Fig 1: Genetic and chemical inhibition of ESCRT/lysosomal protein degradation pathway confirm its role as direct negative regulator of SLC20A1 protein levels.A, left, immunoblot of SLC20A1 protein levels in sgVPS37A-HEK293T and sgCHMP6-HEK293T cells with sgControl (top panel). Immunoblots of VPS37A (middle panel) and CHMP6 (bottom panel) were performed for assessment of gene targeting efficiency. ß-actin was used as loading control. Representative data of two experiments are shown. SLC20A1/ß-actin abundance was determined by densitometry and normalized to the control group (top). Right, SLC20A1 mRNA expression of sgVPS37A and sgCHMP6 cells with sgControl cells was normalized to 36B4. n = 4. Bars represent mean ± SD. B, SLC20A1 immunoblot in HEK293T cells treated with lysosome inhibitor bafilomycin A1 (BafA1) or proteasome inhibitor MG-132 versus DMSO control for 24 h (top panel). ß-actin was used as loading control (middle panel). SLC20A1/ß-actin abundance was determined by densitometry and normalized to the DMSO group (top). Efficiency of BafA1 and MG-132 treatment was assessed using ubiquitin immunoblot, which was exposed for optimized visualization of smaller proteins (bottom panel). C, HEK293T cells were transfected with CHMP6-EGFP expression plasmid for 24 h (green in overlayed image) to induce arrest of ESCRT-mediated protein trafficking. Cells were then stained with SLC20A1 antibody and Alexa 594–coupled secondary antibody (magenta in overlayed image) and subjected to confocal imaging. DAPI was used as nuclear marker. White arrows denote colocalization between SLC20A1 and CHMP6-EGFP. Presented data are representative images of three experiments. The scale bars represent 10 µm. D, phosphate uptake in sgVPS37A and sgCHMP6 HEK293T cells was measured with sgControl cells as control (n = 3). Bars represent mean ± SD. Presented results are representative of two independent experiments. Ct, average cycle threshold; DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EGFP, enhanced GFP; ESCRT, endosomal sorting complexes required for transport; HEK293T, human embryonic kidney 293T cell line; sg, single guide.