Fig 1: Model of BTG3 in the regulation of adipogenesis and the development of skin cancer.Our data are consistent with a role of BTG3 in safeguarding the functional interplay between keratinocytes and adipocytes. In the absence of BTG3, keratinocytes, by releasing IL1α, IL10 and CCL4, promote their own mesenchymal transition by an autocrine mechanism and adipocyte differentiation through paracrine. The latter, in turn, fuels further keratinocyte proliferation and migration by releasing EGF, CCL20, and FGF7, thus forming a feedforward loop to promote skin oncogenesis.
Fig 2: BTG3-KO keratinocytes release IL1α, IL10, and CCL4 to promote 3T3-L1 adipogenic differentiation.a Schematic workflow of cytokine antibody array analysis. CM was collected after 48 h of culture, and analyzed using a human cytokine antibody array. BTG3-KO CM was a mixture from the three KO clones indicated. b The levels of factors and cytokines, including IL1α, IL10, and CCL4, were increased in BTG3-KO CM. CM from (a) was subjected to antibody array analysis. Signals from duplicated spots were quantified and compared between parental and BTG3-KO cells. CCL7 is shown as an unaltered control. c mRNA expression levels of IL1A, IL10, CCL4, and VEGFD, but not CCL7, were increased in BTG3-KO HaCaT cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed and results from three independent experiments are shown. d–i Elevated expression levels of IL1α, IL10, and CCL4 in the back skin of Btg3-KO mice. 8-week-old WT and Btg3-KO mice were shaved and their back skin was collected 3 weeks later and embedded for immunohistochemical (IHC) analysis using antibodies against IL1α d, IL10 f, and CCL4 h. Examples of positively stained cell are indicated by arrows. Quantified results (n = 6) are shown in e, g, and i, respectively. j Addition of recombinant IL1α, IL10, and CCL4 to parental CM promotes 3T3-L1 differentiation. Adipogenesis was measured by Oil Red O staining followed by quantification. k Antibody-mediated neutralization of IL1α, IL10 and CCL4 in BTG3-KO CM reduced the effect on 3T3-L1 adipogenic differentiation. *P < 0.05. **P < 0.01.
Fig 3: NFκB mediates the effect of BTG3 ablation on adipocyte differentiation.a Increased expression of IL1A, IL10, and CCL4 in BTG3-KO HaCaT cells required NFκB. RT-qPCR was performed with RNA prepared from control or RELA-siRNA-transfected parental and BTG3-KO HaCaT cells. Expression levels of IL1A, IL10, and CCL4 were reduced as a result of RELA knockdown. b, c NFκB activity was inhibited by ectopic expression of BTG3. NF-κB luciferase reporter assays were conducted with or without overexpression of myc-BTG3 in the presence or absence of TNFα in parental or BTG3 KO HaCaT cells by transient transfection. *P < 0.05. **P < 0.01. Overexpression of BTG3 was assessed by western blotting c. d BTG3 KO increased nuclear distribution of p65 after UV treatment. Cytosolic and nuclear fractionation was performed with UV-irradiated parental and BTG3-KO (N7 and B6) HaCaT cells. The distribution of p65 was assessed by immunoblotting. Lamin A/C and α-tubulin were used as the nuclear and cytosolic markers, respectively. Numbers indicate relative levels of p65 after normalization to either α-tubulin or Lamin A/C. e Re-expression of BTG3 in BTG3 KO HaCaT cells decreased p65 nuclear distribution. f, g BTG3 interacted with p65 in vivo as demonstrated by coimmunoprecipitation f and in vitro in a GST pulldown assay with recombinant GST-BTG3 proteins g. 293 T cells were transfected with the indicated constructs and immunoprecipitation was conducted with either anti-Flag (M2) or anti-HA antibody. h BTG3 competed with p300 for binding p65 in 293 T cells as revealed by coimmunoprecipitation assays.
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