cck8 detection reagent (cell counting kit-8  (Millipore)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Alpha‐Fetoprotein Stimulates Cleavage of Membranal MICA /B on Liver Cancer Cell Lead to Escape Immune Surveillance of Natural Killer Cells

doi: 10.1111/jcmm.71076

Figure Lengend Snippet: The effect of AFP on the expression of MMP9 and MICA/B shedding on the membrane of HCC cells. (A) Western blotting experiment was performed to detect the expression levels of MMP9; (B) Grey scale values of MMP9 expression were calculated using Image J software and then statistically analysed and plotted; (C) qRT‐PCR experiment was used to check MMP9 mRNA expression levels; (D) Western blotting was performed to detect the expression levels of sMICA/B in HLE, HLE‐NC, HLE‐AFP cells; (E) Western blotting was performed to detect the expression levels of sMICA/B in HuH‐7, HuH‐7‐NC, HuH‐7‐shAFP cells; (F) ELISA were performed to detect the content of sMICA/B in HCC cell supernatants; (G) CCK‐8 experiments were performed to detect the IC50 of GM6001 and TAPI‐1; (H) Detection of MICA/B expression in HCC cells while treated with GM6001 and TAPI‐1 by flow cytometry; (I) Prism GraphPad(10.0) for statistical analysis and graphing of flow cytometry results; (J) Detection of sMICA/B in HCC cells supernatants while treated with GM6001 and TAPI‐1 by ELISA; (K) Western blotting experiment was performed to detect the expression levels of PI3K, AKT, p‐AKT and MMP9; (L) Grey scale values of protein expression were calculated using Image J software and then statistically analysed and plotted. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001. The picture represents at least three repetitions of the experiment. AKT, protein kinase B; GM6001 and TAPI‐1, MMP9 inhibitors; MMP9, matrix metallopeptidase 9; p‐AKT, phospho‐protein kinase B; PI3K, phosphatidylinositol‐3‐kinase; sMICA/B, soluble major histocompatibility complex class I chain‐related proteins A and B.

Article Snippet: The cells were cultured in an incubator for 24 h, after which the medium was discarded, fresh serum‐free medium (100 μL per well) was added, and CCK8 detection reagent (Cat #CK04, DOJINDO, Japan) was added (10 μL per well).

Techniques: Expressing, Membrane, Western Blot, Software, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Flow Cytometry, Immunopeptidomics

Journal: Oxidative medicine and cellular longevity

Article Title: BOP1 Knockdown Attenuates Neointimal Hyperplasia by Activating p53 and Inhibiting Nascent Protein Synthesis.

doi: 10.1155/2021/5986260

Figure Lengend Snippet: Figure 4: p53 activation mediated the biological effects of LV-BOP1 on HVSMCs. (a, b) mRNA and protein expressions of BOP1 after stimulation with different doses of PDGF-BB (n = 3). (c, d) mRNA and protein expressions of BOP1 after stimulation of 20 ng/ml PDGF- BB at different times (n = 3). (e) BOP1 expression in HVSMCs after transfection with LV-BOP1 or LV-SCR detected by western blot (n = 3). (f) CCK-8 assay detecting the HVSMC growth rates of BOP1 knockdown and cotreated with PFT-α (10 μM) or not (n = 3, ∗P < 0:05, the LV-BOP1 group compared with the LV-SCR group) (n = 3, &P < 0:05, the LV-BOP1 group compared with the LV-BOP1+PFT-α group). (g–j) HVSMCs transfected with LV-SCR or LV-BOP1 followed by pretreatment with 10 μM PFT-α or equal DMSO for 12 h. HVSMCs were resuspended and cotreated with 20 ng/ml PDGF-BB and 10 μM PFT-α or equal DMSO for the scratch test. Phase-contrast microscopy images showed the migratory capacity of VSMCs under different treatment. Transwell assay was performed to assess HVSMC migration. Diagram showed quantification of cell migration rate over time. (n = 3),∗P < 0:05. (k, l) The Ki-67 (red) immunofluorescence staining revealed that the BOP1 knockdown markedly restrained the HVSMC proliferation, while PFT-α (10 μM) partly reversed the inhibit effect induced by the BOP1 knockdown, (n = 3). (m, n) HVSMCs transfected with LV-BOP1 or LV-SCR, then pretreated with 10 μM PFT-α or equal DMSO for 12 h. The proportion of cells in each phase of the cell cycle was determined by flow cytometry (n= 3). (o, p) Apoptosis was assessed by Annexin V-PE/7-AAD staining and flow cytometry. (q, r) ROS were detected by 2′,7′ -dichlorodihydrofluorescein diacetate (DCFH-DA) and flow cytometry (n = 3). Data are represented by mean ± SD. ∗P < 0:05; one-way ANOVA followed by Tukey’s post hoc test for (a–f, h, j, l, n, p, r) and Student’s t-test for (e).

Article Snippet: After continuing to treat with the same concentration of PFT-α or DMSO for the corresponding time, 10μl of Cell Counting Kit-8 (CCK8) detection reagent (Beyotime Bio, #C0039, China) was added to each well and the optical density at 450nm of each well was measured by microplate reader (Perkin Elmer, USA).

Techniques: Activation Assay, Expressing, Transfection, Western Blot, CCK-8 Assay, Knockdown, Microscopy, Transwell Assay, Migration, Staining, Cytometry