Fig 2: Ceramide induces lysosome biogenesis. (A) Representative TEM images and corresponding number of total lysosomes in primary isolated trophoblast cells treated with 20 µM CER 16:0 or EtOH vehicle (N = 3 experiments with freshly isolated cells; *P < 0.05). L: lysosomes. (B) LysoTracker® Red IF of JEG3 cells cultured in the presence or absence of 20 µM CER 16:0 or 25 µM 2-OE and corresponding fold change in fluorescence intensity (N = 3 separate experiments; *P < 0.05, **P < 0.01 compared to vehicle control). Arrow: LysoTracker® Red reactivity. (C) Representative WB for TFEB and LAMP1 in JEG3 cells treated with 20 µM CER 16:0 or vehicle. ACTB used as loading control. (D) IF analysis of TFEB (green) in JEG3 cells following exposure to 20 µM CER 16:0 or EtOH vehicle. Nuclei were visualized with DAPI (blue). Data are expressed as mean ± SEM. (E) Representative flow cytometry density and volume plots of Lysotracker® Red stained JEG3 cells exposed for 6 h to either EtOH vehicle, 100 nM Bafilomycin (Baf A1) or 20 mM CER 16:0. Unstained cells were used for gating. Note that DAPI staining, used to mark viability of live cells, indicate no significant shifts between either unstained cells or cells exposed to vehicle EtOH, CER16:0 or Baf A1.

Fig 3: Lysosome biogenesis is increased in E-PE placentae. (A) Representative WB and corresponding densitometry of TFEB and LAMP-1 in E-PE, PTC, and TC placentae normalized to total protein in stain free gel (SFG) (TFEB: E-PE N = 18, PTC N = 16, TC N = 6, ***P < 0.001 E-PE compared to PTC and TC); LAMP-1: E-PE N = 19, PTC N = 12, TC N = 14, ***P < 0.001 E-PE compared to PTC and TC). (B, left panel) WB for TFEB and Lamin A in nuclear (Nu) and cytoplasm (Cy) enriched fractions; (B, right panel) WB for LAMP1 in lysosomal lysates from PTC and E-PE placentae. (C) WB of Beclin-1 (BECN1) in PTC and E-PE placentae (PE, N = 7; PTC, N = 7). (D) Representative WB for ATG9b and corresponding densitometry in lysates of syncytial cells isolated from term and PE placentae. PLAP was used as a syncytial marker and loading control. (PE, N = 3; TC, N = 3; *P < 0.05 compared to TC). Dotted line: lanes were run on the same gel but were non-contiguous. Data are expressed as mean ± SEM.

Fig 4: Multiple commercially available TFEB antibodies fail to show specific staining as demonstrated by lack of reduced staining with TFEB siRNA.HEK293T cells stained with (A) Abcam anti-TFEB ab174745, (B) Bethyl anti-TFEB A303-673A and (C) Proteintech 13372-1-AP in control (left) or transfected with TFEB siRNA (ONTarget-Plus, Dharmacon) (right).

Fig 5: Mitf/TFEB is mislocalized from the nucleus and inactivated.(A) Drosophila larval salivary glands -/+ UAS-30R under the control of vGlut-Gal4 stained with anti-Mitf and DAPI. Dotted lines outline nuclei. Scale bar = 10 µm. (B) Quantification of percent (%) nuclear Mitf (nuclear Mitf fluorescence/total fluorescence) in A. Data are reported as mean ± SEM. Student’s t-test, n = 5 larvae per genotype. (C) Drosophila motor neurons (MNs) expressing UAS-Mitf-HA and UAS-CD8:GFP -/+ UAS-30R under the control of vGlut-Gal4 stained with anti-HA, anti-GFP (membrane), and DAPI to show nuclear localization. Scale bar = 1 µm. (D) Quantification of percent (%) nuclear Mitf in C. Data are reported as mean ± SEM. Student’s t-test, n = 4 and 5 larvae, respectively, with at least 10 motor neurons per larva. (E) Quantitative RT-PCR to assess transcript levels of Mitf and seven target genes from lysates of Drosophila heads expressing control (UAS-LacZ) or UAS-30R driven by daGS in control conditions or with overexpression of Mitf. Data are reported as mean ± SEM. One-way ANOVA, p<0.0001, with Sidak’s multiple comparisons test, n > 4 biological replicates of 30 heads per genotype.