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: 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

Journal: iScience

Article Title: Cytohesin-4/ARF6 facilitates the progression of acute myeloid leukemia through activating PIK3R5/PI3K/AKT pathway

doi: 10.1016/j.isci.2025.112634

Figure Lengend Snippet: CYTH4 silencing alleviates the proliferation of human AML cells and increases the chemosensitivity of AML cells to Ara-C (A) Validation of the downregulated expression of CYTH4 in four shCYTH4-transduced MV4-11 and THP-1 cells by RT-qPCR ( n = 3). β-actin was used as an internal control. Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (B) Validation of the downregulated expression of CYTH4 in shCYTH4-transduced MV4-11 and THP-1 cells by immunoblotting. GAPDH was used as a loading control. (C) CYTH4 silencing inhibits the proliferation of MV4-11 and THP-1 cells. CCK-8 assay ( n = 3) was conducted to detect the viability of MV4-11 and THP1 cells at the indicated time points (0, 24, 48, 72, and 96 h). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (D) Inhibitory effect of CYTH4 silencing on colony formation of AML cell was assessed by microscopically counting the numbers and sizes of colonies at 14 days after plating. The quantification is shown on the right ( n = 3).Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (E) CYTH4 silencing in MV4-11 and THP-1 cells using shRNAs induces cell apoptosis. Following annexin V-APC and 7-AAD staining, the percentages of apoptotic cells were detected by flow cytometry. Representative flow cytometry plots (top) and quantification (bottom) are shown ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (F) CYTH4 silencing in MV4-11 and THP-1 cells using shRNAs induces cell-cycle arrest at the G0/G1 phase. Representative flow cytometry histogram plots (top) and quantification (bottom) are shown ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (G) Immunoblotting assay for c-MYC, BCL-2, BAX, CYCLIN B1, and CYCLIN D1 upon CYTH4 silencing in MV4-11 and THP-1 cells. GAPDH was used as a loading control. (H) CYTH4 silencing sensitizes MV4-11 and THP-1 cells to Ara-C. The growth inhibitory effects of AML cells were evaluated through a CCK-8 assay following treatment with varying concentrations of Ara-C for a duration of 48 h. IC50 values were calculated utilizing GraphPad Prism 8.0 software based on the dose-response curve. Data are represent as mean ± standard deviation. Statistical significance was assessed by unpaired t test. ∗ p < 0.05, ∗ ∗p < 0.01, and ∗∗∗ p < 0.001. Sc, scramble control shRNA; shCYTH4, CYTH4 shRNA.

Article Snippet: Apoptosis was quantified utilizing the Annexin V-allophycocyanin (APC)/7-aminoactinomycin (7-AAD) or DAPI apoptosis detection kit (Elabscience, China) in accordance with the manufacturer’s instructions.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Control, Comparison, Western Blot, CCK-8 Assay, Staining, Flow Cytometry, Software, Standard Deviation, shRNA

Journal: iScience

Article Title: Cytohesin-4/ARF6 facilitates the progression of acute myeloid leukemia through activating PIK3R5/PI3K/AKT pathway

doi: 10.1016/j.isci.2025.112634

Figure Lengend Snippet: Overexpression of CYTH4 has no effect on AML cell proliferation (A) The expression of CYTH4 was validated to be upregulated in CYTH4 -transduced MV4-11 and THP-1 cells by RT-qPCR, with β-actin serving as the internal control ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (B) Immunoblotting confirmed the upregulated expression of CYTH4 in MV4-11 and THP-1 cells transduced with CYTH4, with GAPDH as the loading control. (C) CYTH4 overexpression did not influence the proliferation of MV4-11 and THP-1 cells. A CCK-8 assay was carried out to assess the viability of these cells at the indicated time points (0, 24, 48, and 72 h). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (D) The impact of CYTH4 overexpression on the colony formation of MV4-11 and THP-1 cells was evaluated by counting the number and size of colonies under a microscope at 14th day post-plating. The quantification is depicted on the right ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (E) Overexpression of CYTH4 in MV4-11 and THP-1 cells did not significantly impact cell apoptosis. Apoptotic cell percentages were measured using flow cytometry after annexin V-APC and 7-AAD staining. Representative plots from flow cytometry are on the left, and their quantifications are on the right ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. OE, overexpression.

Article Snippet: Apoptosis was quantified utilizing the Annexin V-allophycocyanin (APC)/7-aminoactinomycin (7-AAD) or DAPI apoptosis detection kit (Elabscience, China) in accordance with the manufacturer’s instructions.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control, Western Blot, Transduction, CCK-8 Assay, Microscopy, Flow Cytometry, Staining

Journal: iScience

Article Title: Cytohesin-4/ARF6 facilitates the progression of acute myeloid leukemia through activating PIK3R5/PI3K/AKT pathway

doi: 10.1016/j.isci.2025.112634

Figure Lengend Snippet: PIK3R5 overexpression effectively counteracts the AML cell growth defect resulting from CYTH4 silencing (A) PIK3R5 overexpression in CYTH4-silenced MV4-11 and THP1 cells were confirmed at the mRNA levels by RT-qPCR ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (B) PIK3R5 overexpression in CYTH4-silenced MV4-11 and THP1 cells were confirmed at the protein levels by immunoblotting. (C) CCK-8 assays showed that the impaired cell proliferations caused by CYTH4 silencing were reversed by PIK3R5 overexpression in MV4-11 and THP-1 cells ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (D) PIK3R5 overexpression could effectively reverse the decrease in clone formation caused by CYTH4 silencing ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (E) The apoptosis of cells in MV4-11 and THP1 lines transduced with shCYTH4 was examined by flow cytometry, and this could be alleviated by overexpressing PIK3R5 with a lentivirus packaging system ( n = 3). Apoptosis was determined with flow cytometry following annexin V-APC and DAPI staining ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (F) Immunoblotting showing that overexpression of PIK3R5 in CYTH4 -silenced MV4-11 and THP-1 cells counteracted the reduction of p-AKT, c-Myc, and Bcl-2. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Sc, scramble control shRNA; shCYTH4, CYTH4 shRNA; pCDH, pCDH-CMV-MCS-EF1-mCherry-P2A-Puro vector.

Article Snippet: Apoptosis was quantified utilizing the Annexin V-allophycocyanin (APC)/7-aminoactinomycin (7-AAD) or DAPI apoptosis detection kit (Elabscience, China) in accordance with the manufacturer’s instructions.

Techniques: Over Expression, Quantitative RT-PCR, Comparison, Western Blot, CCK-8 Assay, Transduction, Flow Cytometry, Staining, Control, shRNA, Plasmid Preparation

Journal: iScience

Article Title: Cytohesin-4/ARF6 facilitates the progression of acute myeloid leukemia through activating PIK3R5/PI3K/AKT pathway

doi: 10.1016/j.isci.2025.112634

Figure Lengend Snippet: Activation of AKT by SC-79 rescues AML cell growth defects induced by CYTH4 silencing (A) Immunoblot confirmation for restoration of AKT activity and the downregulated expression of c-Myc and BCL-2 induced by CYTH4 silencing following SC-79 treatment. (B) SC-79 treatment rescued the growth defect phenotype induced by CYTH4 silencing in MV4-11 and THP-1 cells. The cells were treated with 10 μM SC-79 for 0, 24, 48, 72, and 96 h. CCK-8 assay was performed to evaluate cell viability ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (C) The colony formation defect caused by CYTH4 silencing was rescued by SC-79 in MV4-11 and THP-1 cells ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (D) CYTH4 silencing-induced apoptosis were rescued by SC-79 in MV4-11 and THP-1 cells. Apoptosis was determined with flow cytometry following annexin V-APC and 7-AAD staining ( n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Sc: scramble control shRNA; shCYTH4, CYTH4 shRNA.

Article Snippet: Apoptosis was quantified utilizing the Annexin V-allophycocyanin (APC)/7-aminoactinomycin (7-AAD) or DAPI apoptosis detection kit (Elabscience, China) in accordance with the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Activity Assay, Expressing, CCK-8 Assay, Comparison, Flow Cytometry, Staining, Control, shRNA