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. 2019 Apr 12;11(4):525. doi: 10.3390/cancers11040525

Figure 1.

Figure 1

Transient receptor potential mucolipin (TRPML)-1 expression in glioblastoma (GBM) cell lines. (a) The relative TRPML-1 mRNA expression in normal human astrocytes (NHA), normal human brain (NHB), T98, and U251 glioma cell lines, and in peripheral blood mononuclear cells (PBMCs) used as positive control were evaluated by qRT-PCR. TRPML-1 mRNA levels were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Data are expressed as mean ± SD. * p < 0.05 vs. NHA; # p < 0.05 vs. NHB, PBMCs. (b) Flow cytometric analysis was performed in GBM cells, fixed, permeabilized, and stained with anti-human TRPML-1 Ab followed by phycoerythrin (PE)-conjugated secondary Ab. Isotype control Ab was used as negative control. Numbers represent the percentage of TRPML-1 positive cells. (c) Total lysates were separated on 8% SDS-PAGE and probed with anti-TRPML-1 and anti-GAPDH Abs. Blots are representative of one of three separate experiments. Numbers represent the densitometric analysis as compared with GAPDH. (d) Immunocytochemical stains for TRPML-1 in untransfected (B,F), siTRPML-1 (C,G), and pCMV-pTRPML-1 (D,H) glioma cell lines. Scale bar: 10 μm. (e) Immunocytochemical stain for TRPML-1 in PBMC (A,B). Scale bar: 10 μm. Cells were formaldehyde-fixed, permeabilized, probed with anti-human TRPML-1 Ab, and biotinylated anti-mouse IgG1, ABC reagent, and substrate solution containing DAB. Nuclei were stained with hematoxylin. Representative images are shown. The incubation with the secondary antibody alone was used as negative control (dA, dE, eA). Scale bar: 10 μm.