Human stem cell culture and treatments

Human Adipose-Derived Stem Cells (hADSCs) from 3 different healthy female donors (49–64 years old) were purchased from Lonza Bioscience (Basel, Switzerland) and cultured following manufacturer’s procedures in ADSC growth medium. Cells were incubated at 37 °C 5% CO2. For adipocyte differentiation, 100% confluent cells between passages 3 and 6 were cultured in MesenCult™ Adipogenic Differentiation Medium (Human) (Vancouver, Canada) following the manufacturer’s procedures for 11 days.

BPS and PFOS (Wellington Laboratories, Guelph, Canada) were dissolved in methanol and diluted in culture media to the desired concentration. hADSCs were treated with 0.1 µM BPS and PFOS, either individually or in combination, 48 h before the induction of adipocyte differentiation and every 48 h throughout the entire differentiation process (11 days, D11). Methanol was used as a vehicle control and added at the same concentration (1:1000) to all treatment conditions. Preliminary experiments investigating the effects of methanol were performed comparing the untreated control (in absence of methanol) and the vehicle condition did not reveal detectable differences in adipogenic differentiation and glucose uptake (data not shown). Therefore, the untreated control condition was not included in the present study.

Glucose uptake

Glucose uptake activity was measured using a fluorescent D-glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) buffer (Thermo Fisher Scientific, Waltham, MA, USA). 5000 cell/cm2 were seeded into a 96-well, black, optically clear flat-bottom plate (PhenoPlate, Revvity Waltham, MA, USA) and treated with BPS and/or PFOS as reported above. At day D11 of differentiation, cells were starved in no-glucose DMEM (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h, treated with 100nM insulin (Sigma-Aldrich, St. Louis, MO, USA) for 1 h and then incubated with 60 µM 2-NBDG for 15 min. In all the steps, EDs were present in the media. After washing, the fluorescence intensity of cellular 2-NBDG in each well was measured at an excitation/emission wavelength of 465/540 nm using the Varioskan LUX microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Insulin pathway

To determine alteration of the insulin pathway after ED administration, differentiated cells (D11) were starved in DMEM no glucose DMEM (Thermo Fisher Scientific Waltham, MA, USA) for 1 h and then exposed to 100nM insulin (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. In all the steps, EDs were present in the media. Samples were then collected for protein extraction and analyzed by Western Blot.

Protein extraction and Western blot

Total cell lysate was obtained by lysing the cells with RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with cOmplete mini and PhosSTOP protease and phosphatase inhibitors for 10 min. Lysates were centrifuged at 13,000 g for 15 min.

Proteins were quantified using the Rapid Gold BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Then, 10–15 µg of proteins were separated on a 4–15% polyacrylamide gel (Biorad, Hercules, CA, USA) and transferred to a nitrocellulose membrane (Sigma-Aldrich, St. Louis, MO, USA). Blots were blocked 5% Bovine serum albumin (BSA) in 0.1% TBS-T for 1 h and then incubated overnight at 4 °C with the following anti human primary antibodies: anti-PI3K (1:1000, Cell Signaling Technologies, Danvers, MA, USA), anti pPI3K p85/p55 (Tyr458, Tyr199) Polyclonal Antibody (1:1000, Thermo Fisher Scientific Waltham, MA, USA), anti-pAKT-Ser473 (1:1000, Cell Signaling Technologies, Danvers, MA, USA), anti-pAKT-Thr308 (1:1000, Cell Signaling Technologies, Danvers, MA, USA), anti-AKT (1:1000, Cell Signaling Technologies, Danvers, MA, USA), anti-p-ERK1-2 (1:1000, Cell Signaling Technologies, Danvers, MA, USA), anti-ERK1-2 (1:1000, Cell Signaling Technologies, Danvers, MA, USA), anti GLUT4 (1:1000 Abcam, Cambridge, UK), anti β-actin (1:1000, Thermo Fisher Scientific, Waltham, MA, USA). The appropriate secondary HRP-conjugated antibodies (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) were incubated for 1 h at room temperature. Bands were visualized using Clarity ECL substrate (Bio-Rad, Hercules, CA, USA) imaged using the iBright system (Thermo Fisher Scientific, Waltham, MA, USA) and quantified using ImageJ. Each band intensity was normalized on β-actin, used as housekeeping protein.

Immunofluorescence analysis

Immunofluorescence analysis was performed as previously described [33]. Briefly, cells were fixed with 4% paraformaldehyde for 10 min. After fixation, cells were washed three times with PBS and permeabilized with 0.5% Triton X-100 for 15 min. Blocking was performed in 5% bovine serum albumin (BSA) for 30 min at room temperature. Cells were subsequently incubated overnight with anti-CEBPα (1:100, Cell Signaling Technologies, Danvers, MA, USA), anti-FABP4 (1:100, Thermo Fisher Scientific, Waltham, MA, USA), anti-PPAR-γ (1:100, Thermo Fisher Scientific, Waltham, MA, USA), and anti-GLUT4 (1:100, Abcam, Cambridge, UK) anti IL1β (1:100, Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed three times with PBS and incubated 1 h at room temperature with the appropriate secondary antibody conjugated with Alexa Fluor 488 (1:100; Invitrogen, Carlsbad, CA, USA).

Intracellular lipid drops and F-actin were stained using PhenoVue 493 Lipid stain (Revvity, Waltham MA, USA) and Rhodamine Phalloidin probe, respectively (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s procedure.

Nuclei were counterstained with DAPI (Thermo Fisher Scientific, MA, USA).

Images were acquired using Operetta CLS and analyzed by Harmony (Revvity, Waltham MA, USA).

RNA extraction and reverse transcription

Cells were lysed with QIAzol lysis reagent (QIAGEN, Hilden, Germany) and total RNA was extracted using the miRNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s procedure. For reverse transcription, 1 µg of RNA was retrotranscribed by the High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s procedure.

Real-time PCR (qPCR)

For all the examined mRNAs, qPCR analysis was performed using SYBR green (PowerUp SYBR Green Master mix, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s procedures in QuantStudio 3 (Thermo Fisher Scientific, Waltham, MA, USA). The running method consisted of the following steps: 95 °C for 10 min, 95 °C for 15 s, 60 °C for 1 min. Steps 2 and 3 were repeated for 40 cycles. The authenticity of the PCR products was verified by melt-curve analysis. Each gene expression value was normalized to the 18 S expression. The fold change (FC) of each gene was calculated by the ΔΔCT method and expressed in relation to the CTRL. Primers were designed using Primer Blast, except the ones for CEBPA and 18 S, obtained respectively from Di Ruscio et al. [34] and Rubino et al. [35].

Primer sequences are listed in Table 1.

ELISA assay

Secretion of IL1β was quantified by Human IL-1 beta ELISA Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s procedure. Briefly, standards and samples were analyzed in duplicate in antibody-coated 96-well plates. Following incubation with the biotin-conjugated detection antibody and streptavidin-HRP, TMB substrate was added for color development, and the reaction was stopped with the supplied stop solution. Absorbance was measured at 450 nm. IL-1β concentrations were calculated from a standard curve generated using recombinant human IL-1β standards ranging from 3.9 to 250 pg/mL.

Statistical analysis

One-way analysis of variance (ANOVA) was used to check for differences between experimental conditions. When one-way ANOVA showed statistical significance, Tukey’s post hoc test was used for comparison to CTRL. For qPCR time-course experiments, gene expression data were analyzed using two-way ANOVA, considering treatment and time as independent factors, followed by Dunnett’s multiple comparisons test to compare each treatment with the vehicle control at each time point. All data are reported as mean ± standard deviation of three independent biological experiments. Results were considered statistically significant when p<0.05. All statistical analyses were performed with GraphPad PRISM Version 9.4.1 (GraphPad Software, San Diego, CA).