Journal: bioRxiv

Article Title: Respiratory Airway Secretory Cells act as Immune Sentinels in Human Distal Airways

doi: 10.1101/2025.03.24.644887

Figure Lengend Snippet: a , Schematic of proximal-distal patterning in developing human lung with representative lineage markers. b , Differentiation protocol workflow from human pluripotent stem cells (hPSCs) to distal lung organoids (d-LOs) via anterior foregut endoderm (AFE) spheroids dissociation. c , Immunofluorescence staining of NKX2-1, SOX2 and SOX9 in proximal (p-LO) and distal (d-LO) lung organoids at day 8. White arrows denote SOX2 bright NKX2-1 dim cells; arrowheads indicate SOX9 bright NKX2-1 bright cells. Scale bars, 20 μm. Representative images from three biologically independent experiments. d , Quantitative PCR analysis of NKX2-1 , SOX9 and SOX2 expression in AFE spheroids, d-LOs and p-LOs. Data presented as mean ± s.e.m. (n=3 biological replicates). P -values were calculated using two-tailed Student’s t -test with Welch’s correction. e , UMAP with annotations of different cell types from epithelial cell subclusters. f , Individual conditions of epithelial subclusters shown in ( e ). g , Proportional distribution of cell types between d-LO and p-LO. h , Violin plots showing expression levels of proximal and distal markers in p-LPC and d-LPC cell populations from d-LO and p-LO.

Article Snippet: Detached anterior foregut spheroids (days 5–7) were split into two groups: p-LO: Spheroids were embedded in growth factor-reduced Matrigel (Corning, 354230) and cultured in LPC medium (20 ng/ml BMP4 [MCE, HY-P7007], 10 ng/ml FGF7/KGF [MCE, HY-P70597], 10 ng/ml FGF10 [MCE, HY-P70695], 3 μM CHIR99021, 20 μM DAPT [MCE, HY-13027], and 50 nM retinoic acid [Sigma, R2625]). d-LO: Spheroids were washed with PBS, dissociated into single cells with Accutase, pelleted (300 × g, 5 min), and resuspended in growth factor-reduced Matrigel.

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Respiratory Airway Secretory Cells act as Immune Sentinels in Human Distal Airways

doi: 10.1101/2025.03.24.644887

Figure Lengend Snippet: a , Differentiation protocol workflow for generating proximal lung organoids (p-LOs) from hPSCs. b , Bright-field images comparing organoid morphology under untreated versus dissociation conditions during lung progenitor cell (LPC) induction. Scale bars, 500 μm (far left and middle two panels), 200 μm (right panels). Representative of three biologically independent experiments. c , Phase-contrast images of day 8 p-LOs and d-LOs counterstained with Hoechst. Scale bars, 500 μm. d , Organoid yield quantification (number per Matrigel droplet). e , Organoid diameter measurements. f , Representative flow cytometry analysis of NKX2-1 expression in p-LOs versus d-LOs. n = 3 independent experiments. g , h , Percentage ( g ) and mean fluorescence intensity (MFI) ( h ) of NKX2-1 + cells. i , qPCR analysis of lung-specific and endodermal lineage markers in p-LOs and d-LOs derived from hESC lines (H1, H9) and induced pluripotent stem cells (hiPSCs). d , e , g , h , i , Data are presented as mean ± s.e.m. (n=3 biological replicates). P -values calculated using two-tailed Student’s t -test.

Article Snippet: Detached anterior foregut spheroids (days 5–7) were split into two groups: p-LO: Spheroids were embedded in growth factor-reduced Matrigel (Corning, 354230) and cultured in LPC medium (20 ng/ml BMP4 [MCE, HY-P7007], 10 ng/ml FGF7/KGF [MCE, HY-P70597], 10 ng/ml FGF10 [MCE, HY-P70695], 3 μM CHIR99021, 20 μM DAPT [MCE, HY-13027], and 50 nM retinoic acid [Sigma, R2625]). d-LO: Spheroids were washed with PBS, dissociated into single cells with Accutase, pelleted (300 × g, 5 min), and resuspended in growth factor-reduced Matrigel.

Techniques: Flow Cytometry, Expressing, Fluorescence, Derivative Assay, Two Tailed Test

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Agathisflavone enhances remyelination and induces oligodendrocyte proliferation in organotypic cerebellar slices culture. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Photomicrographs showing the cerebellar white matter stained with MBP (red) and NF (blue); scale bar 20 μm. (B, C) Bar graphs showing the NF + axon index (C) and the percentage of MBP+/NF + myelinated axons (D) per constant field of view (FOV). ( D) Oligodendrocyte lineage Sox10-EGFP + cells (green), immunostained for the proliferating marker Ki67 (red) and counterstained with Hoechst nuclear dye (blue); scale bar 20 μm. (E, F) Bar graphs showing the number of Sox10+ cells per FOV (E) and the percentage of SOX10+/Ki67+ cells (F) in a constant FOV. (G) Photomicrographs of OPCs immunolabelled for NG2; scale bar 50 μm. (H) Bar graph showing the number of NG2 + OPCs per FOV. Data are expressed as the mean ± SEM (n = 6); * p < 0.05, *** p <0.001, **** p < 0.0001 (comparing controls to treatment groups); ‡ p < 0.05, ‡‡‡ p < 0.001, ‡‡‡‡ p < 0.0001 (comparing LPC+DMSO to LPC+FAB5 and LPC+FAB10), One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Staining, Marker

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Agathisflavone increases mature oligodendrocyte cells number and prevents oligodendrocyte apoptosis. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Oligodendrocyte lineage cells identified by expression of the Sox10-EGFP reporter (green), immunolabelling for CC1 for mature oligodendrocytes (yellow) and active Caspase 3 for apoptotic cells (red), and counterstained with Hoechst nuclear dye (blue); scale bar 50 μm. (B, C) Individual values column graphs showing the percentage of CC1+ /Sox10+ cells (B), Caspase+/SOX10+ cells (C) and Caspase+/CC1+ cells (D) in a constant FOV; data are expressed as mean ± SEM (n = 4–6); * p < 0.05, **** p < 0.0001 (comparing controls to treatment groups); ‡‡‡‡ p < 0.0001 (comparing LPC+DMSO to LPC+FAB5 and LPC+FAB10), One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Expressing

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Agathisflavone modifies microglial activation state. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Microglial proliferation was analyzed by immunolabelling for IBA1 (yellow) and Ki67 (red), counterstained with the nuclear dye Hoechst (blue). (B, C) Bar graph showing the number of IBA1+ microglia (B) and the percentage of IBA1+/Ki67+ proliferating microglia (C); data are expressed as the mean ± SEM (n = 5–11) and tested for significance using One-way ANOVA followed by Tukey’s post-hoc test. (D) Photomicrographs and binary and skeletonized IBA + microglia illustrating morphological differences in the different treatment groups; scale bar 50 μm. (E, F, G) Individual values violin plots of microglial soma size per microglial cell (20 microglial cells/image were analyzed) (E) and violin graphs of process endpoints (F) and length (G) per microglial cell; data are expressed as the median ± IQR; * p < 0.05, **** p < 0.0001 (comparing controls to treatment groups); ‡ p < 0.05, ‡‡ p < 0.01, ‡‡‡ p < 0.001, ‡‡‡‡ p < 0.0001 (comparing LPC+DMSO to LPC+FAB5 and LPC+FAB10); †† p < 0.01 (comparing LPC+FAB5 to LPC+ FAB10); Kruskal-Wallis test followed by Dunns. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Activation Assay

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Agathisflavone modulates microglia-oligodendrocyte interactions. Organotypic cerebellar slices from P10-12 Sox10-EGFP mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Photomicrographs of IBA1 immunostaining (red) and SOX10-EGFP+ oligodendrocytes (green) showing oligodendrocytes-microglia contacts in the different treatment groups; scale bar 20 μm. (B) Diagram illustrating microglial processes contacting oligodendrocytes body (Pr-B), or apposition of microglial and oligodendrocyte cell bodies (B—B). (C) Grouped bar graph showing the number of microglial contacts per SOX10+ cells; data are expressed as the mean ± SEM (n = 6), * p < 0.05, ** p < 0.01, *** p < 0.001 (comparing control to treatment groups); ‡‡ p < 0.01, ‡‡‡ p < 0.001 (comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10; two-way ANOVA followed by Tukey’s post-hoc test. (D) Photomicrographs of slices illustrating Sox10-RGFP + oligodendrocytes (green) and immunolabelling for MBP (red) and Iba1 (yellow), showing interrelationships between microglia, oligodendrocytes, and myelinated fibres in the different treatment groups; clusters of IBA1+ microglia around myelin debris and oligodendrocytes are evident following LPCv treatment and are rarely observed in controls or following agathisflavone treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Immunostaining

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Agathisflavone promotes a microglial polarization from a M1 to a M2 profile. Organotypic cerebellar slices from P10-12 mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. (A) Microglial profile analyzed by double immunofluoresecence labelling for the M1 pro-inflammatory marker CD16/32 (red) and M2 anti-inflammatory marker CD206 (green), where co-expression appears yellow; scale bar 50 μm. (B, C) Bar graphs showing the number of CD16/32+, CD206+ and CD206+/CD16/32+ cells (B) and the M1/M2 ratio (C); data are expressed as the mean ± SEM (n = 6), * p < 0.05, ** p < 0.01, **** p < 0.0001 (comparing control to treatment groups); ‡‡‡‡ p < 0.0001 (comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10); One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Marker, Expressing

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Agathisflavone modulates transcript levels of neuroinflammatory genes. Organotypic cerebellar slices from P10-12 mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. ( A, B) Heat map showing the expression of neuroinflammatory genes (A) and respective graphs (B) of RT-qPCR analysis showing the expression of neuroinflammatory genes. (C, D) Heat map showing the expression of regulatory factors (C) and respective graphs (D). Data are expressed as the mean ± SEM or median ± IQR (n = 4); * p < 0.05, ** p < 0.01, *** p < 0.001 (comparing control to treatment groups); ‡ p < 0.05, ‡‡ p < 0.01, ‡‡‡ p < 0.001, ‡‡‡‡ p < 0.001(comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10); †† p < 0.01 (comparing LPC+FAB5 to LPC+FAB10); samples with Gaussian distribution (bar graphs) were analyzed by one-way ANOVA followed by Tukey’s post-hoc test, non-parametric samples (individual values column graphs) by Kruskal-Wallis followed by Dunns.

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Expressing, Quantitative RT-PCR

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Agathisflavone regulates reactive astrogliosis and is neuroprotective. Organotypic cerebellar slices from P10-12 mice were maintained for 7 DIV and then treated with LPC for 15–17 h, followed by agathisflavone (FAB) at 5 or 10 μM for a further 2 DIV, or 0.1% DMSO vehicle. . (A) Photomicrographs illustrating GFAP-EGFP + astrocytes (green) and Hoescht stained nuclei (blue); scale bar 20 μm. (B) Violin graphs showing the mean fluorescence intensity of GFAP in the different treatment groups. (C) Photomicrographs of Purkinje neurons immunolabelled for Calbindin (yellow) and the apoptosis marker cleaved Caspase-3 (red) and counterstained with Hoechst (blue). The panels on the left side show entire cerebellar lobules and the organization of its layers (ML: Molecular layer; PCL: Purkinje cells layer; GL: Granular layer; WM: White matter); scale bar 50 μm. The remaining panels focus on the PCL; scale bar 50 μm. Insets illustrate individual Purkinje cells ; scale bar 20 μm. (D, E) Violin graphs showing the number of Calbindin + cells per FOV (D) and the percentage of Caspase+ /Calbindin + cells (E). (F, G, H) RT-qPCR analysis Cntf (F), Egfr (G) and Gabbr1 mRNA expression in cerebellar slices in the different treatment groups; data are expressed as the mean ± SEM or median ± IQR (n = 5); * p < 0.05, ** p < 0.01, **** p < 0.0001 (comparing control to treatment groups); ‡ p < 0.05, ‡‡ p < 0.01, ‡‡‡‡ p < 0.001 (comparing LPC-DMSO to LPC+FAB5 and LPC+FAB10); † p < 0.05, †† p < 0.01 and †††† p < 0.0001 (comparing LPC+FAB5 to LPC+FAB10); samples with Gaussian distribution (bar graphs) were analyzed by One-way ANOVA followed by Tukey’s post-hoc test, non-parametric samples (individual values column graphs) by Kruskal-Wallis followed by Dunns. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Staining, Fluorescence, Marker, Quantitative RT-PCR, Expressing

Journal: Pharmacological Research

Article Title: The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

doi: 10.1016/j.phrs.2020.104997

Figure Lengend Snippet: Estrogen receptor (ER) activation is required for agathisflavone to inhibit microgliosis and promote remyelination. (A) Root mean square deviation (RMSD) values and rod representation of crystallographic ligand pose (lilac) and the best pose of this ligand generated by DOCK 6.8 (yellow) for each complex. Distances less than 2 Å between the calculated pose and the crystallographic pose indicates that the program was successful in reproducing the experimental data (B) Representation of interactions between agathisflavone (FAB) and retinoic and estrogen receptors; the captions are described in the figure. (C–G) Organotypic cerebellar slices from SOX10-EGFP animals were maintained for 7DIV, then exposed to LPC for 15–17 h, followed by 2 h pretreatment with the selective ER-α antagonist MPP dihydrochloride at 10 nM (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole-dihydrochloride), or the selective ER-β antagonist PHTPP at 1 μM (4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5- a ]pyrimidin-3-yl]phenol), which were kept together with 10 μM FAB for a further 2 DIV. (C) Oligodendrocytes were identified by the Sox10-EGFP reporter (green) and slices were immunolabeled for MBP (red), neurofilament (blue) and Iba-1 (yellow); scale bar: 20 μm. Bar graphs compare LPC and LPC + FAB 10 μM with the effects of the ER antagonists MPP and PHTPP on the NF + axon index (D), the percentage of MBP+/NF + myelinated axons (E), the number of Sox10-EGFP + oligodendrocytes (F) and the number of Iba1+ microglia (G); data are expressed as the mean ± SEM (n = 5); ‡ p < 0.05, ‡‡ p < 0.01, ‡‡‡‡ p < 0.0001 (comparing LPC-DMSO to other treatment groups); †† p < 0.01 and ††† p <0.001 (comparing LPC + FAB10 to LPC + FAB10+MPP); & p < 0.05 (comparing LPC+FAB10+MPP to LPC+FAB10+PHTPP); One-way ANOVA followed by Tukey’s post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After 7 DIV, slices were treated for 15–17 h with medium containing LPC (0.5 mg/mL, Sigma, L4129), after which LPC-medium was removed and replaced with medium containing either agathisflavone at the concentrations of 5 or 10 μM, or 0.1% DMSO vehicle (LPC + DMSO condition), for a further 2DIV; concentrations of agathisflavone used were based on previous studies by our group.

Techniques: Activation Assay, Generated, Immunolabeling

Journal: Nature Communications

Article Title: FoxA and LIPG endothelial lipase control the uptake of extracellular lipids for breast cancer growth

doi: 10.1038/ncomms11199

Figure Lengend Snippet: ( a ) Schematic representation of LIPG action. ( b ) Heat map representation of the downregulated (blue) lipids identified by MS/MS in the cell homogenates of MCF7 or MDA231 LIPG-depleted cells compared with shControl cells. Depicted lipids have a fold change >1.5 and P value<0.05 using the Welch's t -test n =5. ( c ) Downregulated lipid species (previously identified in b ) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG (blue box). P values are <0.05 and calculated using Welch's t -test, n =5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. ( d ) Heat map representation of the upregulated (red) lipids identified by MS/MS in the media of MCF7 or MDA231 LIPG-depleted cells compared with the corresponding shControl cells. Characterized lipids have a fold change >1.5 and P value<0.05 using the Welch's t -test n =5. ( e ) Upregulated lipid species in the media (previously identified in d ) that are common to LIPG-depleted MCF7 and LIPG-depleted MDA231 cells. ShControl cells (red box), and shLIPG cells (blue box). P values are <0.05 and calculated using Welch's t -test, n =5. Whiskers extend to a maximum of 1.5 × IQR beyond the box. ( f ) Heat map representation of the MS/MS downregulated (blue) lipids in the cell media of MCF7/MDA231 LIPG-depleted or shControl cells (as described in d ) compared with fresh medium (without cell incubation). Depicted lipid species have a log 2 fold change>1.5 and P value<0.05 using the Welch's t -test n =5. ( g ) MDA231 and MCF7 cell growth for 48 h in complete medium: medium containing 10% FBS 10%); lipoprotein-free medium: medium containing 10% free lipoprotein FBS; and LPC (18:0): medium containing 10% free lipoprotein FBS and 20 μM of LPC (18:0). P value is the result of T -test. Data are average±s.e.m.; n =3. ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001. ( h ) Above, schematic representation of the experimental protocol used. (bottom) Tumour growth of the indicated cell populations inoculated in Balb/c nude mice treated with high-fat diet (HFD) are determined at the indicated time points. P value is the result of T -test. Data are average±s.e.m.; n = 6–8 tumours. * P ≤0.05, ** P ≤0.01. Inside graph, plasma cholesterol levels of animals treated with standard diet (SD) or HFD. P value is the result of T -test. Data are average±s.e.m.; n = 4 animals per group. ** P ≤0.01, *** P ≤0.001.

Article Snippet: Then, the medium was replace by: complete medium: DMEM supplemented with 10% FBS (Gibco), 100 mg ml −1 streptomycin, 100 U ml −1 penicillin and 2 mM L-glutamine; lipoprotein-free medium: DMEM supplemented with 10% lipoprotein-free FBS (Gibco), 100 mg ml −1 streptomycin, 100 U ml −1 penicillin, and 2 mM L-glutamine, Transferrin (10 μg ml −1 ), Triiodothyronine (0,01 μM) (Sigma), o -phosphorylethanolamine (5 μg ml −1 ) (Sigma), glutathione (0,012 μg ml −1 ) (Sigma), hydrocortisone (0,5 μg ml −1 ) (Sigma), EGF (10 ng ml −1 ) (RD), FGF (10 μg ml −1 ) (Gibco), Insulin (20 μg ml −1 ), medroxiprogesterone (10 nM) (Sigma) and BSA-free fatty acids 0,5% (Sigma); LPC (18:0) medium: lipoprotein-free medium supplemented with 20 μM LPC 18:0 (Avanti polar).

Techniques: Tandem Mass Spectroscopy, Incubation