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. 2024 Jul 4;31(11):1439–1451. doi: 10.1038/s41418-024-01337-y

Fig. 1. TFEB is activated during in vitro syncytiotrophoblast formation.

Fig. 1

A Dot plot displaying TFEB expression (nTPM) in different cell types according to the Human Protein Atlas single-cell datasets. The top-6 cell types that express the highest levels of TFEB are indicated in black, with the Syncytiotrophoblast representing the cell compartment with TFEB highest levels indicated in red. B Heatmap displaying TFEB, TFE3, MITF, and TFEC expression (nTPM) at the single-cell level in human placenta cell types according to Human Protein Atlas clustered single-cell datasets. C (Left) E10.5 placentas derived from wild-type (WT) and TFEB knock-out mice (TFEB-KO) were stained for MCT4 and DAPI. Images shown at different magnifications. Scale bars: 500μm (4×) an 500 μm (10×). Sections from three placentas per genotype were analyzed. (Right) Histograms displaying ImageJ-based quantification of MCT4 signal shown as “Mean Intensity” and “Area” (** one-sided p-value < 0.01 with unpaired Welch’s t-test). D (Up) Representative immunofluorescence images of BeWo cells in DMSO and Forskolin (FRSK) conditions immunostained for TFEB. Nuclei are counterstained blue using DAPI. Scale bar 20 µm. The inset displays a digital magnification of the representative immunofluorence images for a detailed observation of TFEB subcellular localization. (Down) Dot plot showing the ratio relative to the quantification of TFEB fluorescence intensity in the nucleus and the cytoplasm (TFEB N/C) upon indicated treatments (n = 4) measured by High-Content Imaging (*** one-sided p-value < 0.001 with unpaired Welch’s t-test). E Representative image of immunoblot analysis of TFEB levels in BeWo cells in DMSO and Forskolin (FRSK) conditions. A molecular weight shift of TFEB is visible in FRSK-treated cells, suggesting that TFEB is dephosphorylated in this condition. GAPDH was used as a loading control. F (Up) Representative immunofluorescence images of BeWo cells upon DMSO and Forskolin (FRSK) treatments immunostained for human chorion gonadotropin β subunit (CGB). Nuclei are counterstained blue using DAPI. Scale bar 20 µm. (Down) High-content imaging-based quantification of CGB intensity upon indicated treatments (n = 10) shown as dot plots (*** one-sided p-value < 0.001 with unpaired Welch’s t-test). G:(Up) Representative immunofluorescence images of human naive IPSC-derived trophoblast stem cells (TSC) and differentiated syncytiotrophoblast (STB) immunostained for TFEB. Nuclei are counterstained blue using DAPI. Scale bar 20 µm. The inset displays a digital magnification of the representative immunofluorence images for a detailed observation of TFEB subcellular localization. (Down) Dot plot showing the ratio relative to the quantification of TFEB fluorescence intensity in the nucleus and the cytoplasm (TFEB N/C) upon indicated treatments (n = 7) measured by High-Content Imaging (*** one-sided P value < 0.001 with unpaired Welch’s t-test). H Representative image of immunoblot analysis of TFEB levels in human naive IPSC-derived trophoblast stem cells (TSC) and differentiated syncytiotrophoblast (STB). A molecular weight shift of TFEB is visible in STB, suggesting that TFEB is dephosphorylated in this condition. H3 was used as a loading control. I (Up) Representative immunofluorescence images of human naive IPSC-derived trophoblast stem cells (TSC) and differentiated syncytiotrophoblast (STB) immunostained for human chorion gonadotropin β subunit (CGB). Nuclei are counterstained blue using DAPI. Scale bar 20 µm. (Down) High-content imaging-based quantification of CGB intensity upon indicated treatments (n = 7) shown as dot plots (*** one-sided p-value < 0.001 with unpaired Welch’s t-test).