Fig 1: AMPK activation partly involves Metformin inhibition on Cidec and its regulatory factors expression in Ing-ADSCs cultures. (A) Western blot showing relative p-AMPKα protein levels in Ing -ADSCs cultures after metformin treatments at the indicated time points. (B) Western blot showing relative protein levels and (C) Quantitative real-time PCR showing relative mRNA levels of Cidec, Perilipin1, Rab8a, and Pparγ in Ing-ADSCs cultures after metformin treatments with or without CC. (D) Representative images showing the morphology of lipid droplets (Oil Red O staining, upper panel; Bodipy staining, green, DAPI, blue, lower panel) in Ing -ADSCs cultures after metformin treatments with or without CC. Scale bars represent 25 μm for D. (E,F) The diagrams showing the relative optical density of isopropanol extraction (E), average area of lipid droplets (F) and relative mRNA levels of Acc, Fasn, and (G) Dgat2 expression in differentiated adipocytes from Ing-ADSCs cultures after metformin treatments with or without CC. A one-factor or two-factor ANOVA followed by a Tukey’s test was used to make group comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 versus metformin group or group at corresponding indicated time points. # p < 0.01 versus metformin + CC group. Acc, acetyl-CoA carboxylase; CC, Compound C; Cidec, cell death-inducing DFFA-like effector C; Dgat2, diacylglycerol acyltransferase 2; Ing-ADSCs, adipose-derived stem cells (ADSCs) from the inguinal fat pads; Fasn, fatty acid synthesis; LDs, lipid droplets; Pparγ, peroxisome proliferator-activated receptor γ; Rab8a, ras-related protein 8a.
Fig 2: Expression of Cidec and its regulatory factors in ADSCs differentiation after metformin treatments. (A–D) Relative Cidec, Perilipin1, Rab8a, and Pparγ protein levels and mRNA levels in the differentiation process of Epi-ADSCs cultures (A,B) and differentiated adipocytes from Epi-ADSCs cultures after metformin treatments (C,D). (E–H) Relative Cidec, Perilipin1, Rab8a, and Pparγ protein levels and mRNA levels in the differentiation process of Ing-ADSCs cultures (E,F) and differentiated adipocytes from Ing-ADSCs cultures after metformin treatments (G,H). A one-factor ANOVA followed by a Tukey’s test was used to make group comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control group or group at day 0. Cidec, cell death-inducing DFFA-like effector C; Epi-ADSCs, adipose-derived stem cells (ADSCs) from the epididymal fat pads; Ing-ADSCs, adipose-derived stem cells (ADSCs) from the inguinal fat pads; Pparγ, peroxisome proliferator-activated receptor γ; Rab8a, ras-related protein 8a.
Fig 3: Metformin inhibits Cidec and its regulatory factors expression partly via AMPK activation in Epi-ADSCs cultures. (A) Western blot showing relative p-AMPKα protein levels in Epi-ADSCs cultures after metformin treatments at the indicated time points. (B) Western blot showing relative protein levels and (C) Quantitative real-time PCR showing relative mRNA levels of Cidec, Perilipin1, Rab8a, and Pparγ in Epi-ADSCs cultures after metformin treatments with or without CC. (D) Representative images showing the morphology of lipid droplets (Oil Red O staining, upper panel; Bodipy staining, green, DAPI, blue, lower panel) in Epi-ADSCs cultures after metformin treatments with or without CC. Scale bars represent 25 μm for D. (E–G) The diagrams showing the relative optical density of isopropanol extraction (E), area of LDs (F), and relative mRNA levels of Acc, Fasn, and Dgat2 expression in differentiated adipocytes from Epi-ADSCs cultures after metformin treatments with or without CC. A one-factor or two-factor ANOVA followed by a Tukey’s test was used to make group comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 versus metformin group or group at corresponding indicated time points. # p < 0.01 versus metformin + CC group. Acc, acetyl-CoA carboxylase; CC, Compound C; Cidec, cell death-inducing DFFA-like effector C; Dgat2, diacylglycerol acyltransferase 2; Epi-ADSCs, adipose-derived stem cells (ADSCs) from the epididymal fat pads; Fasn, fatty acid synthesis; LDs, lipid droplets; Pparγ, peroxisome proliferator-activated receptor γ; Rab8a, ras-related protein 8a.
Fig 4: Association between gp78 and cidec in hepatic steatosis. (A) Nuclei were stained with DAPI, (a) gp78 was visualized (fluorescein isothiocyanate conjugated secondary antibody; red staining). (b) Cidec was visualized (tetraethyl rhodamine isothiocyanate conjugated secondary antibody; green staining); (c) Colocalization of gp78 and cidec (yellow) visualized on the surface of lipid droplets using immunofluorescent microscopy and identified by coimmunoprecipitation (scale bar=15 µm). (B) Gp78 and Cidec direct interaction detected by coimmunoprecipitation assay. gp78, glycoprotein; cidec, cell death-inducing DFFA-like effector c.
Fig 5: The change of adipocyte‐related gene on obese mice with VitD3 intervention. Protein (right: Western blot) and mRNA (left: RT‐qPCR) expression level of ATGL (a), Fsp27 (b), PPAR γ (c), FABP4 (d) of epiWAT in all groups (n = 4–6 per group). All experiments were repeated at least three times. Data are expressed as the mean ± SD. Intervention groups are analyzed by one‐way ANOVA following Dunnett's multiple comparisons, *p < .05, as compared with HFD + DMSO group. Comparison of CON vs. HFD, significance was determined by Student's t‐test analysis, # p < .05.
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