Fig. 4

Depletion of Foxf2 attenuates TGFβ-induced apoptosis and the expression of the proapoptotic protein Noxa. a Downregulation of Foxf2 promotes cell proliferation. shFoxf2- and shCtrl-expressing NMuMG cells were treated with transforming growth factor (TGF)β for the times indicated and counted using a Neubauer chamber. b Foxf2 depletion decreases apoptosis during TGFβ-induced EMT. shFoxf2- and shCtrl-expressing NMuMG cells were treated with TGFβ for the times indicated, and apoptosis was detected by Annexin-V staining and flow cytometry analysis. c TGFβ induces classical caspase-mediated apoptosis dependent on the upregulation of Foxf2. Immunoblotting analyses of the same experiment as shown in Fig. 1d for cleaved caspase-3 and PARP in shFoxf2- and shCtrl-expressing NMuMG cells treated with TGFβ for the times indicated. Actin was used as a loading control. d Knockdown of Foxf2 attenuates the upregulation of Noxa expression. Noxa mRNA levels in shFoxf2- and shCtrl-expressing NMuMG cells treated with TGFβ for the times indicated were determined by quantitative RT-PCR. Values were normalized to RPL19 and presented as fold changes compared with untreated shCtrl NMuMG cells. e Foxf2 regulates Noxa expression by direct transcriptional activation. Chromatin immunoprecipitation of HA-tagged Foxf2 was performed either on Foxf2-expressing or control NMuMG cells treated for 2 days with TGFβ. Immunoprecipitated DNA fragments were quantified by quantitative PCR using primers covering base pairs −696 to −499 of the noxa promoter region. Enrichment (IP/input) for specific primers was calculated relative to primers covering an intergenic region. f Noxa depletion significantly decreases TGFβ-induced apoptosis. shFoxf2- and shCtrl-expressing NMuMG cells were transfected with control siRNA (siCtrl) and two different siRNAs specific for murine Noxa (siNoxa #1, siNoxa #3) and incubated with TGFβ for 4 days. The extent of apoptosis was measured by Annexin-V staining and flow cytometry. g The impairment of Noxa expression leads to increased cell proliferation. shFoxf2- and shCtrl-expressing NMuMG cells were transfected with control siRNA (siCtrl) and two different siRNAs specific for murine Noxa (siNoxa #1, siNoxa #3) and incubated with TGFβ for the times indicated. Cell numbers were determined using a Neubauer chamber. Results show the mean ± SEM of three independent experiments. Statistical values were calculated using paired/unpaired two-tailed t test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001