Article Title: Secreted Neutrophil Gelatinase-Associated Lipocalin Shows Stronger Ability to Inhibit Cyst Enlargement of ADPKD Cells Compared with Nonsecreted Form
Figure Lengend Snippet: Comparison of NGAL expression between M-1 wild type and 2L3 ADPKD cells. ( A ) Structure of wild type and L3 alleles of Pkd1 gene. PCR genotyping was conducted using primers before and after the first loxP within the intron 30 of Pkd1. Filled triangles: loxP; an mc1 promoter-driven neomycin resistance gene (mc1-neo) flanked by two loxP sites was inserted into intron 34 of Pkd1. ( B ) PCR genotyping of Pkd1 transcripts of M-1 cells (473 bp) and 2L3 cells (507 bp). Pkd1 transcripts of the wild type (+/+), heterozygous (L3/+), and homozygous (L3/L3) mice were used as the control. RT–qPCR showed that 2L3 had significantly lower Pkd1 ( C ) M-1 and 2L3 cells show similar morphology. Scale bar: 100 μm. ( D ) M-1 and 2L3 cells were stained with Lotus Tetragonolobus Lectin (LTL, labeled with Fluorescein, green) and Dolichos biflorus agglutinin (DBA, labeled with Rhodamine, red) and DAPI (blue) and observed by a confocal microscope. Scale bar: 50 μm. RT–qPCR showed that 2L3 had significantly lower Pkd1 ( E ) and higher Lcn2 (NGAL) ( F ) expression levels than that of M-1 wild type cells. ( G ) No significant difference in Slc22a17 (NGAL-R) expression levels between M-1 and 2L3 cells. RQ: relative quantification = 2 −ΔΔCt ; Gapdh was used as the internal control of each sample. Values were normalized to M-1. ( H ) Western blot results showed that 2L3 cells had significantly higher expression of NGAL but not of NGAL-R than M-1 cells. Actin and GAPDH were used as the internal controls of each sample. Values were normalized to the control group of 2L3. ( I ) ELISA showed that 2L3 had higher expression of NGAL compared with M-1 in both cell lysate and supernatant. Student’s t -test was performed to determine the significance between the two groups ( n = 6 in RT–qPCR, n = 3 in western blot and ELISA). * p < 0.05; *** p < 0.001 vs. M-1 of each group; ns: no significant difference.
Article Snippet: Each sample was prepared in a total volume of 20 μL, including gene-specific forward (100 nM), reverse (100 nM) primers , cDNA template (100 ng), ORA TM SEE qPCR Green ROX H Mix (1×, highQu GmbH, Kraichtal, Germany) and PCR water.
Techniques: Expressing, Quantitative RT-PCR, Staining, Labeling, Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay