Fig. 3.

Deficiency of TMEM241 decreased lysosomal NPC2 and increased extracellular NPC2. A: The NPC1 and NPC2 proteins in the whole-cell lysate and culture medium of TMEM241 knockdown cells and control cells. WCL, whole-cell lysate. B: Relative NPC1 and NPC2 mRNA levels in control and TMEM241 knockdown cells. Data are normalized to control cells and represented as mean ± SD. C: The NPC1 and NPC2 proteins in the whole cell lysate and culture medium of HeLa/TMEM241 KO cells and wild type cells. WCL, whole-cell lysate. D: Relative NPC1 and NPC2 mRNA levels in HeLa/TMEM241 KO cells and WT cells. Data are normalized to control cells and represented as mean ± SD. E: HeLa/NPC2 KO cells, HeLa/TMEM241 KO cells, and WT cells were stained with endogenous anti-NPC2 (white) and anti-LAMP1 (green) antibodies. Scale bar, 10 μm. F: Quantification of the relative fluorescence intensity of NPC2 shown in (E). Data are normalized to control cells and represented as mean ± SD (n = 30). ∗∗∗P < 0.001, Student’s t test. G: HeLa/TMEM241 KO cells were transfected with the plasmid expressing TMEM241-3×FLAG (green), fixed and stained with anti-NPC2 antibodies (white). The cells expressing TMEM241-3×FLAG is outlined. Scale bar, 10 μm. H: Quantification of the relative fluorescence intensity of NPC2 shown in (G). Data are normalized to control cells and represented as mean ± SD (n = 20). ∗∗∗P < 0.001, Student’s t test. I: The NPC2 protein in the whole-cell lysate and culture medium of WT cells, HeLa/TMEM241 KO cells, and the TMEM241 KO cells expressing TMEM241-3×FLAG. J: Filipin staining of HeLa/TMEM241 KO cells transfected with the plasmid expressing NPC1 (green) or NPC2 (green). The cells expressing NPC1 or NPC2 are outlined. Scale bar, 10 μm. K: Quantification of the relative fluorescence intensity of filipin staining shown in (J). Data are normalized to control cells and represented as mean ± SD (n = 20). ∗∗∗P < 0.001, Student’s t test.