxO3a in neuronal cells. SH-SY5Y cells were treated with 5 μM Cd for various time periods. ( A ) The cell lysates were subjected to Western Blot analysis to measure FoxO3a protein expression; ( B ) nuclear and cytoplasmic extracts were subjected to Western Blot analysis to measure FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h, and confocal laser scanning microscopy was performed to determine FoxO3a localization. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. The provided scale bar in the merged image represents 20 μm. * p < 0.05, ** p < 0.01 versus the control group.

Article Snippet: The cells were incubated with DAPI (Cell Apoptosis DAPI Detection Kit, KGA215, KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: p38 regulates the Cd-induced nuclear expression of FoxO3a. ( A ) SH-SY5Y cells were treated with 5 μM Cd for various time periods, and phosphorylated and total MAPK proteins were detected by Western Blot analysis. ( B ) SH-SY5Y cells were treated with 5 μM Cd for 6 h after preincubating with 10 μM SB203580 for 1 h. Western Blot analysis was performed to determine FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Confocal laser scanning microscopy was performed to determine FoxO3a expression. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). ( E ) SH-SY5Y cells were treated with 5 μM Cd for various time periods. Western Blot analysis was performed to determine phosphorylation of FoxO3a-Ser7. ( F – H ) SH-SY5Y cells and rat cerebral cortical neurons were incubated with 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Western Blot analysis and confocal laser scanning microscopy were performed to determine phosphorylation of FoxO3a-Ser7 expression. The provided scale bar in the merged image represents 20 μm. ( G – H ) p-FoxO3a is shown in red, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. * p < 0.05, ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the Cd group.

Article Snippet: The cells were incubated with DAPI (Cell Apoptosis DAPI Detection Kit, KGA215, KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Phospho-proteomics, Incubation, Control

Journal: Biologics : Targets & Therapy

Article Title: Investigating the Oncogenic and Immunological Implications of YTHDF1 in Ovarian Cancer

doi: 10.2147/BTT.S542488

Figure Lengend Snippet: YTHDF1 promotes OC progression both in vitro and in vivo. ( A ) The expression of YTHDF1 in A2780 (left) and SKOV3 (right) cells stably transfected with YTHDF1 shRNAS was detected by qPCR. ( B ) The knockdown efficiency of YTHDF1 shRNAs in A2780 (left) and SKOV3 (right) cells was detected by WB. ( C ) CCK-8 assays were used to detect the proliferation ability of cells after YTHDF1 knockdown. ( D ) Representative images of A2780 (up) and SKOV3 (down) cells of EdU assays of every group; and the quantitative data were compared (right). Scale bar, 100μm. ( E ) Representative images of A2780 (up) and SKOV3 (down) cells of apoptosis experiments every group; and the quantitative data were compared (right). ( F–G ) Representative images of migrated (upper) and invasive (lower) A2780 ( F ) and SKOV3 ( G ) cells, along with quantitative analysis using ImageJ software (right). Scale bar, 100μm. ( H ) WB analysis was used to verify the knockdown efficiency of YTHDF1 in ID8 cells (left). The ID8 cells were then subcutaneously implanted into C57BL/6 mice. Thirty-five days after tumor implantation, the tumors were excised and photographed. ( I ) The weight (left) and volume (right) of ID8 tumors were compared between the two groups. ( J ) Expression levels of YTHDF1 and Ki67 in the tumor tissues of each group were detected by IHC staining. Scale bar:100 µm. For A, C-G, data are presented as the means ± SD, unpaired two-sided Student’s t -test. For I, data are presented as the means ± SEM, unpaired two-sided Student’s t -test.

Article Snippet: Apoptosis was assessed using an Annexin V-APC apoptosis detection kit (E-CK-A258, Elabscience, China) following the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Expressing, Stable Transfection, Transfection, Knockdown, CCK-8 Assay, Software, Tumor Implantation, Immunohistochemistry