Fig 2: hSLC39A8 is a Mn-specific transporter and its expression is regulated by Mn. (A) Concentration-dependent Mn uptake (pmol/min/mg protein) by HeLa cells transfected to express hSLC39A8 (closed circles) or empty vector (open circles). Data represent means ± SEM (n = 2 samples/group). #P < 0.05 vs. control; Student’s t-test. (B) Competition assays with HeLa cells transfected to express hSLC39A8. 54Mn uptake was measured in the presence of 10- or 50-fold molar excess of the metals indicated. Ctrl refers to control with no metals added. (C) hSLC39A8 expression under various Mn concentrations, with or without the Mn chelator PAS, and (D) under increasing Mn concentrations for 16 h. Mean relative transcript levels are detected by qPCR. Data represent means ± SEM (n = 3 samples/group). #P < 0.05 vs. control: Student’s t-test. (E) Representative immunoblot of GPP130 in total cell lysates isolated from empty vector- (control), or hSLC39A8-WT expressing cells incubated with 500 µM MnCl2 for 4 h or 100 µM for 16 h. Equal loading was verified by immunoblotting with actin antibody. Full-length blots are presented in Supplementary Figure 3. (F) Quantification of GPP130 relative protein after normalization with actin. Results are means ± SEM (n = 3 samples/group) of three independent experiments. #P < 0.05 vs. control at the same concentration, *P < 0.05 vs. control in the different groups; Student’s t-test.

Fig 3: Disease-associated hSLC39A8 mutants reduce 54Mn uptake and intracellular Mn levels. (A) HeLa cells were transfected with hSLC39A8-WT or hSLC39A8 mutants for 48 h and then incubated at 37 °C for 30 min in the presence of 1 µM of 54Mn. Cells were chilled on ice and washed with ice-cold PBS containing 1 mM EDTA, and cell-associated radioactivity was determined by gamma counting and normalized to control vector-expressing cells. Data represent means ± SEM (n = 3 samples/group). (B) HeLa cells were transfected with hSLC39A8-WT or hSLC39A8 mutants for 48 h. Intracellular metal levels, including Mn, were measured by ICP-MS. Data represent means ± SEM (n = 3 samples/group). (C) Representative immunoblot of GPP130 in total cell lysates isolated from hSLC39A8-WT expressing cells and its mutants incubated with MnCl2 500 µM for 4 h. Equal loading was verified by immunoblotting with actin antibody. Full-length blots are presented in Supplementary Figure 4. (D) Quantification of GPP130 relative protein after normalization with actin. (E) Representative immunoblot of hSLC39A8-WT-HA and its mutants in total cell lysates isolated from cells transfected with empty vector (control), hSLC39A8-WT-HA, or its mutant (hSLC39A8-M1-HA, -M2-HA, -M3-HA, and -M4-HA). GFP is included as a control for transfection efficiency. Equal loading was verified by immunoblotting with actin antibody and the positions of molecular weight markers are shown. Full-length blots are presented in Supplementary Figure 5. (F) Quantification of hSLC39A8-HA relative protein after normalization with actin. Results are means ± SEM of three independent experiments. (G) The endogenous expression of SLC39A8 mRNA in cells expressing SLC39A8-WT or its mutants. Mean relative transcript levels are detected by qPCR. Data represent means ± SEM (n = 3 samples/group). #P < 0.05 between control vector vs. hSLC39A8-WT or *P < 0.05 between hSLC39A8-WT vs. its mutants; Student’s t-test.

Fig 4: Gphr deficiency influences glycosyltransferase trafficking dynamics.(A) Confocal microscopy image. MEFs stably expressing GFP fusion glycosyltransferases B4galt1 or St6gal1 were fixed and stained with the Golgi marker GPP130. Cell nuclei were stained with DAPI. Scale bar: 10 μm. (A, B) Bar graph indicating the quantitative GFP/GPP130 co-localization ratio shown in (A). Data are means ± SD of individual values (n = 6 images, <40 cells examined). P-values were corrected using Welch’s t test. Actual P-values for each condition are indicated. (C) Time-lapse microscopy analysis. MEFs stably expressing either B4galt1-GFP or St6gal1-GFP were photobleached. The recovery of fluorescence was observed using live-cell imaging. Scale bar: 2 μm. (D) The ratio of fluorescence of the bleached area to that of an adjacent unbleached area was measured for each time point, normalized to the initial values, and plotted. Data are presented as mean ± SD of non-photobleached (n = 10) and photobleached (n = 10) samples. Significant differences (**P < 0.01) between the MEFs are shown. P-values were corrected using Welch’s t test. See also Video 1, Video 2, Video 3, and Video 4.