Fig 2: (See previous page). Effects of phase-specific FoxO1 inhibition on adipogenesis. (A and B) Images of cells that were maintained in basal medium (A), and that underwent differentiation induced with the protocol as described in Materials and Methods (B). (C) Images of cells that were treated with AS1842856 during stage BMII (days 7–12) and underwent differentiation induction as in (B). (D) Images of cells that were treated with AS1842856 during stage DMI (days 3–4) and underwent differentiation induction as in (B). (E) Images of cells that were treated with AS1842856 during stage BMI (days 1–2) and underwent differentiation induction as in (B). (F) Images of cells that were treated with AS1842856 during stage DMII (days 5–6) and underwent differentiation induction as in (B). The final concentration of AS1842856 was 0.1 μM. All the images were captured on day 12, and the microscope was set at 100X. (G) Measurement of extracted Oil red O retained in cells by the absorbance (O.D.) at 510 nm (n = 6). Treatments of ‘A’–‘F’ refer to the individual treatments as described in panels A-F. * P < 0 .05; **, P < 0 .01; and ***, P < 0 .0001.

Fig 3: TNF-α reduced the effect of leptin via the FOXO1 pathway. INS-1E cells in six-well plates were treated with leptin (10 nM) and TNF-α (20 ng/ml) alone or in combination, and cells were harvested for mRNA and protein analyses 48 h after incubation. (a) Relative LepRb mRNA levels in INS-1E cells determined by RT-PCR. Data are presented as the means ± SEM of n = 3. (b) Relative LepRb protein in INS-1E cells determined by Western blotting. Data are presented as the means ± SEM of n = 4. (c) Relative phosphorylated STAT3 levels in INS-1E cells determined by Western blotting. Data are presented as the means ± SEM of n = 4. (d) Relative pFOXO1 and SOCS3 levels in INS-1E cells determined by Western blotting. Actin was as an internal control. Data are presented as the means ± SEM of n = 4; ∗P < 0.05 as compared to the vehicle group and #P < 0.05 as compared to the leptin group.

Fig 4: Nuclear FOXO1 and NF-κB. Western blots illustrate protein abundance of signaling molecules in nuclear fraction, using representative samples from each group (A). Graphs show nuclear FOXO1 (B) and NF-κB (C) protein abundance in the groups of Fetal Start (n = 5), Fetal End (n = 5), MV (n = 5), and MV + VARA (n = 6). Values are Mean (SD). ∗indicates p < 0.05. MV, mechanical ventilation; VARA, vitamin A and all-trans retinoic acid.

Fig 5: Inhibition of age‐induced miR‐195 reverses expression of senescence‐associated markers. (A) Transduction of lenti‐scramble or lenti‐miR‐195 inhibitor was monitored by mCherry reporter expression as red fluorescence and confirmed by qRT–PCR. (B, C) Transduction of old SkMs with miR‐195 inhibitor significantly restored mRNA expression of Sirt1 and Tert. Similar pattern was observed in old SkMs with miR‐195 inhibitor, restoring both SIRT1 (P < 0.01 vs. with scramble control) and TERT expressions (P < 0.05 vs. with scramble control). (D) Transduction of miR‐195 inhibitor altered the expression levels of age‐associated proteins such as FOXO1 and P53. Downregulation of acetylated proteins in FOXO1 and P53 was observed in cultured old SkMs with miR‐195, suggesting involvement of deacetylase like SIRT1. (E) The integrated red fluorescence intensity value for each telomere was calculated and presented. Their average fluorescence intensity on cultured SkMs on the slides was higher in old SkMs with miR‐195 inhibitor (n = 10 nuclei each, P < 0.0005 vs. old SkMs with scramble control).