Fig 1: FNDC5 silencing in C3H10T1/2 adipocytes. (A) FNDC5 gene expression inhibition compared to control cells; (B) Representative images of FNDC5 immunofluorescent staining in control and FDNC5 silenced cells (FNDC5 in green; nuclear staining by DAPI in blue); (C) 2-DE immunodetection of FNDC5/irisin in control and FNDC5-KO cell lysates. Note that representative immunoblot images have been cropped from the film in Supplementary Figure 8A and B; (D) 2-DE immunodetection of FNDC5/irisin in control and FNDC5-KO cells secretomes. Note that representative immunoblot images have been cropped from the film in Supplementary Figure 8C and D; (E) Real time proliferation measurement of control vs. FNDC5-KO cells during 6 days; (F) Real time differentiation monitoring of control vs. FNDC5-KO cells from non differentiated state to differentiation day 4; (G) mRNA expression of differentiation markers throughout differentiation of FNDC5-KO C3H10T1/2 cells; (H) Differentiation markers expression fold change of FNDC5-KO cells over wild type; (I) Gene expression fold change at day 8; (J) UCP1 gene expression of control and FNDC5-KO cells differentiated in the presence of intact wild type secretome or blocked with anti-FNDC5 antibody (n = 4 independent experiments; p = 0.174) One way ANOVA Kruskal-Wallis test followed by Dunn’s multiple comparison.
Fig 2: Human obese VAT and SAT secreted factors alters normal adipose cells gene expression during differentiation. (A) FNDC5; (B) UCP1; (C) PGC1a; (D) GLUT4; (E) PPAR?; and (F) Adiponectin gene expression in murine C3H10T1/2 pre-adipocytes (day 2) differentiated in the presence of obese VAT and SAT secreted factors with or without blocking soluble FNDC5/irisin during 24 hours (n = 4 independent human obese secretomes). The effect of plasma from the same obese patients is also shown for each analyzed gene. Histograms show the quantitative expression levels towards control non-treated cells for data normalization [One way ANOVA Kruskal-Wallis test followed by Dunn’s multiple comparison]. *p < 0.05, **P < 0.01, and ***p < 0.001 versus control non-treated cells. CONT: cells without treatment; VAT and SAT SEC: cells treated with 10% obese SAT or VAT secretome; VAT and SAT BLOCK: cells treated with obese VAT and SAT secretome with antibody blocked-irisin; PLASMA: cells treated with 10% obese plasma.
Fig 3: Only differentiated C3H10T1/2 murine mesenchimal stem cells express and secrete FNDC5/irisin. (A,B) mRNA expression and representative protein immunoblots of differentiation markers throughout differentiation of C3H10T1/2 cells. 2E(-??Ct) and error values and representative immunoblot film images from where figures where cropped are shown in Supplementary Figure 5; (C) representative images of pre-adipocytes and mature C3H10T1/2 cells stained with oil red or analyzed for FNDC5 detection by immunocitochemistry (DAB) and immunofluorescence (Cy3) with and without blocked antibody; (D) representative immunoblot against FNDC5/irisin in differentiated and non-differentiated cells showing all the detected bands along differentiation at secreted and intracellular level; (E) FNDC5 immunoblot of secretomes and cellular lysates from pre-adipocytes and mature differentiated cells at day 6 with and without blocked FNDC5 antibody; (F) 2-DE immunoblot of immunoprecipitated FNDC5 from pre-adipocytes and mature differentiated cells secretomes showing a 12 kDa spot only present in adipocytes (pointed with and arrow). Note that representative immunoblot images have been cropped from the film in Supplementary Figure 6A; (G) image showing irisin spot disappearance pointed with and arrow after using a blocked antibody. Note that representative immunoblot images have been cropped from the film in Supplementary Figure 6B. DAB, diaminobenzidine; Pept: commercial irisin peptide.
Fig 4: Functional analysis of FNDC5-KO adipocytes. (A) Oil red quantification of lipid accumulation along differentiation of control vs. FNDC5-KO adipocytes (n = 3 independent differentiation experiments) Two-way ANOVA followed by Bonferroni post hoc; (B) Representative images of control and FNDC5-KO cells stained with oil red until differentiation day 8; (C) Insulin sensitivity of control and FNDC5-KO cells assayed by P-Akt levels immunodetection after insulin time course stimulation; (D) Insulin resistance assay after high glucose/high insulin treatment. Representative immunoblots of control and FNDC5-KO cells stimulated during 10 minutes with insulin. Data is represented towards basal P-Akt levels (time 0) and corrected towards total Akt (n = 3 independent experiments) One way ANOVA Kruskal-Wallis test followed by Dunn’s multiple comparison. (E) Protein phosphorylation array analysis showing the activation of 18 proteins under Akt pathway, after 10 min insulin (100 mM) stimulation (n = 4 independent experiments) Mann-Whitney U test [p-RAS40 p = 0.0265; p-PTEN p = 0.0294; p-Akt p = 0.286; p-P53 p = 0.0286; p-RSK2C p = 0.0286; p-BAD p = 0.286]. Statistical significance is represented as *p < 0.05, **P < 0.01, and ***p < 0.001.
Fig 5: Human obese VAT and SAT secreted factors alters differentiated adipose cells gene expression. (A) FNDC5, and (B) UCP1 gene expression in murine C3H10T1/2 differentiated adipocytes at day 10 after treatment with obese VAT and SAT secreted factors during 24 hours (n = 4 independent human obese secretomes). Histograms show the quantitative expression levels towards control non-treated cells for data normalization [One way ANOVA Kruskal-Wallis test followed by Dunn’s multiple comparison]. *p < 0.05 and **P < 0.01 versus control non-treated cells. CONT: differentiated cells without treatment; OB VAT/SAT: differentiated cells treated during 24 hours with obese VAT and SAT secretomes.
Supplier Page from Abcam for FNDC5 peptide