Fig. 5: Early endosomal trafficking in WT and Trpml3^−/− AMΦ.

a, b Transferrin (Tf) trafficking assay showing the decrease of Tf fluorescence in AMΦ (WT and Trpml3^−/−) within 20 min after the pulse with Tf-AlexaFluor488 (Tf accumulation). Mean ± SEM, 4 biologically independent experiments, each. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Two-way ANOVA followed by Bonferroni’s post hoc test. c Tf fluorescence in AMΦ (WT and Trpml3^−/−) after 20 min pulse with Tf-AlexaFluor488 (0 min timepoint, measures Tf uptake). Mean ± SEM, four biologically independent experiments. *p < 0.05, **p < 0.01, Student’s t test, unpaired, two-tailed. d Representative confocal images and quantification of the colocalization of EEA1 and Tf in AMΦ (WT and Trpml3^−/−). Statistical analysis was performed using Student’s t test, unpaired, two-tailed. Mean ± SEM, three biologically independent experiments. *p < 0.05, ***p < 0.001. e, f TfR expression analysis using Western blot. e Shown are two independent WB blots for TfR (90 kDa) and ß-Actin (45 kDa; loading control) using 5 WT and 5 Trpml3^−/− AMΦ lysates on each blot. f Quantification of WB data as shown in e. TfR protein was normalized to ß-Actin and values from Trpml3^−/− AMΦ were normalized to WT AMΦ. One single dot corresponds to one mouse, each (mean ± SEM). g, h Whole-cell patch-clamp experiments to determine membrane capacitance (measure of cell surface area). GTPγS induces an increase in surface area. Co-application of ML3-SA1 significantly reduces the effect of GTPγS in WT AMΦ, but not in Trpml3^−/− AMΦ ( = loss of membrane surface). Significance: GTPγS vs. GTPγS + ML3-SA1 from 140 to 150 sec *, from 152 to 156 sec **, from 158 to 172 sec ***, from 174 to 194 **, then till 200 sec *** (yellow dots). *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA followed by Tukey’s multiple comparisons test. Shown are mean values ± SEM, n (in parentheses) = biologically independent experiments. i, j Bar diagrams (mean ± SEM) showing the parameters Tau (= time until 2/3 of the maximum amplitude is reached) and Delay (= time until capacitance changes). *p < 0.05, Student’s t test, unpaired, two-tailed. Source data are provided as a Source Data file.