Fig 2: Immunofluorescence showing the distribution of CA XIII, LAMP-1 and LAMP-2 in developing teeth.Images of sections of incisors (A–F’; H, H’) and molars (G, G’, I, I’) at 1 dpp. Incisor sections after single immunostaining showing CA XIII (green fluorescence (GF) in A), LAMP-1 (red fluorescence (RF) in B) and LAMP-2 (RF in C). Incisor sections after double immunostaining showing CA XIII (GF in D) and LAMP-1 (RF in D’) or CA XIII (GF in E) and LAMP-2 (RF in E’). Incisor and molar sections after triple immunostaining showing CA XIII (GF in F and G) and LAMP-1+ LAMP-2 (RF in F’ and G’). Negative controls processed without the primary antibodies (H, H’, I, I’). The GF and RF spots are artifacts due to autofluorescence of red blood cells, somes areas in preameloblasts (PA) as well as areas with tissue folds. G”, G’’’, I” and I”’ are high magnifications views of areas in G, G’, I and I’, respectively. Abbreviations: Am, differentiating ameloblasts; DM, dental papilla mesenchyme. Scale bars: 200 µm (G–G’, I, I’), 100 µm (A–F’, H, H’, G”, G”’, I”, I”’).

Fig 3: Effect of ML2-SA1 on chemokine release from primary mouse macrophages.(A) Shown are data obtained from primary WT and TRPML2-/- mouse bone marrow macrophages (BMDMF) with and without LPS treatment for 4 hr and 8 hr, respectively. The fraction treated with LPS and 10 µM ML2-SA1 showed significantly increased CCL2 secretion compared to WT controls treated with LPS only. TRPML2-/- cells displayed strongly reduced CCL2 secretion. Shown are normalized mean values ± SEM of 5 mice each. * indicates p<0.05, Student’s t test, unpaired. (B) Lysosomal exocytosis assay showing the increase in beta-hexosaminidase release upon stimulation with either ionomycin, ML-SA1, or ML2-SA1 (conc. as indicated) from LPS (6 hr) stimulated BMDMF. *** indicates p<0.001, one-way ANOVA test followed by Tukey’s post-hoc test. (C) Lysosomal exocytosis assay by flow cytometry showing the percentage of cells which show an increase in LAMP1 fluorescence on the plasma membrane. Cells were treated with DMSO, calcium ionophore A23187 (calcimycin), and 30 µM ML2-SA1. (D–E) Recycling endosome assay showing the decrease of Tf mean fluorescence in LPS stimulated RAW264.7 cells, treated with either DMSO or 30 µM ML2-SA1. Scale bar (identical for all images)=10 µm. Plot shows the normalized Tf intensity (shown is the average of 3 independent experiments, each). **p<0.01, two-way ANOVA, repeated measures, followed by Bonferroni post-hoc test. (F) Cartoon showing organelles with functional TRPML2 expression as confirmed by endolysosomal patch-clamp analysis (EE, RE, LE/LY). CCL2 (MCP-1) is hypothesized to be trafficked and secreted via the EE/RE pathway, based on the observation that ML2-SA1 promotes Tf trafficking in the EE/RE compartment, while no effect on lysosomal exocytosis was found. No secretory vesicles are reported to exist in macrophages.

Fig 4: GFP-labeled mitochondria in injured hypoglossal motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice Whole-mount observation of hypoglossal nerve of Atf3:BAC2 Tg mouse without staining.Immunostaining of hypoglossal nerve at 5 and 10 days after injury, using anti-GFP, Lamp1, and neurofilament (NF) antibodies. Arrows show Lamp1 positive signals in injured nerve.Graph shows mitochondria number in hypoglossal (XII) axon at 5 days and 10 days after injury (n = 4 mice).Graph shows lysosome number in hypoglossal (XII) axon at 5 days and 10 days after injury (n = 4 mice).The GFP expression of injured hypoglossal motor neurons at 5 days following injury. XII, hypoglossal nucleus; cc, central canal.The percentage of GFP-positive injured motor neurons compared with ChAT-positive uninjured motor neurons (n = 3 mice per group).Super-resolution images of GFP-stained mitochondria in injured hypoglossal motor neuron. Stacked image of 2 µm thickness.Single layer image corresponding to boxed area in (G) and plot profile of the fluorescence intensity corresponding to the line in the image. Data information: Dashed lines outline axon in (A) and hypoglossal nucleus in (E). Data are shown as the mean ± s.e.m. *P = 0.0247, **P = 0.0011 in (C), ***P < 0.0001 in (D), P = 0.7854 in (F), determined by one-way ANOVA followed by Tukey post hoc test. Scale bars, 300 µm in (A; left two panels) and 10 µm in (A; right panel), 5 µm in (B), 150 µm in (E), 4 µm in (G), and 2 µm in (H). Source data are available online for this figure.

Fig 5: Loss of function of Igf2r (Igf2rI1565A/+p) shortens survival and promotes intestinal adenoma proliferation in conditional Apcloxp/loxp. The effects of Igf2rI1565A/+p was determined following conditional generation of intestinal adenoma. Igf2rI1565A/+p was first combined with homozygote Apcloxp/loxp, and an inducible intestinal Lgr5-Cre (Lgr5CreERT2). Tamoxifen injection induced Cre recombinase (injection at 6–8 weeks of age) in these mice, with survival and intestinal adenoma formation quantified. (a) Significant shortening of survival was observed (upto day 65) for Igf2rI1565A/+p compared to Igf2r+m/+p (Kaplan-Meier plot, long-rank test p = 0.0468). (b) Intestinal adenoma number in the distal small intestine was significantly lower in number when comparing Igf2rI1565A/+p to Igf2r+m/+p (*Distal p = 0.0149), whereas in the proximal small intestine they were signgicantly greater (*Proximal p = 0.0469). The differences in colonic adenoma between genotypes were not significant. All comparisons One-way ANOVA with Fishers LSD. (Note colonic adnoma differences were borderline with an unpaired one-tailed t-test, p = 0.0423). (c) Ki-67 proliferation labelling was significantly higher (p = 0.0302) in adenoma from Igf2rI1565A/+p compared to Igf2r+m/+p mice (Mann Whitney, two-tailed test). (d) Adenoma diameter were not significantly different between Igf2rI1565A/+p compared to Igf2r+m/+p. (Two-way ANOVA). Percentage comparisons relative to total adenoma number per mouse. (e) No significant differences in the immuno-localisation of IGF2R protein were observed between Igf2rI1565A/+p and Igf2r+m/+p adenoma, in relation to nuclei (DAPI) and LAMP1 (endosomal compartment). Scale bar 100 µm.