EMBO/soda-nel huggingface dataset:
Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo (2019)
Figure 1

Figure 1. LV interact with HF in a functional HFSC niche dependent manner

A. Adult mouse back skin sections immunostained for LYVE1 (red), αSMA (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. LV, lymphatic vessels; apm, arrector pili muscle.

B. Back skin sections of P55 mouse skin immunostained for LYVE1 (red), Tenascin-C (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. TNC, Tenascin-C.

C. Adult mouse back skin sections from different postnatal (P) days immunostained for LYVE1 (red), Lhx2 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.

D. Histogram of the quantification of the LV distance to HFSC positive for Lhx2. ). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Data represent the mean value ± SEM. ***p < 0.001. (Mann-Whitney U test).

E. Back skin sections of P49 mouse skin immunostained for LYVE1 (red), CD34 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. White arrowheads denote the proximity of lymphatic capillaries to HFSC.

F. Histogram of the quantification of the LV distance to HFSC positive for CD34. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. C, Catagen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen; A, Anagen. Data represent the mean value ± SEM. (Kruskal - Wallis test, Tukey's test).

G. Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.

H. Histogram of the quantification of the LV angle relative to the epidermis. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Data represent the mean value ± SEM. ***p < 0.001. (Mann-Whitney U test).
Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo (2019)
Figure 2

Figure 2. LV associate with HF during development

A-C. Distribution of LV and HF at the mouse embryo stage E15.5 (A), E16.5 (B) and E17.5 (C). Maximum projection images of whole mount immunofluorescence analyses using LYVE1 (red) as lymphatic endothelial marker and counterstained with DAPI (blue). Red arrowheads denote HF placodes. n= 3 - 4 embryos. Scale bar, 50 μm.
Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo (2019)
Figure 4

Figure 4. Dynamic reorganization of LV during the HF cycle

A. Adult back skin sections from different postnatal (P) days immunostained for LYVE1 (red) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 10 μm. White arrowheads denote LV membrane protrusion and fenestrated areas. LV, lymphatic vessels; HF, hair follicle.

B. Histogram of the caliber (μm) of LV at different postnatal days. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. A, Anagen; C, Catagen; T, Telogen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen. The data shown represent the mean value ± SEM. *p< 0.05; **p < 0.01; ***p < 0.001. (One way ANOVA, Tukey's test).

C. H&E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. epi, epidermis; der, dermis.

D - E. LYVE1 immunofluorescence (red) counterstained with DAPI (blue) (D) and Histogram of the caliber (μm) of LV (E) in back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. The data shown represent the mean value ± SEM. ****p < 0.0001. (Mann-Whitney U test).

F. Back skin sections of Controls and K14ΔNβ-cateninER+/T mice immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.

G. Histogram of the LV caliber in skin sections from Controls and K14ΔNβ-cateninER+/T mice. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Data represent the mean value ± SEM. ****p < 0.0001. (Mann-Whitney U test).

H. Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Data represent the mean value ± SEM. *p< 0.05; **p < 0.01. (Mann-Whitney U test).
Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo (2019)
Figure 5

Figure 5. Transcriptome analysis of LV in late Telogen compared to mid Telogen

A. Gene set enrichment plots (GSEA) of LV using public annotations showing the enrichment of selected gene signatures involved in different pathways in late Telogen (P69, Tl) compared with mid Telogen (P55, Tm). NES: normalized enrichment score. The false discovery rate (FDR; q-value) is indicated.

B. Table of differentially expressed genes (DEGs), in LV during late Telogen compared to mid Telogen, relative to biological processes, analyzed using the Enrichr platform (Chen et al., 2013; Kuleshov et al., 2016). Upregulated (red), downregulated (blue). Data shown represent the fold change of the genes.

C. Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen, relative to biological processes, analyzed using the Enrichr platform (Chen et al., 2013; Kuleshov et al., 2016). Data shown represent the log2 (FPKM+1).

D - G. Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 (D), DCN (E), PKD1 (F) and PLXND1 (G), assessed by the number of mRNA spots present in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= average of 30 LYVE1+ cells / sample, n= 3 mice. Data represent the mean value ± SEM. *p< 0.05; **p < 0.01. (D, F and G, unpaired Student's t-test; E, Mann-Whitney U test).

H - I. Histograms represent the mean fluorescence intensity for Jup (H) and Emilin1 (I) in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Data represent the mean value ± SEM. **p < 0.01, ***p < 0.001. (H, Mann-Whitney U test; I, unpaired Student's t-test).
Lymphatic vessels interact dynamically with the hair follicle stem cell niche during skin regeneration in vivo (2019)
Figure 6

Figure 6. Depletion of LV blocks both the pharmacological induction and the physiological HF growth

A - B. H&E staining (A) and histogram of the HF length (B) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal Diphtheria Toxin (CSA+DT) starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM. ****p < 0.0001. (One way ANOVA, Tukey's test).

C - D. Ki67 immunostaining (C) and histogram of the number of Ki67+ cells per HF (D) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle; and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 20 μm. Data represent the mean value ± SEM. ****p < 0.0001. (One way ANOVA, Tukey's test).

E - F. LYVE1 immunostaining (E) and histogram of the LV caliber (F) analyzed in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). Scale bar, 10 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Data represent the mean value ± SEM. ***p < 0.001. (Mann-Whitney U test).

G - H. H&E staining (G) and histogram of the HF length (H) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM. ****p < 0.0001. (unpaired Student's t-test).

I - J. Cleaved caspase 3 immunostaining (I) and histogram of the number of cleaved caspase 3+ cells per HF (J) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). Scale bar, 50 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Data represent the mean value ± SEM. ****p < 0.0001. (Mann-Whitney U test).

Figures - Expanded View
TREM2-deficiency reduces the efficacy of immunotherapeutic amyloid clearance (2016)
Figure 2

Figure 2. Increased Fcγ-receptors expression and enhanced Syk phosphorylation in TREM2-deficient BMDM

(A) Representative histograms for Fcγ-receptors-PE (FcγR-PE) expression levels as used for quantification. Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR (I, IIB/III and IV).

(B) Relative quantification of cell surface levels of FcγR molecules. Absolute number of cell surface FcγR-PE molecules was determined by the BD QuantiBRITE® method (see methods section for details) and normalized to expression levels of the respective wt control. (n=4, +/- SEM, t test, two-tailed; wt vs. ko: FcγRI-PE P=0.0008, FcγRIIB/III-PE P=0.0033, FcγRIV-PE P=0.7001).

(C) mRNA levels of FcγR are increased in Trem2 ko BMDM. Fold changes of the respective FcγR (I, IIB, III, IV) mRNA levels in Trem2 ko BMDM were determined by quantitative real-time PCR. (n=6, +/- SEM; one-sample t test, two-tailed; wt vs. ko: FcγRI P=0.0118, FcγRIIB P=0.0006, FcγRIII P=0.0004, FcγRIV P=0.0284)

(D) Phosphorylated Syk (P-Syk) and total Syk (T-Syk) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone, together with antibody 2D8 (Aβ-2D8), or an isotype control (Aβ-IC). Actin was used as a loading control.

(E) Quantification of P-Syk normalized to T-Syk. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0490, genotype P=0.0898, treatment P<0.0001. Bonferroni-corrected pair-wise post hoc tests, ** P =0.0075 vs. wt).
TREM2-deficiency reduces the efficacy of immunotherapeutic amyloid clearance (2016)
Figure 3

Figure 3. Engulfment of fibrillar Aβ by BMDM and primary microglia

(A) A schematic shows 10 µm cryosections of unfixed brain cryosections from 6-months old APP/PS1 mice, which show a high amyloid plaque burden (right panel with methoxy-X04 staining), were incubated with antibody for 1 h, followed by adding BMDM or primary microglia on top of the sections. After 24 h incubation, sections were analyzed by immunostaining or immunobloting.

(B) mAb11 but not the IC co-localized with methoxy-X04.

BMDM (C) or primary microglia (D) were cultured on cryosections pre-incubated with mAb11 (1 µg/ml). After 24 h, sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy-X04 staining to visualize Aβ. Note that both cell types internalize Aβ into intracellular vesicles (right panels show enlargement of insets). Scale bar: 10µm.
TREM2-deficiency reduces the efficacy of immunotherapeutic amyloid clearance (2016)
Figure 4

Figure 4. TREM2-deficiency reduces antibody-mediated amyloid plaque clearance

(A) BMDM from wt or Trem2 ko mice were cultured on APP/PS1 mice brain cryosections incubated with or without mAb11 (1 µg/ml) or an isotype control (IC; 1 µg/ml) for 24 h. Sections were then probed with methoxy-X04. Scale bar, 500 µm.

(B) The amyloid plaque load was quantified from the entire sagittal section. Sections incubated with medium (no cell) were set as baseline. (n = 6, +/- SEM; 2 way ANOVA, interaction P<0.0001, genotype P<0.0001, treatment P<0.0001; Tukey’s multiple comparisons tests; wt vs. ko for the following conditions: no antibody P=0.0304, IC P=0.0049, mAb11 P=0.0212; wt : IC vs. wt : mAb11 P=0.0008; ko : IC vs. ko : mAb11 P=0.0001).

(C, D) Equal numbers of wt and Trem2 ko BMDM were added and cell numbers were analyzed after termination of experiments by quantifying the CD68 positive cells on top of the sections. (n = 4, +/-SEM; t-test; n.s., nonsignificant, P=0.5004). Scale bar, 200 µm.

(E) Aβ was extracted by urea buffer from replicate slices of the experiment shown in (A) and total Aβ was identified by western blotting.
TREM2-deficiency reduces the efficacy of immunotherapeutic amyloid clearance (2016)
Figure 5

Figure 5. Compensation of reduced amyloid plaque clearance by elevated antibody dose

(A) Cryosections from unfixed brain of 6-months old APP/PS1 mice were pre-incubated with increasing concentrations of mAb11 (0.001, 0.01, 0.1, 1, 5 µg/ml). BMDM from wt or Trem2 ko mice were added for 24 h. Sections were stained with methoxy-X04. Scale bar, 500 µm.

(B) Methoxy-X04 signals were quantified from the entire sagittal section. (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.0082, genotype P<0.0001, treatment P<0.0001. Fisher’s LSD post hoc comparisons; * show statistics between wt and ko under the same experimental condition. # in black show wt compares to no antibody stimulation; # in grey show ko compares to no antibody stimulation; wt vs. ko for the following conditions: no antibody P=0.0053, mAb11 0.001 µg/ml P=0.0003, mAb11 0.01 µg/ml P<0.0001, mAb11 0.1 µg/ml P=0.0001, mAb11 1 µg/ml P=0.0007, mAb11 5 µg/ml P=0.0011; Following conditions compare to wt / no antibody: wt / mAb11 0.01 µg/ml P=0.0166, wt / mAb11 0.1 µg/ml P=0.0002, wt / mAb11 1 µg/ml P<0.0001, wt / mAb11 5 µg/ml P<0.0001; Following conditions compare to ko / no antibody: ko / mAb11 0.1 µg/ml P=0.0099, ko / mAb11 1 µg/ml P<0.0001, ko / mAb11 5 µg/ml P<0.0001.
Neuropilin-1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction (2020)
Figure 1

Figure 1. Neuropilin-1 expression is selectively suppressed in GnRH neurons in Gnrh::cre; Nrp1loxP/loxP mice in vivo.

(A) Genetic strategy to selectively knock out Nrp1 in GnRH-expressing cells, using Nrp1loxP/loxP mice crossed with Gnrh::cre mice. These mice were also crossed with Gnrh::gfp mice expressing green fluorescent protein under the control of the GnRH promoter in order to generate triple transgenic mice.

(B) Isolation of GFP-positive cells by FACS: schematic diagram showing the selection of the GFP-positive population (green).

(C) Relative mRNA expression from real-time PCR analysis of the GnRH transcript in GFP-positive cells, in comparison with GFP-negative cells. The GnRH transcript appears to be selectively expressed in GFP-positive cells. Mann-Whitney U test, n=4 mice from 2 litters.

(D) Relative mRNA expression from real-time PCR analysis of Neuropilin-1 (Nrp1), Nrp2, Plexin A1 (PlxnA1), and PlxnA4 transcripts in FACS-isolated GFP-positive GnRH neurons from control (Nrp1loxP/loxP; grey) and mutant (Gnrh::cre; Nrp1loxP/loxP; red) P0 mice. Unpaired t-test, n=5 to 6 mice per group from 3 litters.

(E) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose-brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50μm.

Data information: Bar graphs show individual values and means ± SEM. ** P<0.01, * P<0.05.
Neuropilin-1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction (2020)
Figure 2

Figure 2. Mice lacking Neuropilin-1 expression in GnRH neurons show an increased number of migrating GnRH cells and abnormal GnRH neuronal migration in the accessory olfactory bulb.

(A) Quantification of total number of GnRH neurons during embryogenesis, at E12.5, E14.5 and E18.5. More GnRH neurons were found in E14.5 and E18.5 mutant embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 to 6 mouse embryos from at least 3 litters.

(B) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14.5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose, the olfactory bulb (OB) and the ventral forebrain (VFB) in control and mutant embryos. Scale bar: 50μm. VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulbs; VFB, ventral forebrain.

(C) Quantitative analysis showing distribution of GnRH neurons in the nose, OB and VFB of mutant and control embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=5 and 6 mouse embryos for mutants and controls, respectively, from at least 3 litters.

(D) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs, in a control (left panel) and a mutant (right panel) postnatal brain. Knock-out animals show an accumulation of GnRH neurons in the accessory olfactory bulb (circle formed by the GnRH labelling, right panel). AOB, accessory olfactory bulb. Scale bar: 300μm.

(E) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis (OVLT) in the median preoptic area, from a control (left) and a mutant (right) female postnatal brain. Scale bar: 500μm. Oc, optic chiasma.

(F) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates. OB, olfactory bulb; DBB, diagonal band of Broca; 3V, periventricular region of the preoptic area; Tot, total. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 adult mice from 3 litters.

Data information: Bar graphs show individual values and means ± SEM. * P<0.05, *** P<0.001
Neuropilin-1 expression in GnRH neurons regulates prepubertal weight gain and sexual attraction (2020)
Figure 3

Figure 3. Neuropilin-1 signaling in GnRH neurons controls their survival and migration.

(A) Schematic representation of the transwell assay used to assess the effect of Sema3A, which was placed in the lower chamber, on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst (grey) in the lower part of the transwell membrane after overnight incubation in SFM (left panel), SFM with 100ng/ml Sema3A (middle panel) and SFM with 100ng/ml Sema3A + 1μg Nrp1Ab (right panel). Scale bar: 100μm.

(B) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber (SFM) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber. Kruskal-Wallis test, n= 5 to 6 wells in two independent experiments.

(C) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11.5 mouse embryos after 8 days in vitro. NE, nasal epithelium; NMC, nasal midline cartilage. Scale bar 500μm (150μm in inset).

(D) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos, quantified after 8 days in culture. Mann-Whitney U test, n=3 to 5 embryos from at least 3 litters.

(E) Distance travelled by GnRH neurons from the olfactory epithelia; Cells were quantified within a radius of 1 to 6 mm. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 to 5 explants.

(F) Representative immunofluorescence for GnRH- (red) and BrdU- (green) positive cells in control and mutant E14.5 embryos arising from progenitors born between E9.5 and E10.5. BrdU-positive GnRH neurons (white arrowheads) were more abundant in mutant than in control embryos. Scale bar: 20μm.

(G) Number of BrdU-positive GnRH neurons in the nose, olfactory bulb (OB) and ventral forebrain (VFB) at E14.5 and total number of cells in control and mutant embryos. Double-labeled cells were more numerous in the mutant VFB. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 embryos from 2 litters.

(H) Schematic diagram of intra-embryonic nasal injection in wild-type E11.5 embryos and collection of embryos at E14.5.

(I) Number of GnRH neurons in E14.5 embryos injected with neutralizing antibodies to Neuropilin-1 (Nrp1Ab) compared to an isotype goat antibody, or the antiapoptotic compound ZVAD compared to its vehicle, together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age. Note that the data are represented as fold change, as raw values are not comparable with transgenic animals that did not undergo surgery. Unpaired t-test, n=5 to 7 embryos from at least 2 litters.

Data information: Bar graphs show individual values and means ± SEM. * P<0.05, ** P<0.01, *** P<0.001.
TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1 and Batten disease (2022)
Figure 1

Figure 1. TPC2 agonist effect on lactosylceramide trafficking. (A) Colocalization of LacCer and LysoTracker (LyTr) in different CTR and LSD patient fibroblasts. Mander´s coefficients were calculated using the Fiji JACoP plugin. (B-C) Confocal images (B) and statistical analysis (C) showing colocalization of LacCer and LyTr in human CTR and MLIV fibroblasts, treated with TPC2-A1-P (30 µM, 16h). (D-E) Confocal images (D) and statistical analysis (E) showing colocalization of LacCer and LyTr in human CTR and NPC1 fibroblasts, treated with TPC2-A1-P (30 µM, 48h). (F-G) Confocal images and statistical analysis showing LacCer/LyTr colocalization in MLIV patient fibroblasts which were mock-electroporated and treated with DMSO or electroporated with a gain-of-function hTPC2(M484L/G734E):mCherry TOPO 3.1 vector and treated with either DMSO or TPC2-A1-P (30 µM, 16h). (H-I) Ca2+ chelation (BAPTA-AM) dose-dependently impairs LacCer trafficking in CTR fibroblasts. (J-K) Confocal images (J) and statistical analysis (K) of NPC1 patient fibroblasts treated with 50 nM mock siRNA (siSCR) or siRNA targeting TPCN2 (siTPC2) for 72 hours. Cells were then treated with DMSO or TPC2-A1-P (30 µM). Statistics: Shown are mean values ± SEM. n > 3 technical and biological replicates for each tested condition (each dot represents an imaged frame containing several cells); one-way ANOVA, post hoc Bonferroni's (A, C, E, G, I) or Tukey's (K) multiple comparisons test. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001.