Effects of Exposure to Tobacco Cigarette, Electronic Cigarette and Heated Tobacco Product on Adipocyte Survival and Differentiation In Vitro

Figure 1. Effect of extracts on 3T3-L1 pre-adipocyte viability. The effect of extracts on metabolic activity and thus, viability of 3T3-L1 pre-adipocytes, after 24 h and 48 h of treatment, was assessed by the MTT-based chromogenic assay. Dilutions of 5% v/v and 10% v/v of the extracts were administered. Data are presented as the mean ± SEM of 3 independent experiments done in triplicate (n = 3). Statistical analysis was performed using one-way ANOVA with post-hoc LSD test for multiple comparisons. Statistical differences are indicated by * p < 0.05 and ** p < 0.01.

Figure 2. At the end of the differentiation process (10 days), cells were stained with Oil Red O. (A). Stained adipocytes were observed under the light microscope, revealing the lipid droplet formation in beige adipocytes. (B) Pictures of stained monolayers of adipocytes were obtained using a dissection microscope.

Figure 3. Effect of extracts on lipid content in beige adipocytes. 3T3-L1 pre-adipocytes were differentiated in the presence of each treatment for 10 days, whereupon cells were fixed and stained with Oil Red O. Undifferentiated 3T3-L1 cells maintained in parallel were also undergone the staining process. The extracted Oil Red O solution was quantified by spectrophotometric analysis at 550 nm. To determine relative lipid accumulation, data were normalized to the ambient air extract-treated cells. Histograms display the mean of the ratios ± SEM of 3 independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA with post-hoc least significant difference (LSD) test for multiple comparison. Statistical differences are indicated by ** p < 0.01.

Figure 4. Expression of Ucp1 in undifferentiated and differentiated 3T3-L1 cells, either unstimulated or treated with 10 μM forskolin, for 4 h prior to harvesting for RNA isolation. Values display the mean ± SEM of 3 independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA. * p < 0.05.

Figure 5. Expression of markers of adipogenic differentiation. At day 5 and day 10, total RNA was isolated and mRNA levels of Pgc-1α, Ppar-γ and Resistin were analyzed. Rps-18 was used as the reference gene (endogenous control) and data were normalized to the ambient air extract-treated cells. Values are mean ± SEM of 3 independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA with post-hoc LSD correction. Statistical differences are indicated by * p < 0.05 or ** p < 0.01.