Fitc annexin 5 apoptosis detection kit 2 with propidium iodide av pi

Cell proliferation ability was measured at 0, 24, 48 and 72 h after transfection using Counting Kit-8 (CCK-8) (Sangon Biotech, China) according to the instruction of manufacturer. Cell apoptosis rate was measured at 48 h by FITC Annexin V Apoptosis Detection Kit II with propidium iodide (AV/PI) (BD, USA) according to the instruction of manufacturer. Apoptotic protein Cleaved Caspase 3 (C-Caspase 3) and anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) were measured at 48 h using Western Blot. Cell migration rate was calculated by dividing migration distance across the gap at 24 h by that at 0 h under microscope (Olympus, Japan) using wound healing assay referring to a previous report (12 (link)). In addition, invasive cell count was calculated using Matrigel invasion assay with Matrigel basement membrane matrix (BD, USA) and 8-um Polyvinylidene Fluoride contained transwell filter chamber (Coring, USA) referring to a previous report (12 (link)).

Negative control (NC) shRNA and lncRNA Dlx6os1 shRNA plasmids were constructed by Genewiz, Inc. and transfected into a DN cellular model (SV40 MES13 cells under 30 mmol/l glucose culture), then termed the sh-NC group and sh-lncRNA group. Subsequently, i) lncRNA Dlx6os1 expression in the two groups was detected by reverse transcription-quantitative (RT-q) PCR at 48 h; ii) proliferation ability was detected by Cell Counting Kit-8 (Sigma-Aldrich; Merck KGaA) at 0, 24, 48 and 72 h according to the manufacturers' instructions; iii) the mRNA and protein expression levels of proliferative markers (cyclin D1 and proliferating cell nuclear antigen) as well as fibrosis markers (fibronectin and collagen I) were detected by RT-qPCR and western blotting at 48 h; iv) the cell apoptosis rate was detected by FITC Annexin V Apoptosis Detection kit II with propidium iodide (AV/PI; BD Biosciences) at 48 h according to the manufacturer's protocol (Fig. S1). Data were analyzed using a flow cytometer (BD FACSCalibur) with FlowJo 7.6 software (FlowJo, LLC). A wavelength of 488/535 nm was used.