Fig. 7: MitoTam inhibits differentiation of adipocytes.
3T3-L1 pre-adipocytes were differentiated into mature adipocytes (ADP) while treated with MitoTam (MT, 0.32 μM), tamoxifen (TX, 0.32 μM) or the vehicle at time points as indicated. a Time schedule of 3T3-L1 cell exposure to MitoTam or tamoxifen during adipogenesis. b Quantification of intracellular lipid accumulation using Oil Red O staining (n = 9; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p < 0.001; ADP vs. MT 2–9 p < 0.001); c representative images are shown, the bar indicating 100 μm. Transcripts of d Pparγ (3T3-L1, ADP, MT 0–2, MT 2–9 n = 9; TX 0–2, TX 2–9 n = 6; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p < 0.001; ADP vs. MT 2–9 p = 0.004); e Cebpα (3T3-L1, ADP, MT 0–2, MT 2–9 n = 9; TX 0–2, TX 2–9 n = 6; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p < 0.001; ADP vs. MT 2–9 p < 0.001); f AdipoQ (3T3-L1, ADP, MT 0–2, MT 2–9 n = 9; TX 0–2, TX 2–9 n = 6; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p < 0.001; ADP vs. MT 2–9 p < 0.001) and g leptin (3T3-L1 n = 9; ADP n = 6; MT 0–2 n = 8; MT 2–9, TX 0–2, TX 2–9 n = 5; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p = 0.001; ADP vs. MT 2–9 p = 0.01; ADP vs. TX 2–9 p = 0.04) were quantified by qRT-PCR. h Mitotic clonal expansion shown as number of 3T3-L1 cells after the first phase of differentiation (0–2/x) (n = 9; 3T3-L1 (day 2) vs. ADP (0–2/x) p < 0.001; 3T3-L1 (day 2) vs. TX (0–2/x) p < 0.001). i Mitochondrial morphology documented by Tomm20 immunofluorescent staining, DAPI denoting cell nuclei. The bar indicates 10 μm. j Basal and uncoupled (+oligomycin) cellular respiration of pre-adipocytes in the absence or presence of MitoTam was evaluated (3T3-L1 n = 6; ADP, MT 0–2 n = 5; MT 2–9 n = 4; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p < 0.001; ADP vs. MT 2–9 p = 0.01). k Representative images of mitochondrial NAD(P)H signal measured by two-photon FLIM microscopy. Fluorescence lifetime is color-coded as shown. The bar indicates 10 μm (top). Dots in the phasor display (bottom) represent average values from individual fields of view (3T3-L1 n = 8; ADP n = 19; MT 0–2 n = 10; MT 2–9, TX 0–2 n = 8; TX 2–9 n = 9). G and S values correspond to real and imaginary parts of first components of Fourier transformation. l Quantification of senescent cells assessed by SA-β-gal activity (n = 9; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p < 0.001; ADP vs. MT 2–9 p < 0.001); and m The level of p21waf1 assessed by qRT-PCR (3T3-L1, ADP n = 9; MT 0–2 n = 8; MT 2–9 n = 9; TX 0–2, TX 2–9 n = 6; ADP vs. 3T3-L1 p < 0.001; ADP vs. MT 0–2 p < 0.001; ADP vs. MT 2–9 p = 0.006). b, d–h, l, m One-way ANOVA, Dunnett’s multiple comparisons test was used. j Two-way ANOVA, Dunnett’s multiple comparisons test was used. The results are derived from at least two or three independent biological experiments. Data in all experiments are expressed as mean ± SD; *p < 0.033; **p < 0.002; ***p < 0.001. Source data are provided as a Source Data file.
