Figure 6

Blocking Fstl1-mediated ciliogenesis inhibits 3T3-L1 preadipocyte differentiation. (A) Western blot of Fstl1 showing upregulation of the protein upon induction to differentiate and the levels of the protein in Fstl1 KD 3T3-L1 cells. The data is representative of two independent experiments. Full-length blots are shown in Figure S9B. (B) Clonal expansion in 3T3-L1 cells upon induction to differentiate was measured by a pulse of BrdU and immunofluorescence. The difference in the percentage of BrdU positive cells between induced an uninduced cultures is shown. Fstl1 KD does not inhibit clonal expansion. Result of two independent experiments with three biological replicates each. Approximately 500 cells were counted per condition, per experiment. (C and G) The percentage of ciliated cells was analyzed in 3T3-L1 cells at day 4 (C) and day 10 (G) of differentiation. Data are shown as a line at the mean and error bars represent 95% confidence intervals. (D and H) Cilia length was measured in 3T3-L1 cells at day 4 (D) and day 10 (H). Approximately 100 cilia were measured per condition, per experiment. Scatter plots with a line at the mean are shown and error bars represent 95% confidence intervals. (E and I) Micrographs showing examples of cilia used in the analysis at D4 and D10 respectively. Basal bodies (γ-tubulin) are in cyan, cilia (acetylated tubulin) are in red and nuclei (DAPI) are in blue. Scale bars represent 5μm. (F and J) qRT-PCR analysis of the adipocyte differentiation markers Pparγ, Cebpα, Fabp4, Scd1 and Atgl in 3T3-L1 cells at day 4 (F) and day 10 (J) of differentiation. Bars represent the fold change upon induction of each gene relative to Gapdh comparing induced with uninduced cells. Error bars represent standard deviation. (K) Oil red staining comparing lipid accumulation between control and Fstl1 KD cells. The data shown are representative of two independent experiments. ns: P > 0.05; **P = 0,001-0,01; ***P = 0,0001–0,001 and ****P < 0,0001, hypothesis test for proportions, Kruskal Wallis or t-test.