Journal: bioRxiv

Article Title: Tanycytic annexinA1-containing extracellular vesicles control thermogenesis by orchestrating microglial and neuronal functions

doi: 10.1101/2025.10.12.681867

Figure Lengend Snippet: (A-B) Series of scanning electron microscopy micrographs showing the ultrastructure of multivesicular cargos (MVC, green) within tanycyte process (red) (A) and their 3D-representation (B) in the tanycyte process in adult male mice (mitochondria in yellow, blood vessel in blue). (C-D) Mean size of multivesicular cargos (C) and internal vesicles (D) observed in tanycyte processes. A total of 6 different MVCs in 2 different tanycytes and 3/6 vesicles per MVC were analyzed. (E) Series of scanning electron microscopy micrograph showing the ultrastructure of a multivesicular cargo (green) in primary tanycyte cultures, with a high magnification picture (a, insert in 3) and 3D-representation (b). (F) Representative particle size distribution profiles for extracellular vesicles obtained from primary tanycyte culture media by ultracentrifugation analyzed by Nanoparticle Tracking Analysis (NTA). (G) Mean size of secreted EV analyzed by NTA. Two technical replicates in 2 different cultures were analyzed (n=3-4). (H) Volcano plot displaying enriched proteins in the tanycyte EV versus soluble secretome fraction obtained from primary tanycyte culture media. Three technical replicates in 3 cultures were analyzed (n=9 per group). (I) Western blot confirming the presence or absence of ANXA1, ALIX, CD9, and CD81 in tanycyte-derived EV fraction and soluble secretome.

Article Snippet: To test the impact of glucose, primary tanycytes were incubated in no-glucose tanycyte culture medium (TCM) (DMEM no glucose (Gibco, P04-01548S1), 10% pen-strep, 2% L-glutamine) for 24h.

Techniques: Electron Microscopy, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Tanycytic annexinA1-containing extracellular vesicles control thermogenesis by orchestrating microglial and neuronal functions

doi: 10.1101/2025.10.12.681867

Figure Lengend Snippet: (A) Representative bright-field microscope images of primary tanycyte culture showing elongated and cuboidal cells. (B) Representative immunofluorescence of vimentin-positive, GFAP-positive, and both vimentin- and GFAP-positive cells in primary tanycyte culture. Arrows indicate GFAP + /Vimentin + cells; Arrowheads indicate GFAP - /Vimentin + cells. (C-F) Morphological and molecular characterization of primary tanycyte cultures, indicating the cell shape (C), the cell area (D), the process length (E), and the marker distribution (F). 15 pictures per well were analyzed in three technical replicates in one culture (n=3). (G) GO_extracellular_exosomes in different cell type clusters from the Brunner/Lopez dataset . (H-I) Gene set enrichment analysis on the Brunner/Lopez dataset using Top 100 exocarta (H) and vesiclepedia (I) genes enriched in extracellular vesicles. (J) Principal component analysis plot representing proteomics data. (K-L) Venn diagram presenting the number of detected proteins (K) and dotplot with selected top markers in each fraction (i.e., cell lysate, EV, and soluble secretome fractions) (L). (M) Anxa1 expression in the mouse hypothalamus using single-cell RNA sequencing (Mouse HypoMap ). (N) Representative images of ANXA1 staining with HuC/D, GFAP, IBA1, or CD31 staining in the mediobasal hypothalamus in fed male mice. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To test the impact of glucose, primary tanycytes were incubated in no-glucose tanycyte culture medium (TCM) (DMEM no glucose (Gibco, P04-01548S1), 10% pen-strep, 2% L-glutamine) for 24h.

Techniques: Microscopy, Immunofluorescence, Marker, Expressing, RNA Sequencing, Staining

Journal: bioRxiv

Article Title: Tanycytic annexinA1-containing extracellular vesicles control thermogenesis by orchestrating microglial and neuronal functions

doi: 10.1101/2025.10.12.681867

Figure Lengend Snippet: (A) Representative images of ANXA1 immunostaining (green) along the ventricle (arrows). ANXA1 staining (green) colocalizes with tdTomato-positive tanycytes (red). Cell nuclei are counterstained using DAPI (white). (B) Representative images of Anxa1 mRNA expression (green) along the third ventricle (brackets). (C) Representative images of ANXA1 immunostaining (green) along tdTomato-positive tanycyte process (red) along the VMH. (D) Schematic representation of ANXA1 expression (green) along the third ventricle on the ventrodorsal and anteroposterior axis based on in situ hybridization and immunohistochemistry analysis. (E) Schematic representation of ANXA1 subcellular localization within each tanycyte subtype based on immunohistochemistry analysis. (F-H) Representative images of Anxa1 mRNA expression (green, F) along the third ventricle during fasting-refeeding paradigm and quantification on the entire region (G) and the anteroposterior axis (H) (n=4-7 mice/group in two cohorts). (I-K) Representative images of ANXA1 expression (green, I) along the third ventricle during fasting-refeeding paradigm and quantification on the entire region (J) and the anteroposterior axis (K) (n=3-7 mice/group in two cohorts) (L-M) Quantification of ANXA1 distribution in tanycyte processes during fasting-refeeding paradigm on the entire region (L) and the anteroposterior axis (M) (n=4 mice/group in one cohort) during fasting-refeeding paradigm. ARH, arcuate nucleus; DMH, dorsomedial nucleus; VMH, ventromedial nucleus; 3v, third ventricle. *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To test the impact of glucose, primary tanycytes were incubated in no-glucose tanycyte culture medium (TCM) (DMEM no glucose (Gibco, P04-01548S1), 10% pen-strep, 2% L-glutamine) for 24h.

Techniques: Immunostaining, Staining, Expressing, In Situ Hybridization, Immunohistochemistry

Journal: bioRxiv

Article Title: Tanycytic annexinA1-containing extracellular vesicles control thermogenesis by orchestrating microglial and neuronal functions

doi: 10.1101/2025.10.12.681867

Figure Lengend Snippet: (A) Representative images of ANXA1 immunostaining (ANXA1 in green, Vimentin in red) in primary tanycyte cultures in the presence of glucose. (B) Representative images of ANXA1 (green) within nuclei (white, DAPI-positive), multivesicular bodies (red, CD9-positive), cell membrane (red, WGA-positive), and lysosomes (red, Lysotracker-positive) in primary tanycyte cultures following glucose treatment. (C-F) Quantifications of ANXA1 within nuclei (DAPI-positive, C), multivesicular bodies (CD9-positive, D), early endosomes (EEA1-positive, E), and lysosomes (Lysotracker-positive, F) in primary tanycyte cultures following glucose treatment for 1h after glucose deprivation for 24h. 6-7 tanycytes per well were analyzed in three technical replicates in three cultures (n=9 per condition; light gray points indicate each tanycyte analyzed). (G-I) Time-course quantification of ANXA1 redistribution from the nucleus (G) to CD9-positive vesicles (H) and cell membrane (I) following glucose exposure (after glucose deprivation for 24h). 6-7 tanycytes per well were analyzed in three technical replicates in three cultures (n=9 per condition). (J-K) Time-course quantification of ANXA1 redistribution from the nucleus to CD9-positive vesicles following glucose exposure (after glucose deprivation for 24h) in the absence or presence of BAPTA (J) and free-calcium Krebs medium (K). 6-7 tanycytes per well were analyzed in three technical replicates in three cultures (n=9 per condition). (L) ANXA1 redistribution from the nucleus to CD9-positive vesicles in the absence or presence of thapsigargin during glucose deprivation in free-calcium Krebs medium. 6-7 tanycytes per well were analyzed in three technical replicates in three cultures (n=9 per condition). (M) ANXA1 Tyr21 phosphorylation in primary tanycyte cultures following glucose treatment for 2h after glucose deprivation for 24h. 3 wells were analyzed in one culture (n=3 per condition). *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To test the impact of glucose, primary tanycytes were incubated in no-glucose tanycyte culture medium (TCM) (DMEM no glucose (Gibco, P04-01548S1), 10% pen-strep, 2% L-glutamine) for 24h.

Techniques: Immunostaining, Membrane, Phospho-proteomics

Journal: bioRxiv

Article Title: Tanycytic annexinA1-containing extracellular vesicles control thermogenesis by orchestrating microglial and neuronal functions

doi: 10.1101/2025.10.12.681867

Figure Lengend Snippet: (A-D) Anxa1 expression (by qPCR) in primary tanycyte cultures following glucose treatment after glucose deprivation for 24h (A) (n=4-17 per condition in 5 cultures), with dexamethasone (B) (n=4-18 per condition in 5 cultures), insulin (C) (n=4 per condition in one culture), or dexamethasone/cAMP/insulin (D) (n=4 per condition in one culture). (E-F) ANXA1 expression (by western blot) in primary tanycyte cultures following dexamethasone/cAMP/insulin (E) (n=3 TR per condition in one culture) or glucose treatment after glucose deprivation for 24h (F) (n=3-9 per condition in three cultures). (G) Percentage of tanycyte with ANXA1 staining within the nucleus following glucose treatment for 1h after glucose deprivation for 24h (n=17 pictures in one culture). (H-I) Glucose dose-dependent effect on ANXA1 localization while removing glucose for 1h (H) or adding glucose for 1h after glucose deprivation for 24h (I). (J) Time-course formation of CD9-positive vesicles following glucose treatment after glucose deprivation for 24h. 6-7 tanycytes per well were analyzed in three technical replicates in three cultures (n=9 per condition). *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To test the impact of glucose, primary tanycytes were incubated in no-glucose tanycyte culture medium (TCM) (DMEM no glucose (Gibco, P04-01548S1), 10% pen-strep, 2% L-glutamine) for 24h.

Techniques: Expressing, Western Blot, Staining

Journal: bioRxiv

Article Title: Tanycytic annexinA1-containing extracellular vesicles control thermogenesis by orchestrating microglial and neuronal functions

doi: 10.1101/2025.10.12.681867

Figure Lengend Snippet: (A) Fpr1 and Fpr2 expression in the mouse hypothalamus using single-cell RNA sequencing (Mouse HypoMap). (B) Distribution in percentage of Fpr1 and Fpr2 in different subpopulations in the Mouse HypoMap (C) Co-expression of FPR2 with different neuronal markers (i.e., Sf1 , Pomc , Npy, Avp , Crh , Trh ) using single-cell RNA sequencing (Mouse HypoMap). (D-G) Representative images of FPR1 protein (D, red), FPR2 protein (E, red), Fpr1 mRNA (F, red), and Fpr2 mRNA (G, red) distribution in the mediobasal hypothalamus. (H-I) Schematic representation of FPR1 (H) and FPR2 (I) expression (red) within the mediobasal hypothalamus on the anteroposterior axis, based on in situ hybridization and immunohistochemistry analysis. (J-K) Quantitative analysis of FPR1 (J) and FPR2 (K) expression in the different nuclei (n=3 mice). (L-M) Quantification of FPR1 (L) and FPR2 (M) in different hypothalamic nuclei in fed, 24h-fast, and 4h-refed conditions (n=3-5 mice per condition). (N) Quantification of FPR2 in the tanycyte layer in fed, 24h-fast, and 4h-refed conditions (n=3-5 mice per condition). *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To test the impact of glucose, primary tanycytes were incubated in no-glucose tanycyte culture medium (TCM) (DMEM no glucose (Gibco, P04-01548S1), 10% pen-strep, 2% L-glutamine) for 24h.

Techniques: Expressing, RNA Sequencing, In Situ Hybridization, Immunohistochemistry

Journal: Journal of Proteome Research

Article Title: Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection

doi: 10.1021/acs.jproteome.5c00100

Figure Lengend Snippet: Initial data set analysis. (A) Normality assessment using log 2 intensity histograms, shown exemplarily for Standard media, Advanced DMEM/F12, and TCM Serum Replacement for either passage 1 or passage 20. (B) Exemplary protein rank plot. Distribution of all proteins found in Standard media, passage 1, demonstrating the dynamic range and the signal distribution of drug-metabolizing enzymes and transporters. (C) Sample correlation matrix using the Pearson correlation. (D) Exemplary pairwise correlation plots to demonstrate biological and technical reproducibility.

Article Snippet: TCM serum replacement , MP Biomedicals , serum-free , BSA, bovine transferrin , <0.1% (at final concentration).

Techniques:

Journal: Journal of Proteome Research

Article Title: Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection

doi: 10.1021/acs.jproteome.5c00100

Figure Lengend Snippet: Comparison between serum-free cultivated cells and cells cultivated under FBS-containing conditions. (A) Volcano plots generated from log 2 fold changes and −log 10 ( p -values) for either 20 or 3 passages. (B) Venn diagram showing overlap between significantly regulated proteins in Advanced DMEM/F12 and TCM Serum Replacement when compared to cells cultivated under standard conditions based on 20 passages. (C) Venn diagram showing overlaps between significantly regulated proteins in all serum-free media when compared to cells cultivated under standard conditions based on the first three passages. (D) Heatmap of all proteins identified as significantly regulated in any of the analyzed serum-free conditions. Hierarchical clustering was performed using the Euclidean distance metric with the average linkage.

Article Snippet: TCM serum replacement , MP Biomedicals , serum-free , BSA, bovine transferrin , <0.1% (at final concentration).

Techniques: Comparison, Generated

Journal: Journal of Proteome Research

Article Title: Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection

doi: 10.1021/acs.jproteome.5c00100

Figure Lengend Snippet: Canonical pathways analysis. (A) Bubble plot of canonical pathways differentially regulated in Advanced DMEM/F12 when considering all 20 passages. Only pathways with absolute z -scores ≥ 2 and p -values <0.05 are shown. Pathways are clustered according to functionality. Size indicates the protein ratio of regulated to unregulated proteins, while color indicates z -scores. (B) Bubble plot of canonical pathways differentially regulated in TCM Serum Replacement when considering all 20 passages. Only pathways with absolute z -scores ≥ 2 and p -values <0.05 are shown. Pathways are clustered according to functionality. Size indicates the protein ratio of regulated to unregulated proteins, while color indicates z -scores. (C) Bar plots visualizing p- values, z -scores, and protein ratios of canonical pathways related to oxidative stress protection or drug metabolism for the comparison of cells cultivated in Advanced DMEM/F12 in comparison to cells cultivated in FBS-containing medium based on 20 passages in culture. (D) Bar plots visualizing p -values, z -scores, and protein ratios of canonical pathways related to oxidative stress protection or drug metabolism for the comparison of cells cultivated in TCM Serum Replacement in comparison to cells cultivated in FBS-containing medium based on 20 passages in culture. (E) Heatmap of canonical pathways related to oxidative stress protection or drug metabolism for comparisons between serum-free media and the standard based on the first three passages only.

Article Snippet: TCM serum replacement , MP Biomedicals , serum-free , BSA, bovine transferrin , <0.1% (at final concentration).

Techniques: Comparison

Journal: Journal of Proteome Research

Article Title: Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection

doi: 10.1021/acs.jproteome.5c00100

Figure Lengend Snippet: NRF2-mediated oxidative stress response. (A) Simplified schematic depiction of NRF2-mediated oxidative stress response overlaid with measurement and prediction data for Advanced DMEM/F12 vs Standard medium based on the whole experimental timeline (20 passages). Pink-labeled molecules were upregulated in the data set. Orange (arrows and molecules) indicates a predicted upregulation. Gray molecules were found in the data set but did not pass the threshold. The pathway was adapted from IPA. (B) Hierarchically clustered heatmap visualization of 133 proteins involved in NRF2-mediated oxidative stress response for Advanced DMEM/F12 versus Standard medium and TCM Serum Replacement versus Standard medium based on the whole experimental timeline. (C) Hierarchically clustered heatmap visualization of 131 proteins involved in NRF2-mediated oxidative stress response for all serum-free media versus standard medium based on the first three passages. (D) Log 2 fold changes of proteins determined to be up- or downregulated in at least one passage in Advanced DMEM/F12. (E) Log 2 fold changes of proteins determined to be up- or downregulated in at least one passage in TCM Serum Replacement.

Article Snippet: TCM serum replacement , MP Biomedicals , serum-free , BSA, bovine transferrin , <0.1% (at final concentration).

Techniques: Labeling

Journal: Journal of Proteome Research

Article Title: Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection

doi: 10.1021/acs.jproteome.5c00100

Figure Lengend Snippet: Expression analysis of antioxidant enzymes under serum-free conditions compared to cells cultivated under FBS-containing conditions. (A) Log 2 fold changes of antioxidant enzyme families for Advanced DMEM/F12 vs Standard. (B) Log 2 fold changes of antioxidant enzyme families for TCM Serum Replacement versus Standard.

Article Snippet: TCM serum replacement , MP Biomedicals , serum-free , BSA, bovine transferrin , <0.1% (at final concentration).

Techniques: Expressing

Journal: Journal of Proteome Research

Article Title: Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection

doi: 10.1021/acs.jproteome.5c00100

Figure Lengend Snippet: Antioxidant enzyme activity in cell lysates after 10 passages in a serum-free culture. Enzyme activity was normalized to protein concentration to allow for comparison between samples. Ordinary one-way ANOVA with Dunnett’s correction for multiple comparisons was used to assess statistical significance ( N = 3; n = 2). (A) GST activity in nmol/mL/mg protein. Serum-free cultivated cells displayed higher GST activity. The highest enzyme activity was observed for cells cultivated in TCM Serum Replacement. (B) GPX activity in nmol/mL/mg protein. Serum-free cultivated cells show a significantly increased GPX activity. The highest enzyme activity was observed for cells cultivated in Advanced DMEM/F12.

Article Snippet: TCM serum replacement , MP Biomedicals , serum-free , BSA, bovine transferrin , <0.1% (at final concentration).

Techniques: Activity Assay, Protein Concentration, Comparison

Journal: Journal of Proteome Research

Article Title: Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection

doi: 10.1021/acs.jproteome.5c00100

Figure Lengend Snippet: Expression analysis of selenoproteins expressed in serum-cultivated HepG2 cells in comparison to cells cultured in standard medium. (A) Log 2 fold changes of selenoproteins for Advanced DMEM/F12 versus Standard. (B) Log 2 fold changes of selenoproteins for TCM Serum Replacement versus Standard.

Article Snippet: TCM serum replacement , MP Biomedicals , serum-free , BSA, bovine transferrin , <0.1% (at final concentration).

Techniques: Expressing, Comparison, Cell Culture