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. 2017 Nov 27;29(3):869–879. doi: 10.1681/ASN.2016121322

Figure 7.

Figure 7.

APOL1-G0, -G1, and -G2 proteins, induced with low doses of tetracycline, do not localize to plasma membrane, cause cell death, or stimulate p38 MAPK phosphorylation. (A) APOL1-G0, -G1, or -G2 clones were cultured without tetracycline (0); with low (L) concentrations of tetracycline adjusted to induce equivalent amounts of G0, G1, and G2 protein (G0, 5 ng/ml; G1, 5 ng/ml; G2, 10 ng/ml); or with high (H) tetracycline concentrations (1 μg/ml, all genotypes). Cell surface proteins were biotinylated and cells were collected in lysis buffer. APOL1 was detectable in the input lysates of tetracycline-stimulated but not untreated cells (left panel). Biotinylated APOL1 was only identified in membranes from cells treated with high concentrations of tetracycline (right panel, labeled IP). (B) Fluorogenic cytotoxicity/viability assay demonstrates dose-dependent cytotoxicity with increasing tetracycline doses across APOL1 genotypes. An increased cytotoxicity/viability ratio was observed in APOL1-G0 and APOL1-G1 clones at the “1×” tetracycline dose compared with the no tetracycline control, despite using doses chosen to provide matched APOL1 expression. Cells were treated for 24 hours with no tetracycline (0), or increasing doses of tetracycline (1×–5×); these doses correspond to 5, 10, 15, 20, and 25 ng/ml for APOL1-G0 and -G1; and, 10, 20, 30, 40, and 50 ng/ml for APOL1-G2, respectively. (C) MTT assays also show no significant cytotoxicity after treatment with low concentrations of tetracycline. Bars represent mean±SD (n=2). (D) Immunoblots of APOL1, phosphorylated p38 MAPK, total p38 MAPK, and GAPDH from one of three experiments yielding similar results. Lanes 1–3, lysates from 293 cells used to generate the stable cell lines (293 No. Tet); lanes 4–6, lysates from G0 (0), G1 (1), and G2 (2) cells treated for 24 hours with low concentrations of tetracycline, as above; lanes 7–9, lysates from G0 (0), G1 (1), and G2 (2) cells treated for 8 hours with tetracycline, 1 μg/ml. IB, immunoblot; IP, immunoprecipitate; M.W., molecular weight; Phospho-p38, phosphorylated-p38 mitogen-activated protein kinase; Tet, tetracycline.