Fig 2: Mitochondrial function and glycolysis are impaired in HGPS fibroblasts. (A) Schematic representation of Seahorse XF Cell Mito stress test and calculated values are indicated. Oxygen consumption rates (OCR) (B) and extracellular acidification (ECAR) (E) were determined with a Seahorse XF96 Flux analyzer in basal and stimulated conditions (n = 3). Additional parameters like basal respiration (C), spare respiratory capacity (D), and basal ECAR levels (F) were calculated with Wave software v2.6.1.53 (Agilent Technologies, Santa Clara, CA, USA). (G) Cellular ATP levels were measured using a CellTiter-Glo luminescence ATP assay. (H) Intracellular ROS levels were determined by measuring oxidized dichlorofluorescein (DCF) levels using a DCFDA cellular ROS detection assay (n = 3). Graphs show mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 3: Senescence index of SKPs: SA-β-gal and p21 staining of original fibroblasts and dissociated SKPs at day 4. (a) SA-β-gal test performed using initial fibroblast cultures and on dissociated spheroids at day 4 for both control and HGPS groups starting from 5 and 15% SNS. (b) Immunofluorescence staining for p21 and lamin A/C in dissociated spheroids at day 4, from control and HGPS SKPs with 5 and 15% SNS. Cells were counterstained with DAPI. (c) Quantification of the percentage of p21-positive nuclei in the initial fibroblast cultures and in dissociated SKPs at day 4 in both control and HGPS groups. Values are presented as mean ± SD (n = 3), * p < 0.05, (c) two-way ANOVA with Tukey’s multiple comparisons test.

Fig 4: Differentiation of trypsinized SKPs into adipocytes. (a) Panel showing the optimized protocol for SKP trypsinization at day 4 and differentiation into adipocytes. The spheroids were trypsinized at day 4 and cultured directly in ADM. The differentiated cells were observed for 21 days and then stained with ORO and Bodipy at day 21. (b) Bodipy staining of lipid vesicles for control (GMO5567A) and HGPS (HGADFN127 and HGADFN003) cells, starting from 5% SNS fibroblast cultures. Staining was performed at days 7, 14, and 21 of differentiation. Cells were counterstained with DAPI. Scale bar: 100 µm; for the magnified images, scale bar: 20 µm. (c) Quantification of area with Bodipy signals at the three time points for both control and HGPS adipocytes. Values are presented as mean ± SD (n = 3), *** p < 0.001, (c) two-way ANOVA with Tukey’s multiple comparisons test.

Fig 5: Co-expression of progerin with Bodipy and IL-8 in HGPS differentiated SKPs. (a) Immunofluorescence staining for progerin and Bodipy at days 7 and 14 of adipogenesis in HGPS (HGADFN127). (b) Immunofluorescence staining for progerin and IL-8 at days 7 and 14 of adipogenesis in HGPS. Cells were counterstained with DAPI. (c) Percentage of positive nuclei expressing either progerin, Bodipy, or both (progerin/Bodipy 493/503). (d) Percentage of progerin-positive nuclei, IL-8-positive or progerin/IL-8-positive cells. Values are presented as mean ± SD (n = 3), not significant (ns), ** p < 0.01, (c,d) unpaired t-test.